WO2021155775A1

WO2021155775A1 - Method and kit for dectecting target nucleic acid

Info

Publication number: WO2021155775A1
Application number: PCT/CN2021/074772
Authority: WO
Inventors: 吴尧; 史才雪; 李秋实
Original assignee: 苏州克睿基因生物科技有限公司
Priority date: 2020-02-03
Filing date: 2021-02-02
Publication date: 2021-08-12

Abstract

A method for detecting a target nucleic acid in a sample, comprising: amplifying a target nucleic acid in a sample to obtain a single-stranded amplification product; contacting the single-stranded amplification product with a V-type CRISPR/Cas effector protein, a gRNA, and an indicator nucleic acid, the gRNA comprising a region bonded to the V-type CRISPR/Cas effector protein and a guide sequence hybridized to a target sequence in the target nucleic acid, the sequence adjacent to the 5' end or the 3' end of the target sequence in the target nucleic acid not comprising a PAM sequence, the indicator nucleic acid being a single stranded nucleic acid molecule and not hybridized to the guide sequence of the gRNA; and detecting a detectable signal generated by the indicator nucleic acid cleaved by the V-type CRISPR/Cas effector protein, thereby detecting the target nucleic acid.

Description

Method and kit for detecting target nucleic acid

Technical field

This application relates to the field of biomedicine, in particular to a method and kit for detecting target nucleic acid in a sample.

Background technique

Nucleic acids contain the universal information characteristics of organisms. The ability to quickly detect nucleic acids with high sensitivity and single-base specificity on a portable platform is of great significance for the diagnosis and monitoring of many diseases, providing valuable epidemiological information, and can be used as a general scientific tool. Although many nucleic acid detection methods have been developed (Du et al. 2017; Green et al. 2014; Kumar et al. 2014; Pardee et al. 2014; Pardee et al. 2016; Urdea et al. 2006), all There will inevitably be pros and cons between sensitivity, specificity, simplicity and speed. For example, the qPCR method is sensitive but expensive, and relies on complex instruments, thus limiting its usability in non-laboratory environments. Other methods, such as the new method that combines isothermal nucleic acid amplification with a portable platform (Du et al. 2017; Pardee et al. 2016), provide high detection specificity in point-of-care (POC) settings, but some are due to The sensitivity is low and the application is limited.

In addition, based on the progress of the CRISPR/Cas system in recent years, Zhang Feng’s team used Cas13a and RPA isothermal amplification technology to develop the SHERLOCK system to detect Zika and Dengue viruses (Zhang et al. Science. 2017 Apr 28; 356( 6336):438–442); Jennifer’s research group found that Cas12a and other V-type Cas effector proteins have the characteristics of target-activated non-specific ssDNase cleavage. The DETECTR nucleic acid detection method was created by combining Cas12a with isothermal amplification, which can Detection of human papillomavirus in patient samples (Doudna JA et al. Science. 2018 Apr 27; 360(6387):436-439).

However, the target nucleic acid targeted by the SHERLOCK system is RNA. After the target nucleic acid undergoes exponential amplification at the DNA level, the amplified product must be transcribed into RNA to trigger the detection module of the SHERLOCK system for detection. This step introduces unnecessary non-specificity, increases the reaction time, aggravates the complexity of the reaction system, and also increases the detection cost. Although the DETECTR system does not need to transcribe the amplified product into RNA, the cleavage of the nucleic acid to be detected by Cas12a will greatly affect the amplification reaction of the target nucleic acid. Therefore, in the DETECTR nucleic acid detection method, the amplification of the nucleic acid to be detected The detection using V-type Cas effector protein needs to be performed separately to avoid the reduction of amplification efficiency due to the cleavage of the amplification template by the V-type Cas effector protein, which increases the complexity of the operation and inevitably brings expansion. Increase the risk of product contamination and cross-contamination; and if the amplification of the nucleic acid to be detected in the DETECTR system is carried out at the same time as the detection using the V-type Cas effector protein in the same system, it will be due to the V-type Cas effector protein. The cleavage of the enhanced template thus affects the sensitivity and specificity of the detection.

Summary of the invention

This application provides a method for detecting target nucleic acid in a sample, which includes:

a) Amplifying the target nucleic acid in the sample to obtain a single-stranded amplification product;

b) contacting the single-stranded amplification product with i) a type V CRISPR/Cas effector protein, ii) gRNA, and iii) an indicator nucleic acid, wherein the gRNA includes a region that binds to the type V CRISPR/Cas effector protein and A guide sequence that hybridizes with the target sequence in the target nucleic acid, the sequence adjacent to the 5'end or 3'end of the target sequence in the target nucleic acid does not contain a PAM sequence, and the indicator nucleic acid is a single-stranded nucleic acid molecule and does not interact with The guide sequence of the gRNA hybridizes; and

c) Measure the detectable signal generated by the indicator nucleic acid after being cleaved by the V-type CRISPR/Cas effector protein, so as to detect the target nucleic acid.

In some embodiments, the a) and the b) are carried out in the same reaction system.

In some embodiments, the V-type CRISPR/Cas effector protein, the gRNA and the indicator nucleic acid are added to the reaction system containing the single-stranded amplification product.

In certain embodiments, the amplification includes asymmetric amplification.

In some embodiments, the target nucleic acid comprises double-stranded DNA and/or single-stranded DNA.

In some embodiments, a forward amplification primer and a reverse amplification primer are used in the amplification, and the concentration ratio of the forward amplification primer and the reverse amplification primer is 1:10-1 :40.

In some embodiments, a forward amplification primer and a reverse amplification primer are used in the amplification, and the ratio of the reverse amplification primer and the forward amplification primer is 1:10-1:40. .

In certain embodiments, the amplification comprises isothermal amplification.

In certain embodiments, the amplification comprises recombinase polymerase amplification (RPA).

In some embodiments, the sample contains target nucleic acids derived from one or more of the following: cells of an organism, body fluids of an organism, and/or nucleic acid molecules of an organism.

In some embodiments, the sample contains target nucleic acid derived from a virus, and the virus is selected from the group consisting of African swine fever virus, HCV and HIV.

In certain embodiments, the contacting occurs under in vivo, in vitro, or ex vivo conditions.

In some embodiments, the type V CRISPR/Cas effector protein includes Cas12 protease.

In certain embodiments, the Cas12 protease is selected from the group consisting of Cas12a protease and Cas12b protease.

In some embodiments, the type V CRISPR/Cas effector protein comprises the amino acid sequence set forth in any one of SEQ ID NO. 1-11.

In some embodiments, the indicator nucleic acid comprises a detectable label.

In some embodiments, the detectable label includes a fluorescent label.

In some embodiments, the detectable signal includes a fluorescent signal.

In some embodiments, the concentration of the target nucleic acid is at least 1*1E2 copies or more.

On the other hand, the present application also provides a kit for detecting target nucleic acid in a sample, which comprises i) type V CRISPR/Cas effector protein, ii) gRNA and iii) indicator nucleic acid; wherein the gRNA contains the V The region where the type CRISPR/Cas effector protein binds and the guide sequence that hybridizes to the target sequence in the target nucleic acid; and the guide sequence is designed to be in the 5'end or 3'end sequence of the target sequence to which it hybridizes No PAM sequence; the indicator nucleic acid is a single-stranded nucleic acid molecule and does not hybridize with the guide sequence of the gRNA.

In some embodiments, the kit further includes reagents for obtaining a single-stranded amplification product containing the target nucleic acid.

In some embodiments, the reagents for obtaining single-stranded amplification products include reagents required for asymmetric amplification.

In some embodiments, the reagent for asymmetric amplification includes an upstream primer that amplifies the target nucleic acid and/or a downstream primer that amplifies the target nucleic acid.

In some embodiments, the reagents for obtaining single-stranded amplification products include reagents required for isothermal amplification.

In some embodiments, the reagents required for the isothermal amplification include RPA recombinase, RPA polymerase and/or RPA buffer.

In some embodiments, the reagent for the single-stranded amplification product of the target nucleic acid and the V-type CRISPR/Cas effector protein and the gRNA are located in the same container.

In some embodiments, the indicator nucleic acid comprises a detectable label.

In some embodiments, the detectable label includes a fluorescent label.

Those skilled in the art can easily perceive other aspects and advantages of the present application from the detailed description below. In the following detailed description, only exemplary embodiments of the present application are shown and described. As those skilled in the art will recognize, the content of this application enables those skilled in the art to make changes to the disclosed specific embodiments without departing from the spirit and scope of the invention involved in this application. Correspondingly, the drawings and descriptions in the specification of the present application are only exemplary, and not restrictive.

Description of the drawings

The specific features of the invention involved in this application are shown in the appended claims. The characteristics and advantages of the invention involved in this application can be better understood by referring to the exemplary embodiments and the accompanying drawings described in detail below. A brief description of the drawings is as follows:

Figure 1 shows the VP72-gRNA/cas12a target nucleic acid detection result in this application.

Figure 2 shows the K205R-gRNA/cas12a target nucleic acid detection result in this application.

Figure 3 shows the target nucleic acid detection results of the combination of primer-F/primer-R upstream and downstream in different ratios in this application.

Figure 4 shows the target nucleic acid detection results of the primer-F1/primer-R1 upstream and downstream combinations in different ratios in this application.

Figure 5 shows the detection results of different concentrations of target nucleic acid when the upstream and downstream ratio of primer-F/primer-R in the present application is 20:1.

Figure 6 shows the detection results of different concentrations of target nucleic acid when the upstream and downstream ratio of primer-F/primer-R is 1:1 in the present application.

Figure 7 shows the comparison results of detection of target nucleic acids at different concentrations when the upstream and downstream ratios of primer-F/primer-R in the present application are 1:1 and 20:1.

Figure 8 shows the detection results of different concentrations of target nucleic acid when the upstream and downstream ratio of primer-F1/primer-R1 in the present application is 20:1.

Fig. 9 shows the detection results of different concentrations of target nucleic acid when the upstream and downstream ratio of primer-F1/primer-R1 is 1:1 in the present application.

Figure 10 shows the comparison results of the detection of target nucleic acids at different concentrations when the upstream and downstream ratios of primer-F1/primer-R1 in the present application are 1:1 and 20:1.

Detailed ways

The following specific examples illustrate the implementation of the invention of this application. Those familiar with this technology can easily understand the other advantages and effects of the invention of this application from the content disclosed in this specification.

The application is further described as follows: In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology related terms and laboratory procedures used herein are all terms and routine procedures widely used in the corresponding fields. At the same time, in order to better understand the present invention, definitions and explanations of related terms are provided below.

In this application, the terms "CRISPR/Cas system", "CRISPR/Cas system" or "CRISPR system" are used interchangeably, and generally refer to a group of molecules including RNA-guided nucleases or other effector molecules and gRNA molecules, The molecule can direct and realize that the RNA-guided nuclease or other effector molecules modify the nucleic acid at the target nucleic acid, for example, create a gap in the target nucleic acid or cause the target nucleic acid to degrade.

In this application, the term "CRISPR" generally refers to clustered regularly interspaced short palindromic repeats. This sequence usually refers to the first gene sequence found in prokaryotes, which contains the gene fragments of viruses that have attacked the prokaryotes. The organism uses these gene fragments to recognize and resist the same virus attack and destroy it. Nucleic acid molecule. This type of genome constitutes a key part of the prokaryotic immune system. CRISPR usually includes multiple highly conserved repeats and spacers that are different from each other, and the two appear alternately. The length of the repetitive sequence is generally 23 to 50 bp, and the average length is about 31 bp. The repetitive sequence is highly conserved in the same CRISPR site, and there can be a difference of 1 to 5 bases; but between microorganisms, or between CRISPR sites at different positions on the genome of the same microorganism, the conserved sequence of the repetitive sequence big difference. The spacers distributed between the repeats generally consist of 17 to 84 bp, with an average length of about 36 bp. Spacers are poorly conserved. Even in the same CRISPR site, there are basically no identical spacers.

In this application, the terms "Cas", "Cas protein" or "CRISPR/Cas effector protein" are used interchangeably, and generally refer to CRISPR-associated protein. The Cas protein is the main executor to realize the functions of the CRISPR system, such as the acquisition of spacers or the shearing of DNA molecules. In some embodiments, "CRISPR/Cas effector protein can be an enzyme with DNA cleavage activity in the CRISPR/Cas system, which can cut double-stranded DNA molecules or/and single-stranded DNA molecules. The Cas protein is a larger Polymorphism family proteins. Genes encoding Cas proteins are generally located downstream of the CRISPR sequence, and sometimes scattered in the genome. Current studies have found that the CRISPR-Cas system mainly includes two categories, including multi-subunit protein effectors Type 1 of the complex and type 2 of the single-subunit protein effector complex. The CRISPR-Cas system of type 1 is more common in bacteria and archaea (including all hyperthermophiles), and this type of protein accounts for about all 90% of the identified Cas proteins (Makarova KS, et al. An updated evolutionary classification of CRISPR-Cas systems. Nat. Rev. Microbiol. 2015; 13:722-736). The Cas proteins available for this system mainly include I, Type III and IV effector proteins. The type 2 CRISPR-Cas system is almost exclusively found in bacteria. The Cas proteins available for this system mainly include type II, V, and VI effector proteins, which account for about 10% of the Cas protein. Commonly used Cas9 protein (type II), and Cas12 (type V), Cas13 (VI) type, and Cas14 (type V) proteins (Chylinski K. et al., Nucleic Acids Res. 2014; 42:6091-6105; Shmakov S, etc.) , Mol.Cell.2015,60:385–397; Sergey Shmakov et al., Nat Rev Microbiol.2017 March; 15(3):169–182; Doudna J. et al., Science.2018 Nov16;362(6416):839- 842); Among them, V-type CRISPR/Cas system and V-type effector protein (V-type CRISPR/Cas effector protein) can be found in Shmakov et al., Nat Rev Microbiol. 2017 March; 15(3): 169-182, Koonin et al., CurrOpin Microbiol .2017 June; 37:67-78.

In this application, the terms "gRNA molecule" or "guide RNA", "guide RNA molecule", and "gRNA" are used interchangeably, and generally refer to nucleases or other effector molecules that can promote specific guidance of RNA guidance (generally with gRNA molecule complex) to the nucleic acid molecule on the target nucleic acid. In certain embodiments, the guidance is achieved by hybridizing a portion of the gRNA to DNA (eg, via a gRNA steering domain or guide sequence) and binding a portion of the gRNA molecule to an RNA-guided nuclease or other effector molecule. In some embodiments, the gRNA molecule is composed of a single continuous polynucleotide molecule, such as crRNA; in some embodiments, the gRNA molecule may be composed of multiple (such as two ) Polynucleotide molecular composition.

In the present application, the "leader sequence" generally refers to a nucleic acid sequence in the gRNA molecule that can bind to the target sequence in whole or in part by Watson-Crick base pairing and/or G/U base pairing.

In this application, the "PAM sequence" generally refers to a nucleic acid sequence in the target nucleic acid that can be recognized by the Cas protein in the CRISPR system, and can usually be located at the 3'end or the gRNA complementary sequence in the target sequence according to the difference of the Cas protein. 5'end. For example, the PAM sequence for Cas12 can be located at the 5'end of the target sequence. The PAM sequence can usually consist of 2-6 nucleotides.

In this application, the terms "target nucleic acid" and "target polynucleotide" generally refer to a target nucleotide sequence that needs to be identified, detected or located. When the CRISPR system is used as a method of recognition, detection or positioning, the target nucleic acid usually contains a continuous or discontinuous sequence that is complementary to the guide sequence, which can become the target sequence. The hybridization between the target sequence and the guide sequence can promote the formation of the CRISPR complex. The target nucleic acid may be double-stranded DNA (dsDNA) or single-stranded DNA (ssDNA). The complete complementarity of the target sequence and the guide sequence is not necessary, and the required condition is that there is sufficient complementarity to cause hybridization and promote the formation of a certain CRISPR complex. The target nucleic acid can be a polynucleotide from any source. For example, the target polynucleotide can be an exogenous polynucleotide that resides in the nucleus of a eukaryotic cell (for example, the target polynucleotide can be a viral genome sequence). ); For example, the target polynucleotide may be a sequence encoding a gene product (for example, a protein) or a non-coding sequence (for example, a regulatory polynucleotide or useless DNA). The CRISPR complex generally refers to a complex formed by gRNA and Cas protein.

In this application, the term "amplification" generally refers to a process in which the copy number of the target nucleic acid is selectively increased while the copy numbers of other genes are not increased proportionally. That is to say, the amplification usually refers to the purposeful amplification of the target nucleic acid, and the non-specific increase in the copy number of sequences other than the target nucleic acid accompanying the process cannot be ruled out. Normally, the non-specific increase is not enough to prevent the detection, recognition or localization of the target nucleic acid.

In this application, the term "single-stranded amplification product" generally refers to a single-stranded DNA sequence obtained by amplification of a target nucleic acid. Generally, the product obtained by amplifying the target nucleic acid contains single-stranded DNA sequence and double-stranded DNA sequence, and the single-stranded DNA sequence can be used as a target for recognition and detection of the CRISPR complex. For example, the single-stranded amplification product may be obtained by asymmetric amplification.

In this application, the term "indicator nucleic acid" generally refers to a single-stranded DNA fragment connected with a pair of fluorescent signal molecules. The emission spectrum of one signal molecule overlaps the region of the absorption spectrum of the other signal molecule in the pair. The fragmentation of the DNA fragment (for example, under the shearing action of the Cas protein) causes the connected fluorescent semi-colon molecules to emit fluorescence or to diminish the fluorescence emitted by the fluorescent signal molecules, thereby achieving the purpose of detection. For example, the fluorescent signal molecule may be a fluorescence resonance energy transfer (FRET) pair or a quencher/fluorescer pair.

In the present application, the terms "hybridization", "hybridizable" or "complementary" generally refer to the nucleus contained in nucleic acid (e.g., RNA, DNA) under conditions in vitro and/or in vivo at a suitable temperature and ionic strength of the solution. The nucleotide sequence enables it to specifically non-covalently bind (ie form Watson-Crick base pairs and/or G/U base pairs) to another nucleic acid sequence. Watson-Crick base pairing includes: adenine/adenosine (A) paired with thymidine/thymine (T), A paired with uracil/uridine (U), and guanine/guanosine (G) paired with cytos. Pyrimidine/cytidine (C) pairing. In some embodiments, the hybridization between two RNA molecules (for example, dsRNA), or the hybridization between a DNA molecule and an RNA molecule (for example, when a DNA target nucleic acid base is paired with a guide RNA, etc.), G can also be U base pairing.

Hybridization requires that the two nucleic acids contain complementary sequences, but possible mismatches between bases cannot be ruled out. The conditions suitable for hybridization between two nucleic acids depend on the length and degree of complementarity of the nucleic acids, which are well known in the art. The greater the degree of complementarity between two nucleotide sequences, the greater the value of melting temperature (Tm) of hybrids of nucleic acids having these complementary sequences. Generally, the length of a hybridizable nucleic acid is 8 nucleotides or more (e.g., 10 nucleotides or more, 12 nucleotides or more, 15 nucleotides or more, 20 nucleotides Acid or more, 22 nucleotides or more, 25 nucleotides or more or 30 nucleotides or more). Those skilled in the art know that the sequence of a polynucleotide need not be 100% complementary to the sequence of its target nucleic acid. For example, it can be 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more More, 98% or more, 99% or more, 99.5% or more complementary. The remaining non-complementary nucleotides can be clustered or interspersed with complementary nucleotides and need not be adjacent to each other or complementary nucleotides. For example, a polynucleotide can hybridize on one or more segments so that no intermediate or adjacent segments are involved in the hybridization event (e.g., loop structure or hairpin structure, "bulge", etc.).

Any available method can be used to determine the percentage of complementarity between fragments of a particular nucleic acid sequence within a nucleic acid. For example, BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi); for example, the PowerBLAST program (Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res .,1997,7,649-656); For example, Gap program (Wisconsin sequence analysis software package, Unix version 8, Genetics Computer Group, University Research Park, Madison Wis).

Complementarity can also be expressed by identity, for example, the Needle program of the EMBOSS software package (EMBOSS: European Molecular Biology Open Software Suite, Rice et al., 2000, Trends in Genetics 16:276-277), version 3.0.0 or more The Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453) executed in the higher version is determined. The optional parameters used are gap penalty 10, gap extension penalty 0.5 and EBLOSUM62 replacement matrix (EMBOSS version of BLOSUM62). Use the output of Needle marked as "longest identity" (obtained with the nobrief option) as the percent identity, and calculate it as follows: (same residue×100)/(length of alignment-in alignment The total number of vacancies in).

In this application, the term "asymmetric amplification" generally refers to a method of setting the concentration or length of upstream and downstream primers to be different to obtain single-stranded nucleic acid (single-stranded DNA or single-stranded RNA) amplification products. Generally, the asymmetric amplification can include the following types: 1) Asymmetric amplification is performed using upstream and downstream primers of different concentrations. As the circulation increases, the primers with a small amount are gradually depleted, and the primers with an excessive amount can continue to amplify to generate single-stranded DNA (Gyllensten and Erlich, Proc, Natl. Acad Sci. USA, 1988, 85: 7652-7656) . 2) Use upstream and downstream primers of different lengths for asymmetric amplification. For example (see Peng Xiaomou et al., Chinese Laboratory Diagnosis, 2002, 6: 206-208) using 34 bases upstream primers and 20 bases downstream primers to asymmetrically amplify the S gene of HBV. In the temperature cycle, increase the annealing temperature, short primers cannot anneal, and long primers can continue the extension reaction to achieve the purpose of preparing single-stranded nucleic acids. 3) Perform a symmetric amplification reaction (ie double-stranded product amplification reaction) first, and then purify the amplified product. Using the purified symmetrical amplified product as a template, add a single primer or unequal primers for asymmetric amplification (Gorelov, et.al.,Biochem.Biophys.Res.Commun.,1994,200:365-369; Scott,et.al.,Lett.Appl.Microbiol.,1998,27:39-44; Guo,et.al. , Genome Res., 2002, 12:447-457). In the above several ways, in some embodiments, it is possible to make adaptive selection, adjustment or modification according to different amplification purposes or amplification conditions, and select an appropriate concentration ratio through routine experiments.

In this application, the term "isothermal amplification" generally refers to a method of performing nucleic acid molecule amplification under constant temperature conditions. The traditional polymerase chain reaction (PCR) usually involves denaturation (about 95°C), annealing (about 5°C lower than the primer Tm value, generally 45-55°C), and extension (about 72°C). , A specific reaction instrument (such as a PCR machine) is required for temperature control, and isothermal amplification does not require the above-mentioned temperature change process, and is completed under a certain constant temperature condition, so there is no need for a PCR machine, and the operation is simple. Isothermal amplification was first published in 2000 by Japanese scholar Notomi on Nucleic Acids Res. Loop-mediated isothermal amplification technology (Loop-mediated isothermal amplification) was developed. Later, a variety of methods for amplifying nucleic acid molecules under isothermal conditions have been developed. For example, Recombinase Polymerase Amplification (RPA) and so on.

In this application, the term "recombinase polymerase amplification (RPA)" usually refers to a method that utilizes recombinase, single-stranded DNA-binding protein (SSB), and single-stranded displacement polymerization. Enzyme (strand-displacing polymerase) isothermal amplification method. RPA was developed and released by the British biotechnology company TwistDx Ltd. (formerly known as ASM Scientific Ltd.). The above-mentioned recombinase, single-stranded DNA binding protein, and single-stranded displacement polymerase are used as the core factors in the RPA process. Among them, The recombinase can combine with the primer to form a protein-DNA complex, find homologous sequences in the double-stranded DNA and make the primer pair with the homologous sequence in the double-stranded DNA. When the primer is positioned to the homologous sequence, stranding will occur. Exchange reaction and start DNA synthesis under the action of single-strand displacement polymerase, and exponentially amplify the target region on the template. The replaced DNA strand binds to the SSB to prevent further replacement. Like PCR, the target nucleic acid is amplified by using two opposite primers. In view of the general properties of enzymes, RPA can be performed under suitable temperature conditions (e.g. 37-42°C), but it is still effective even if it runs slower under other temperature conditions (e.g. room temperature). In some embodiments, other compounds (such as enzymes or indicators) can be used to provide additional functions. For example, the detection of RNA and DNA can be achieved by adding reverse transcriptase to the RPA reaction system without the need for a separate step to produce cDNA; for example, by adding a CRISPR system and a fluorescent indicator to achieve the detection of target nucleic acids.

In this application, the term "Cas12 protease" is also referred to as "Cas12", "Cas12 protein", "Cas12 nuclease", and generally refers to a type of CRISPR-related protein that can detect single-stranded DNA or double-stranded DNA under the guidance of gRNA. DNA is cut. This protein was originally called Cpf1 and was first proposed by Zhang Feng's team (see "Cpf1Is a Single RNA-Guided Endonuclease of a Class 2CRISPR-Cas System", Cell, 2015). In addition, Professor Doudna’s team found that when Cas12a binds to and cleaves the target nucleic acid DNA, it can unexpectedly cut the single-stranded DNA in the system at random, which allows a single-stranded "reporter" nucleic acid molecule to be combined with the Cas12a protein. When Cas12a finds and cuts the target, the reporter molecule will generate a signal reflecting the cutting action.

In this application, the term "comprising" generally refers to the inclusion of explicitly specified features, but not excluding other elements.

In this application, the term "about" generally refers to a range of 0.5%-10% above or below the specified value, such as 0.5%, 1%, 1.5%, 2%, 2.5%, above or below the specified value. Variation within the range of 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10%.

On the one hand, the present application provides a method for detecting target nucleic acid in a sample, which includes:

For example, the above a) and the above b) are carried out in the same reaction system.

For example, the V-type CRISPR/Cas effector protein, the gRNA and the indicator nucleic acid are added to the system for amplifying the target nucleic acid in the sample.

For example, the V-type CRISPR/Cas effector protein, the gRNA and the indicator nucleic acid are added while preparing the system for the amplification of the target nucleic acid in the sample.

For example, the V-type CRISPR/Cas effector protein, the gRNA and the indicator nucleic acid are added to the reaction system containing the single-stranded amplification product.

gRNA (guide RNA or guide RNA), target sequence and protospacer proximity motif (PAM)

In this application, the type V CRISPR/Cas effector protein, like many known CRISPR/Cas endonucleases, can specifically bind and/or cleave the target nucleic acid including: 1) Type V CRISPR/Cas effect The protein recognizes the protospacer-adjacent motif (PAM) in the target nucleic acid and/or 2) guides the base pairing between the RNA and the target sequence.

In the present application, the gRNA may include a type V CRISPR/Cas effector protein (such as Cas12 protein, such as Cas12a, Cas12b, Cas12c, Cas12d, Cas12e) combined to form a ribonucleoprotein complex (RNP), and the complex A single-stranded nucleic acid molecule that targets a specific target sequence within the target nucleic acid. For example, the gRNA may include RNA molecules, DNA/RNA hybrid molecules, that is, the gRNA may include DNA bases in addition to RNA bases.

In the present application, the guide RNA may include a guide sequence and a region that binds to the V-type CRISPR/Cas effector protein (also referred to as a protein binding region or a constant region), and the guide sequence hybridizes with the target sequence of the target DNA. , The constant region can bind to the V-type CRISPR/Cas effector protein.

In the present application, the gRNA can reduce the cleavage/degradation effect of the V-type CRISPR/Cas effector protein on the double-stranded target nucleic acid, and realize the amplification of the double-stranded target nucleic acid and the recognition/cutting of the single-stranded amplification product by the CRISPR/Cas effector protein In the same system.

For example, the ribonucleoprotein complex formed by the gRNA and the V-type CRISPR/Cas effector protein cannot cleave a double-stranded nucleotide sequence, and at the same time can cleave a single-stranded nucleotide sequence.

For example, the guide sequence in the gRNA is complementary to the target sequence (target DNA fragment), and the sequence adjacent to the 5'end or 3'end of the target sequence does not contain the PAM sequence in the target nucleic acid.

For example, no PAM sequence in the 5'end or 3'end of the target sequence may include a PAM sequence that recognizes a double-stranded target nucleic acid by the ribonucleoprotein complex formed by the V-type CRISPR/Cas effector protein and the gRNA, The guide sequence in the gRNA cannot interact with the sequence adjacent to the PAM sequence to initiate the cleavage of the double-stranded target nucleic acid.

For example, the ribonucleoprotein complex can realize the cleavage of the single-stranded amplification product through the recognition of the gRNA and the target sequence in the single-stranded nucleotide that is complementary to the gRNA, without relying on PAM sequence that recognizes single-stranded nucleotides.

For example, the absence of a PAM sequence in the sequence adjacent to the 5'end or 3'end of the target sequence may include: between the PAM sequence and the first nucleotide at the 5'end or 3'end of the target sequence at least There is 1 nucleotide, such as at least 2 nucleotides, such as at least 3 nucleotides, such as at least 4 nucleotides, such as at least 5 nucleotides, such as at least 6 nucleosides Acid, for example, at least 7 nucleotides, for example, at least 8 nucleotides, for example, at least 9 nucleotides, for example, at least 10 nucleotides, for example, at least 11 nucleotides, for example, at least 12 nucleotides, such as at least 13 nucleotides, such as at least 14 nucleotides, such as at least 15 nucleotides, such as at least 16 nucleotides, such as at least 17 nucleotides , Such as at least 18 nucleotides, such as at least 19 nucleotides, such as at least 20 nucleotides, such as at least 21 nucleotides, such as at least 22 nucleotides, such as at least 23 Nucleotides, such as at least 24 nucleotides, such as at least 25 nucleotides, such as at least 26 nucleotides, such as at least 27 nucleotides, such as at least 28 nucleotides, For example, there are at least 29 nucleotides, for example, at least 30 nucleotides.

For example, different Type V CRISPR/Cas effector proteins (e.g., from different species) may require different PAM sequences in the target nucleic acid. Therefore, for the selected specific V-type CRISPR/Cas effector protein, the corresponding PAM sequence is selected. Various methods (including computer methods and/or experimental methods) for identifying suitable PAM sequences are known and conventional in the art, and any convenient method.

For example, the PAM sequence may include: 5'-TTTN-3' (when the V-type CRISPR/Cas effector protein is LbCas12a and/or AsCas12a), 5'-TTN-3' (when the V When the type CRISPR/Cas effector protein is FnCas12a, PmCas12a, MbCas12a, Mb2Cas12a, Mb3Cas12a, TsCas12a, BsCas12a and/or AacCas12a).

For example, the length of the guide sequence is 15-28 nucleotides (nt), such as 15-26 nucleotides, 15-24 nucleotides, 15-22 nucleotides, 15-20 nuclei. Nucleotides, 15-18 nucleotides, 16-28 nucleotides, 16-26 nucleotides, 16-24 nucleotides, 16-22 nucleotides, 16-20 nucleotides , 16-18 nucleotides, 17-26 nucleotides, 17-24 nucleotides, 17-22 nucleotides, 17-20 nucleotides, 17-18 nucleotides, 18 -26 nucleotides, 18-24 nucleotides or 18-22 nucleotides in length.

For example, the length of the leader sequence is 18-24 nucleotides.

For example, the leader sequence is at least 15 nucleotides, for example, at least 16, 18, 20, or 22 nucleotides.

For example, the guide sequence and the target sequence of the target nucleic acid have 80% or more (eg, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more , 98% or more, 99% or more, or 100%) complementarity. For example, the leader sequence may include at least 15 nucleotides that are complementary to the target sequence.

In this application, the constant region may include a V-type CRISPR/Cas effector protein (such as Cas12 protein, such as Cas12a, Cas12b, Cas12c, Cas12d, Cas12e, for example, LbCas12a, AsCasi2a, Frcas12a, PoCas12a, MbCas12a, Mb2Cas12a, Mb3Cas12a, IsCas12a, BsCas12a) are used together with the nucleotide sequence.

For example, the constant region may be 15 or more nucleotides (nt) in length, for example, 18 or more, 20 or more, 21 or more, 22 or more, 23 or more, 24 or more. More, 25 or more, 26 or more, 27 or more, 28 or more, 29 or more, 30 or more, 31 or more, 32 or more, 33 or more, 34 or More, or 35 or more.

For example, the length of the constant region may range from 12 to 100 nucleotides, for example, 12 to 90, 12 to 80, 12 to 70, 12 to 60, 12 to 50, 12 to 40, 15 to 100, 15 To 90, 15 to 80, 15 to 70, 15 to 60, 15 to 50, 15 to 40, 20 to 100, 20 to 90, 20 to 80, 20 to 70, 20 to 60, 20 to 50, 20 to 40 , 25 to 100, 25 to 90, 25 to 80, 25 to 70, 25 to 60, 25 to 50, 25 to 40, 28 to 100, 28 to 90, 28 to 80, 28 to 70, 28 to 60, 28 To 50, 28 to 40, 28 to 100, 29 to 100, 29 to 90, 29 to 80, 29 to 70, 29 to 60, 29 to 50 or 29 to 40 nucleotides.

For example, the constant region of the gRNA may be truncated relative to the corresponding region of the corresponding wild-type gRNA.

For example, the constant region of the gRNA may be extended relative to the corresponding region of the corresponding wild-type gRNA.

For example, the constant region may be located at the 5'end or 3'end of the guide sequence.

For example, the constant region may comprise a complementary RNA sequence, which forms an RNA duplex (dsRNA) by self-folding.

For example, the length of the RNA duplex (dsRNA) can be 2 to 12 base pairs, for example, 2 to 10, 2 to 8, 2 to 6, 2 to, 2 to 4, 2 to 3 base pairs ; For example, the length of the RNA duplex (dsRNA) can be 3 to 12 base pairs, 3 to 10 base pairs, 3 to 8 base pairs, 3 to 6 base pairs, 3 to Base pairs, 3 to 4 base pairs, 4 to 12 base pairs, 4 to 10 base pairs, 4 to 8 base pairs, 4 to 6 base pairs or 4 to 5 bases Base pair.

For example, the constant region may include a length of 2 or more base pairs, such as 3 or more, 4 or more, 5 or more, 6 or more or 7 DsRNA duplexes of two or more base pairs.

For example, the constant region of the guide RNA may include a dsRNA duplex that is longer than the dsRNA duplex of the corresponding wild-type guide RNA.

For example, the constant region of the guide RNA may include a dsRNA duplex that is shorter than the dsRNA duplex of the corresponding wild-type guide RNA.

For example, the length of the constant region of the guide RNA can be 12 to 100 nucleotides, such as 12 to 90, 12 to 80, 12 to 70, 12 to 60, 12 to 50, 12 to 40, 15 to 100, 15 to 90, 15 to 80, 15 to 70, 15 to 60, 15 to 50, 15 to 40, 20 to 100, 20 to 90, 20 to 80, 20 to 70, 20 to 60, 20 to 50, 20 to 40, 25 to 100, 25 to 90, 25 to 80, 25 to 70, 25 to 60, 25 to 50, 25 to 40, 28 to 100, 28 to 90, 28 to 80, 28 to 70, 28 to 60, 28 to 50, 28 to 40, 29 to 100, 29 to 90, 29 to 80, 29 to 70, 29 to 60, 29 to 50, or 29 to 40 nucleotides and the constant region of the guide RNA The length of can be in the range of 28 to 100 nucleotides, for example, the length of the constant region of the guide RNA is in the range of 28 to 40 nucleotides.

For example, the constant region sequence of the guide RNA may include the nucleotide sequence shown in any one of SEQ ID NOs. 12-18.

For example, the constant region of the gRNA may also include the constant region sequence shown in any one of SEQ ID NO. 12-18 having 70% or higher identity (for example, 80% or higher, 85% Or higher, 90% or higher, 95% or higher, 98% or higher, 99% or higher, or 100% identity).

For example, the guide RNA (gRNA) may include a nucleotide sequence targeting African swine fever virus VP72 and/or African swine fever virus K205R.

For example, the guide RNA (gRNA) may include a nucleotide sequence as shown in any one of SEQ ID NOs. 19-26.

V-type CRISPR/Cas effector protein

In this application, type V CRISPR/Cas effector protein is a subtype of the 2 types of CRISPR/Cas effector protein, including any non-target ssDNA cleavage once activated (by hybridizing with its related guide RNA and target DNA) Active V-type CRISPR/Cas effector protein (see Doudna JA et al., Science. 2018 Apr 27; 360(6387):436-439). The V-type CRISPR/Cas effector protein may be derived from different bacterial genera, and its enzyme activity may also be different. For example, the V-type CRISPR/Cas effector protein may include Cas12 effector protein, for example, Cas12a, Cas12b (C2c1), Cas12c (C2c3), C2c4, C2c8, C2c5, C2c10, C2c9, and CasX (Cas12e), CasY (Cas12d )Wait. For the definition of the above Cas12 effector protein, see Shmakov et al., Nat Rev Microbiol. 2017 March; 15(3): 169-182, Koonin et al., CurrOpin Microbiol. 2017 June; 37: 67-78.

For example, the Cas12 effector protein may include Cas12a, Cas12b, Cas12c, Cas12d and/or Cas12e.

For example, the Cas12 effector protein may include Cas12a, Cas12b, and Cas12c.

For example, the type V CRISPR/Cas effector protein includes Cas12 protease.

For example, the Cas12 protease is selected from the group consisting of Cas12a protease and Cas12b protease.

For example, the Cas12 protease is selected from the group consisting of LbCas12a, AsCasi2a, Frcas12a, PoCas12a, MbCas12a, Mb2Cas12a, Mb3Cas12a, IsCas12a, BsCas12a.

For example, the V-type CRISPR/Cas effector protein comprises the amino acid sequence described in any one of SEQ ID NOs 1-11.

For example, the type V CRISPR/Cas effector protein is a naturally occurring protein (e.g., naturally occurring in prokaryotic cells). For example, the type V CRISPR/Cas effector protein is a non-naturally-occurring protein (for example, a variant protein, a chimeric protein, a fusion protein, etc. of a naturally-occurring protein).

For example, the non-naturally-occurring protein and its corresponding naturally-occurring protein have functional conservation, and the functional conservation means that the non-naturally-occurring protein maintains a certain degree of function of its corresponding naturally-occurring protein (e.g. 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 98% or more, 100%). The function can be detected by conventional technical means in the art, for example, the activity of the V-type CRISPR/Cas effector protein to cleave the target nucleic acid and/or non-specifically cleave ssDNA as described in this application.

For example, the naturally-occurring protein may be from different genus, or from the same genus (for example, the same bacterial species), and the naturally-occurring protein has the function (for example, the type V CRISPR described in this application). /Cas effector protein activity) conservative (e.g. 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more , 95% or more, 98% or more, 100%), the naturally occurring protein has 20% or more amino acid sequence identity (for example, 30% or more, 40% or more, 50% or more High, 60% or higher, 70% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher,) amino acid sequence.

For example, the type V CRISPR/Cas effector protein may include the Cas12a, the Cas12b (C2c1), the Cas12c (C2c3), the C2c4, the C2c8, the C2c5, the C2c10, the The amino acid sequence of C2c9, the CasX (Cas12e) or the CasY (Cas12d) has at least 80%, 83%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical polypeptide sequences.

For example, the Cas12a protein may be Cas12a proteins derived from different species, such as FnCas12a, AsCas12a, LbCas12a, Lb5Cas12a, HkCas12a, OsCas12a, TsCas12a, BbCas12a, BoCas12a and/or Lb4Cas12a.

For example, the Cas12a protein may include an amino acid sequence as shown in any one of SEQ ID NO. 1-11.

For example, the Cas12a protein may include at least 80%, 83%, 85%, 86%, 87%, 88%, 89%, 90% of the amino acid sequence shown in any one of SEQ ID No: 1-11. , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical Cas12a protein, or any one of SEQ ID No: 1-11 The amino acid sequence shown has a variant of Cas12a with one or several amino acid deletions, substitutions or additions, and the variant has the functional conservation of Cas12a. For example, the deletion, substitution or addition of one or several amino acids can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid deletions, substitutions Or add.

For example, the Cas12a protein is derived from Lachnospiraceae, Acidaminococcus, Porphyromonas macacae, Moraxella bovoculi and/or Thiomicrospira sp.).

For example, the Cas12 protein can also form a fusion protein with a heterologous polypeptide (fusion ligand).

For example, the heterologous polypeptide can provide subcellular localization, that is, the heterologous polypeptide contains a subcellular localization sequence (for example, a nuclear localization signal (NLS) for targeting the nucleus, for keeping the sequence of the fusion protein outside the nucleus. Output sequence (NES), a sequence that keeps the fusion protein retained in the cytoplasm, is used to target the mitochondrial mitochondrial localization signal, is used to target the chloroplast-targeted chloroplast localization signal, the Golgi localization signal, etc.). For example, the NLS may include the NLS sequence derived from the following: the NLS of the SV40 virus large T antigen, with the amino acid sequence PKKKRKV; the NLS from the nucleoplasmic protein (for example, the nucleoplasmic protein dyad NLS with the sequence KRPAATKKAGQAKKKK); c -myc NLS, with the amino acid sequence PAAKRVKLD or RQRRNELKRSP; hRNPA1M9NLS, with the sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY.

For example, the heterologous polypeptide may be provided with a tag (a detectable label) to facilitate tracking and/or purification. For example, the tag may include fluorescent protein (eg, green fluorescent protein (GFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), blue fluorescent protein (CFP), etc.), mCherry, tdTomato, histidine Labels (for example, 6×His label), hemagglutinin (HA) label, FLAG label, Myc label, biotin label, nemycin label, etc.

For example, the positioning sequence or tag contained in the fusion protein may be any one or more of the above, and each tag or positioning sequence may be one or more repeats.

Indicator nucleic acid

In this application, the indicator nucleic acid contains a detectable label. For example, the detectable label includes a fluorescent label. For example, the fluorescent label is a fluorescent signal molecule pair, such as a fluorescence resonance energy transfer (FRET) pair or a quencher/fluorescent agent pair. The fluorescent signal molecule pair is respectively labeled on the 5'end and 3'end of the single-stranded DNA molecule, which is the indicator nucleic acid.

For example, after the indicator nucleic acid is cleaved, the strand of the indicator nucleic acid molecule is broken so that the relative position of the fluorescent signal molecule pair changes, thereby generating detectable signal changes before and after cleavage.

For example, the indicator nucleic acid may be cleaved by the V-type CRISPR/Cas effector protein; the cleavage of the indicator nucleic acid by the V-type CRISPR/Cas effector protein may be the same as that of the V-type CRISPR/Cas effector protein on the target nucleic acid. The cleavage of the single-stranded amplification product is associated.

For example, the signal change can be used as an indicator for detecting the target nucleic acid.

For example, the signal change may include: the indicator nucleic acid generates a certain amount of detectable signal before being cleaved, and when the indicator nucleic acid is cleaved, the amount of the detectable signal is reduced or quenched.

For example, the signal change may include: the indicator nucleic acid generates a first detectable signal before being cleaved, and when the indicator nucleic acid is cleaved, a second detectable signal is generated.

For example, the signal change may include: indicating that the nucleic acid does not generate a detectable signal before being cleaved, and when the indicating nucleic acid is cleaved, generating the detectable signal.

For example, the signal change may include: the indicator nucleic acid generates a certain amount of detectable signal before being cleaved, and when the indicator nucleic acid is cleaved, the amount of the detectable signal increases.

For example, the fluorescence resonance energy transfer (FRET) pair may include a donor and an acceptor. The donor and acceptor of the fluorescence resonance energy transfer (FRET) pair are known to those skilled in the art, and the donor and acceptor applicable to this application can be selected adaptively. For specific examples, please refer to Baja et al., Sensors ( Basel). 2016 Sep 1416(9); and Abraham et al., PLoS One. 2015 Aug 3; l0(8): e0l34436. The above two documents will be incorporated into this article by reference.

For example, the fluorescence resonance energy transfer (FRET) pair (donor/acceptor) may include:

Tryptophan/Dansyl, IAEDANS((5-(2-iodoacetylaminoethyl)aminonaphthalene-1-sulfonic acid)/DDPM(N-(4-dimethylamino-3, 5-Dinitrophenyl)maleimide), BFP/DsRFP, Dansyl/FITC, Dansyl/Octadecylrhodamine, Cyan fluorescent protein )/Green Fluorescent Protein (GFP), CF (Carboxyfluorescein Succinimidyl Ester)/TexasRed, Fluorescein/Tetramethylrhodamine, Cy3/Cy5, Green Fluorescent Protein (GFP)/Yellow Fluorescent protein (YFP), Rhodamine 110/Cy3, Rhodamine 6G/Malachite Green, FITC/Eosin Thiosemicarbazide, B-Phycoerythrin/Cy5, Cy5 /Cy5.5.

For example, the quencher/fluorescer pair may include a quencher group and a fluorescent group. The fluorescent group can emit a detectable signal. When the quenching group and the fluorescent group are close to each other (for example, when they are respectively labeled at the 5'end and 3'end of the single-stranded DNA molecule), so The detectable signal is completely or partially quenched by the quenching group. The quenching groups and fluorescent groups are known to those skilled in the art, and the quenching groups and fluorescent groups applicable to this application can be selected adaptively. For specific examples, please refer to: Bao et al., Annu Rev Biomed Eng .2009; 11:25-47, US8822673, US8586718, US20140378330, US20140349295, US20140194611, US20130323851, US20130224871, US20110223677, US20110190486, US20110172420, US20060179585, US20030003486, W0200142505, WO200186001, etc. The above documents are incorporated herein by reference.

For example, the fluorescent group may include: carboxyfluorescein (FAM, Carboxy fluorescein), fluorescein isothiocyanate (FITC, Fluorescein isothiocyanate), tetrachloro-6-carboxyfluorescein (TET, Tetrachloro fluorescein), hexachlorofluorescein -6-Methyl fluorescein (HEX, Hexachloro fluorescein), 2,7-dimethyl-4,5-dichloro-6-carboxy fluorescein (JOE), rhodamine dyes, such as R110, TAMRA, Texas Red, etc.), ROX, AlexaFluor dyes (e.g. Alexa

350, Alexa

405,Alexa

430,Alexa

488,Alexa

500, Alexa

514,Alexa

532,Alexa

546,Alexa

555,Alexa

568,Alexa

594,Alexa

610,Alexa

633,Alexa

635,Alexa

647,Alexa

660,Alexa

680, Alexa

700, Alexa

750, Alexa

790), ATTO dyes (e.g. ATTO 390, ATTO 425, ATTO 465, ATTO 488, ATTO 495, ATTO 514, ATTO520, ATTO 532, ATTO Rho6G, ATTO 542, ATTO 550, ATTO 565, ATTO Rho3B, ATTO Rho11, ATTO Rho12 ,ATTO Thio12,ATTO Rho101,ATTO 590,ATTO 594,ATTO Rho13,ATTO 610,ATTO 620,ATTO Rho14,ATTO 633,ATTO 647,ATTO 647N,ATTO 655,ATTO Oxa12,ATTO665,ATTO 680,ATTO 700,ATTO 725 ,ATTO 740), DyLight dyes, cyanine dyes (such as Cy2, Cy3, Cy3.5, Cy3b, Cy5, Cy5.5, Cy7, Cy7.5), FluoProbes dyes, SulfoCy dyes, Seta dyes, IRIS dyes, SeTau dyes, SRfluor dye and/or Square dye, etc.

For example, the quenching group may include DABCYL, TAMRA, MGB, BHQ-0, BHQ-1, BHQ-2, and/or BHQ-3.

For example, the single-stranded DNA molecule may have any length capable of realizing the signal change before and after cutting. For example, the length of the single-stranded DNA molecule can be 3-180 nucleotides, for example, 5-100, 5-80, 10-50, 5-30, 10-60, 10- 70, 10-30, 15-50, 12-40, 8-80, 12-28, 18-40, 100-180, 80-180, 70-100, 30- 80 nucleotides.

For example, the single-stranded DNA molecule does not hybridize to the guide sequence of the gRNA. The non-hybridization with the guide sequence of the gRNA usually means that the identity of the single-stranded DNA molecule and the guide sequence is not sufficient to form a double-stranded structure through complementary base pairing between the two. For example, the single-stranded DNA molecule has 40% or less identity with the guide sequence, such as 30% or less, such as 20% or less, such as 15% or less, such as 10% or less, such as 5 % Or less, for example, no identity.

For example, the single-stranded DNA probe is 5-FAM/TTATTAATTATA/BHQ1-3.

Sample, target nucleic acid

In the present application, the sample contains target nucleic acids derived from one or more of the following: cells of an organism, body fluids of an organism, and/or nucleic acid molecules of an organism. For example, the sample may contain nucleic acid sequences other than the target nucleic acid.

For example, the sample contains target nucleic acid derived from cells of the organism. For example, the cells of the organism may be in vitro cells (for example, an established cultured cell line), or may be isolated cells (cultured cells from an individual, primary cells). The cell may be a cell in the body (a cell in a biological individual).

For example, the sample may be a biological cell, a lysate of biological cells, or a homogenate of biological tissues, and the lysate or homogenate may be prepared by a method known to those skilled in the art (e.g., RIPA lysis method). , Ultrasonic crushing, grinding homogenate, etc.). For example, the sample may also be a product of further purification of the cell lysate, for example, some ions or organics are removed to reduce its influence on subsequent operations.

For example, the biological cells may include animal cells, plant cells, and microbial cells. For example, the plant cells may include Arabidopsis thaliana cells, and may also include cells of agricultural crops, such as plant somatic cells such as wheat, corn, rice, sorghum, millet, soybeans, etc.; the plant cells may also include cells of fruit and nut plants , For example, produce apricots, oranges, lemons, apples, plums, pears, almonds, walnuts and other plants. For example, the plant cell may be a cell derived from any part of the plant body, for example, root cells, leaf cells, xylem cells, phloem cells, cambium cells, apical meristem cells, parenchyma cells.

For example, the microbial cells may include bacteria (e.g. Escherichia coli, archaea), fungi (e.g. yeast), actinomycetes (e.g.), rickettsiae, mycoplasma, chlamydia, spirochetes, and the like.

For example, the animal cell may include invertebrate (e.g., Drosophila, nematode, planarian, etc.) cells, and vertebrate (e.g., zebrafish, chicken, mammalian) cells.

For example, the mammalian cells may include mice, rats, rabbits, pigs, dogs, cats, monkeys, humans, and the like.

For example, the animal cells may include cells from any tissue in the organism, such as stem cells, induced pluripotent stem (iPS) cells, germ cells (eg oocytes, egg cells, sperm cells, etc.), adult stem cells, somatic cells ( For example, fibroblasts, hematopoietic cells, cardiomyocytes, neurons, muscle cells, bone cells, liver cells, pancreatic cells, epithelial cells, immune cells and those derived from lung, spleen, kidney, stomach, large intestine, small intestine and other organs or tissues Any cell) and embryos at any stage in vitro or in vivo.

For example, the sample contains the target nucleic acid derived from the body fluid of the organism. For example, the body fluid of the organism may include cerebrospinal fluid, aqueous humor, lymph, digestive juice (e.g., saliva, gastric juice, small intestinal fluid, bile, etc.), breast milk, blood, urine, sweat, tears, feces, respiratory secretions, reproduction Organ secretions (such as semen, cervical mucus), etc.

For example, the sample may include nucleic acid molecules of the organism. The nucleic acid molecule can be isolated and extracted from any organism by the technical means known to those skilled in the art to separate nucleic acid molecules, including DNA and RNA. For example, the nucleic acid molecule is extracted from the above-mentioned biological cells or body fluids of the biological body.

In the present application, the target nucleic acid may be a polynucleotide from any source, for example, it may include double-stranded DNA and/or single-stranded DNA.

For example, the polynucleotide may include an exogenous polynucleotide that resides in the nucleus of a eukaryotic cell (for example, the target polynucleotide may be a viral genome sequence); for example, the target polynucleotide may include a coding gene The sequence of the product (e.g., protein) or a non-coding sequence (e.g., regulatory polynucleotide or useless DNA).

For example, the genome sequence of the virus may include papillomavirus (for example, human papilloma virus (HPV), polyoma virus, etc.), and hepatitis virus (for example, hepatitis B virus (HBV), hepatitis C virus (HCV) Etc.), herpes virus (such as herpes simplex virus (HSV), varicella-zoster virus (VZV), cytomegalovirus (CMV), herpes lymphovirus, pityriasis erythema, Kaposi’s sarcoma-associated herpes virus, etc.), Adenovirus, adeno-associated virus, poxvirus (such as smallpox, vaccinia virus, monkeypox virus), African swine fever virus and/or human immunodeficiency virus (HIV).

For example, the sequence encoding the gene product may include a sequence encoding a tumor antigen, for example, the tumor antigen may include a TNF receptor family member B cell maturation antigen (BCMA), Tn antigen (such as Tn'Ag, GalNAcα-Ser/Thr) , Prostate-specific membrane antigen (PSMA); receptor tyrosine kinase-like orphan receptor 1 (ROR1), Fms-like tyrosine kinase 3 (FLT3); tumor-associated glycoprotein 72 (TAG72), CD38, CD44v6, cancer Embryonic antigen (CEA), epithelial cell adhesion molecule (EPCAM), B7H3 (CD276), KIT (CD117), interleukin-13 receptor subunit α-2 (IL-13Ra2 or CD213A2), mesothelin, interleukin 11 receptor α (IL-11Ra), prostate stem cell antigen (PSCA), p53, p53 mutant, prostate specific protein (prostein), prostate cancer tumor antigen-1 (PCTA-1 or galectin 8), T cell recognition Melanoma antigen 1 (MelanA or MART1); rat sarcoma (Ras) mutant, human telomerase reverse transcriptase (hTERT), sarcoma translocation breakpoint, melanoma inhibitor of apoptosis protein (ML-IAP) And/or Glypican-3 (GPC3).

For example, the target nucleic acid may also include a sequence containing a SNP site in the genome of an organism.

For example, the target nucleic acid may also include the gene sequence of pathogenic bacteria, parasites and the like.

For example, the concentration of the target nucleic acid is at least 1*1E2 copies or more, 1*1E3 copies or more, 1*1E4 copies or more, for example, 0.2*1E5 copies or more, 0.3*1E5 copies or Above, 0.4*1E5 copies or more, 0.5*1E5 copies or more, 0.6*1E5 copies or more, 0.7*1E5 copies or more, 0.8*1E5 copies or more, 0.9*1E5 copies or more, 1*1E5 copies or more, 3*1E5 copies or more, 6*1E5 copies or more.

For example, the concentration of the target nucleic acid is at least 1 amol or more, 10 amol or more, 30 amol or more, 80 amol or more, 150 amol or more ( amol) or more, 200 amol or more, 300 amol or more, 500 amol or more, 700 amol or more, and 900 amol or more.

For example, the concentration of the target nucleic acid is at least 1 femtomole (fmol) or more, 10 femtomole (fmol) or more, 30 femtomole (fmol) or more, 80 femtomole (fmol) or more, 150 femtomole (fmol) or more ( fmol) or more, 200 femtomole (fmol) or more, 300 femtomole (fmol) or more, 500 femtomole (fmol) or more, 700 femtomole (fmol) or more, and 900 femtomole (fmol) or more.

For example, the concentration of the target nucleic acid is at least 1 picomoles (pmol) or more, 10 picomoles (pmol) or more, 30 picomoles (pmol) or more, 80 picomoles (pmol) or more, 150 picomoles (pmol) or more ( pmol) or more, 200 picomoles (pmol) or more, 300 picomoles (pmol) or more, 500 picomoles (pmol) or more, 700 picomoles (pmol) or more, 900 picomoles (pmol) or more.

For example, the concentration of the target nucleic acid is at least 1 nanomole (nmol) or more, 10 nanomole (nmol) or more, 30 nanomole (nmol) or more, 80 nanomole (nmol) or more, 150 nanomole ( nmol) or more, 200 nanomole (nmol) or more, 300 nanomole (nmol) or more, 500 nanomole (nmol) or more, 700 nanomole (nmol) or more, 900 nanomole (nmol) or more. For example, the concentration of the target nucleic acid is at least 1 micromole (μmol) or more, for example, 30 micromole (μmol) or more, 50 micromole (μmol) or more, and 80 micromole (μmol) or more.

Nucleic acid amplification and fluorescence detection

In this application, the method for detecting target nucleic acid in a sample includes:

c) measuring the detectable signal generated after the indicator nucleic acid is cleaved by the V-type CRISPR/Cas effector protein, so as to detect the target nucleic acid;

For example, the contact may occur under in vivo, in vitro or ex vivo conditions, for example, the V-type CRISPR/Cas effector protein can bind to the gRNA, the gRNA may include a guide sequence, and the single-stranded amplification product A target sequence may be included, and the guide sequence can hybridize to the target sequence so that the V-type CRISPR/Cas effector protein targets the single-stranded amplification product.

For example, the interaction of the V-type CRISPR/Cas effector protein and the single-stranded amplification product can correlate with the non-specific cleavage of the indicator nucleic acid by the V-type CRISPR/Cas effector protein.

For example, the V-type CRISPR/Cas effector protein can cleave the single-stranded amplification product.

For example, the cleavage of the single-stranded amplification product enables the V-type CRISPR/Cas effector protein to perform non-specific cleavage of the indicator nucleic acid.

For example, the indicator nucleic acid molecule strand breaks can cause changes in the relative positions of the fluorescent signal molecule pairs respectively connected to the 5'and 3'ends of the indicator nucleic acid DNA strand, thereby generating detectable signal changes before and after cleavage. .

For example, the detection of the target nucleic acid can be achieved by detecting the change in the signal.

For example, the double-stranded target nucleic acid and/or its double-stranded amplification product can avoid being cut or degraded by the V-type CRISPR/Cas effector protein, so that the amplification and detection of the target nucleic acid can be achieved in the same system conduct.

For example, the double-stranded target nucleic acid and/or its double-stranded amplification product can avoid being cleaved or degraded by the V-type CRISPR/Cas effector protein by being adjacent to the 5'end or 3'end of the target sequence in the target nucleic acid This is achieved without PAM sequence in the sequence.

For example, the above a) and the above b) are carried out in the same reaction system. Wherein, the amplification may include any amplification method that can produce a single-stranded amplification product, and the V-type CRISPR/Cas effector protein can still be maintained under the amplification conditions (such as temperature, ion concentration, etc.) Its enzymatic properties, such as cleaving the single-stranded amplification product and the activity of the indicator nucleic acid, for example, can maintain at least 30%, for example, at least 40% of its activity under conventional conditions (such as the best known activity conditions) , At least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or 100%, the conventional conditions are known to those skilled in the art, such as those reported in the known technical literature Use conditions of different CRISPR/Cas effector proteins. For example, the CRISPR/Cas effector protein is Cas12, and the conventional use conditions of Cas12 can be found in Doudna J. et al., Science. 2018 Nov 16; 362(6416):839-842.

For example, the CRISPR/Cas effector protein can realize the function of detecting the target nucleic acid described in the present application under the amplification conditions (such as temperature, ion concentration, etc.).

For example, different V-type CRISPR/Cas effector proteins (that is, V-type CRISPR/Cas effectors from various species) can be selected according to the amplification conditions to facilitate the use in various provided amplification methods to take advantage of all Desired characteristics (for example, specific enzymatic properties).

For example, different amplification methods can be selected according to the enzymatic characteristics of different V-type CRISPR/Cas effector proteins (that is, V-type CRISPR/Cas effector proteins from various species), so as to facilitate the selection of various amplification methods provided. Use to take advantage of the specific enzymatic properties required.

For example, the amplification may include asymmetric amplification.

For example, the asymmetric amplification may include the following types: 1) Upstream and downstream primers of different concentrations are used for asymmetric amplification. As the circulation increases, the primers with a small amount are gradually depleted, and the primers with an excessive amount can continue to amplify to generate single-stranded DNA (Gyllensten and Erlich, Proc, Natl. Acad Sci. USA, 1988, 85: 7652-7656) ; 2) Upstream and downstream primers of different lengths are used for asymmetric amplification. For example (see Peng Xiaomou et al., Chinese Laboratory Diagnosis, 2002, 6: 206-208) using 34 bases upstream primers and 20 bases downstream primers to asymmetrically amplify the S gene of HBV. In the temperature cycle, increase the annealing temperature, short primers cannot anneal, and long primers can continue to extend the reaction to achieve the purpose of preparing single-stranded nucleic acid and/or 3) Perform a symmetric amplification reaction (ie double-stranded product amplification reaction) first, and then Purify the amplified product, use the purified symmetrical amplified product as a template, add a single primer or unequal primers for asymmetric amplification (Gorelov, et.al., Biochem. Biophys. Res. Commun., 1994, 200:365 -369; Scott, et. al., Lett. Appl. Microbiol., 1998, 27: 39-44; Guo, et. al., Genome Res., 2002, 12: 447-457). For example, the above-mentioned several methods can be adaptively selected, adjusted or modified according to the amplification purpose or amplification conditions, and the appropriate concentration ratio can be selected through routine experiments.

For example, a forward amplification primer and a reverse amplification primer are used in the amplification, and the concentration ratio of the forward amplification primer and the reverse amplification primer is 1:10-1:80, for example, The concentration ratio is 1:10-1:70, 1:10-1:60, 1:10-1:50, 1:10-1:40, 1:15-1:40, 1:20-1 :40, 1:10-1:30, 1:10-1:20, 1:15-1:30. For example, the concentration ratio is 1:10-1:40.

For example, a forward amplification primer and a reverse amplification primer are used in the amplification, and the ratio of the reverse amplification primer and the forward amplification primer is 1:10-1:80, for example, the The concentration ratio is 1:10-1:70, 1:10-1:60, 1:10-1:50, 1:10-1:40, 1:15-1:40, 1:20-1:40 , 1:10-1:30, 1:10-1:20, 1:15-1:30. For example, the concentration ratio is 1:10-1:40.

For example, the amplification includes isothermal amplification. The isothermal amplification refers to a method of amplifying nucleic acid molecules under constant temperature conditions. For example, it may include loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), and recombinase polymerase. Amplification (RPA), strand displacement amplification (SDA), nucleic acid sequence-based amplification (NASBA), transcription-mediated amplification (TMA), nickase amplification reaction (NEAR), rolling circle amplification (RCA) , Multiple displacement amplification (MDA), branching (RAM), circular helicase dependent amplification (cHDA), single primer isothermal amplification (SPIA), RNA technology signal-mediated amplification (SMART), self-sustaining Sequence replication (3SR), genomic exponential amplification reaction (GEAR) and isothermal multiple displacement amplification (IMDA).

For example, the amplification includes recombinase polymerase amplification (RPA). The amplification system amplified by the recombinase polymerase may include recombinase, single-stranded DNA-binding protein (SSB), and single-stranded displacement polymerase.

For example, the amplification temperature of the recombinase polymerase may be 10-50°C, such as 15-50°C, 18-50°C, 20-50°C, 25-50°C, 30-50°C, 35-50°C , 10-45℃, 20-45℃, 25-45℃, 28-45℃, 30-45℃, 35-45℃, 37-42℃.

For example, the amplification is an amplification method in which the asymmetric amplification and the isothermal amplification are combined. The combined amplification method is to obtain the amplification product under isothermal conditions.

In the present application, it is possible to determine whether there is a target nucleic acid or the abundance of the target nucleic acid in the sample by detecting the signal change.

For example, the detection of the signal change is a fluorescence detection system, and the fluorescence detection system may include the following modules: 1) A temperature control module, the temperature control range is 0-100°C, and a specific temperature can be selected according to the temperature conditions of the amplification reaction. Temperature control conditions, for example, 25-50 ℃, for example, 37-42 ℃; 2) fluorescence detection module, according to the different pairs of fluorescent signal molecules set different excitation wavelength and/or emission wavelength, for example, excitation light wavelength 490nm, emission wavelength is 520nm, for example, excitation light wavelength is 535nm, emission wavelength is 560nm; 3) Timing detection function module, can detect the set fluorescence every 0.5-120 minutes, for example, every 2 Perform a test every 15 minutes, for example, perform a test every 2-5 minutes, for example, the duration of the test is 10 minutes to 3 hours, for example, 15 minutes to 2 hours.

For example, the fluorescence detection system may be BioTekCytation 3; for example, the fluorescence detection system may be ThermoVarioskan ^TM LUX; for example, the fluorescence detection system may be fluorescence quantitative PCR, for example, the fluorescence detection system may be Applied Biosystems ^TM 7500 Real- Time PCR System.

Reagent test kit

The application also provides a kit for detecting target nucleic acid in a sample, which comprises i) a type V CRISPR/Cas effector protein, ii) a gRNA, and iii) an indicator nucleic acid; wherein the gRNA comprises the same type as the type V CRISPR/Cas The region where the effector protein binds and the guide sequence that hybridizes to the target sequence in the target nucleic acid; and the guide sequence is designed such that the sequence at the 5'end or 3'end of the target sequence to which it hybridizes does not contain a PAM sequence ; The indicator nucleic acid is a single-stranded nucleic acid molecule and does not hybridize with the guide sequence of the gRNA.

For example, the kit further includes reagents for obtaining a single-stranded amplification product containing the target nucleic acid. For example, the reagents for obtaining single-stranded amplification products include reagents required for asymmetric amplification. For example, the reagent may include a single primer or primer pair designed for the target nucleic acid to be detected, and the single primer or primer pair can hybridize with the target nucleic acid and initiate the amplification of the target nucleic acid. For example, the primer pair includes amplification The upstream primer of the target nucleic acid and/or the downstream primer that amplifies the target nucleic acid, for example, the upstream primer and the downstream primer of the primer pair may be located in different containers, or may be located in the same container, and set to be different concentration. For example, the upstream primer and the downstream primer may have different lengths, and the length difference between the upstream primer and the downstream primer is sufficient to enable the longer primer to anneal and hybridize to the target nucleic acid, while the shorter primer cannot Annealing and hybridizing with the target nucleic acid.

For example, the reagents for obtaining single-stranded amplification products include reagents required for isothermal amplification. The reagent may include any substance capable of achieving the isothermal amplification, and may be selected according to the enzymatic characteristics of the V-type CRISPR/Cas effector protein used in combination therewith to achieve the purpose of detecting the target nucleic acid. For example, the reagents required for the isothermal amplification may include RPA recombinase, RPA polymerase and/or RPA buffer. For example, the RPA polymerase may include a single-strand displacement polymerase, for example, the RPA buffer may include a single-stranded binding protein and metal ions, for example, magnesium ions.

For example, the reagents for the single-stranded amplification product and/or the reagents required for isothermal amplification may also include dATP, dGTP, dTTP, and dCTP. For example, the reagents for the single-stranded amplification product of the target nucleic acid and/or the reagents required for isothermal amplification and the V-type CRISPR/Cas effector protein and the gRNA are located in the same container. For example, the type V CRISPR/Cas effector protein includes Cas12 protease. For example, the Cas12 protease is selected from the group consisting of Cas12a protease and Cas12b protease. For example, the V-type CRISPR/Cas effector protein comprises the amino acid sequence described in any one of SEQ ID NOs. 1-11.

For example, the indicator nucleic acid contains a detectable label. For example, the detectable label includes a fluorescent label. For example, the fluorescent label can be adapted to select the fluorescence resonance energy transfer (FRET) pair and/or the quencher/fluorescent agent pair.

For example, the kit may also include other compounds (such as enzymes or indicators, etc.) to provide additional functions. For example, the detection of RNA and/or DNA can be achieved by adding reverse transcriptase.

Without intending to be limited by any theory, the following examples are only to illustrate the chimeric antigen receptor, preparation method, and use of the present application, and are not used to limit the scope of the present application. The examples do not include detailed descriptions of traditional methods, such as those used to construct vectors and plasmids, methods of inserting genes encoding proteins into such vectors and plasmids, or methods of introducing plasmids into host cells. Such methods are well known to those of ordinary skill in the art, and are described in many publications, including Sambrook, J., Fritsch, EF and Maniais, T. (1989) Molecular Cloning: A Laboratory Manual , 2nd edition, Cold spring Harbor Laboratory Press.

Example

Example 1. Design of amplification primer pair and gRNA

If cas12a cuts the amplified template, the amplification will fail, and the one-step method of first amplifying and then cutting cannot be realized. Therefore, in order to realize the one-step method, the gRNA that only cuts ssDNA is designed, and the asymmetric RPA amplification method is used to generate a large number of ssDNA targets.

According to the African swine fever protein VP72 sequence (shown in SEQ ID NO.31), design two gRNA (gRNA1 and gRNA2) sequences (shown in SEQ ID NO.19-20, respectively) on the sense strand, and on the antisense strand Design two gRNA (gRNA3 and gRNA4) sequences (as shown in SEQ ID NO. 21-22, respectively), and the corresponding amplification primer pair (upstream primer and downstream primer for RPA amplification): primer-F/primer-R (Respectively shown in SEQ ID NO.27-28).

According to the African swine fever protein K205R sequence (shown in SEQ ID NO.36), design two gRNA (gRNA5 and gRNA6) sequences (shown in SEQ ID NO.23-24, respectively) on the sense strand, and design on the antisense strand Two gRNA (gRNA7 and gRNA8) sequences (shown in SEQ ID NO.25-26), and the corresponding amplification primer pair: primer-F1/primer-R1 (shown in SEQ ID NO.29-30, respectively) .

Example 2. cas12a cutting ssDNA reaction

Using the 4 gRNAs designed in Example 1, select more than 1E8 copies of ssDNA (as shown in any one of SEQ ID Nos. 32-35 and 37-40) as the cleavage template.

The cas12a cleavage ssDNA reaction system (50μl) is shown in Table 1:

Table 1

Among them, the 2xNA buffer formula is: 80mM Tris-HCl, 120mM NaCl, 12mM MgCl, PH 7.3; For the preparation method of cas12a, refer to JSChen, E.Ma, LB Harrington, M. Da Costa, X. Tian, JMPalefsky, et al. .; CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity; Science, 360(6387)(2018), pp.436-439; gRNA is synthesized by GenScrip; signal reporter probe (5'-FAM-TTATT-BHQ1 -3') was synthesized by Suzhou Hongxun Biotechnology Co., Ltd.; ssDNA was synthesized by Suzhou Jinweizhi Biotechnology Company; NTC was nuclease-free water, purchased from Invitrogen. The concentration in Table 1 refers to the original solution concentration of each reagent before being added to the reaction system.

The above 50μl reaction system is reacted at 37°C for 60 minutes, and the FAM fluorescence value is read every 2 minutes (Fluorescence Quantitative PCR Instrument, ABI 7500). Each gRNA is set with two replicates and an NTC negative control group. When the concentration of cas12a in the system is 250nM, The concentration of gRNA is 250nM, the concentration of the signal reporter probe (5'-FAM-TTATT-BHQ1-3') is 1uM, and the concentration of ssDNA is 1E8 copies/ul. The gRNA detection result of VP72 sequence is shown in Figure 1. The gRNA test results are shown in Figure 2. Figure 1-Figure 2 show that all 8 gRNAs can achieve the cutting effect, and gRNA4 and gRNA6 are selected for downstream verification.

Example 3. Cas12a/gRNA4, cas12a/gRNA6 cleavage combined with asymmetric RPA amplification

Select gRNA4 and gRNA6, design a combination of four upstream and downstream primer concentrations, primer-F: primer-R=1:1 or 10:1 or 20:1 or 40:1; primer-F1: primer-R1=1:1 Or 1:10 or 1:20 or 1:4. Screen out the primer concentration combination with the best detection effect.

50μl of cas12a cutting combined with asymmetric RPA reaction system is shown in Table 2:

Table 2

成分Element	体积volume	浓度concentration
RPA上游引物RPA upstream primer	2μl2μl	0.1μM-10μM0.1μM-10μM
RPA下游引物RPA downstream primer	2μl2μl	0.1μM-10μM0.1μM-10μM
cas12acas12a	2.5μl2.5μl	5uM-10uM5uM-10uM
gRNAgRNA	2.5μl2.5μl	5uM-10uM5uM-10uM
RPA酶预混液RPA enzyme master mix	33.5μl33.5μl	n/an/a
乙酸镁Magnesium acetate	2.5μl2.5μl	n/an/a
待测DNA(质粒)DNA to be tested (plasmid)	1μl1μl	1E6拷贝/μl1E6 copies/μl
无核酸酶水Nuclease-free water	补充至50μlSupplement to 50μl	To

Among them, RPA upstream primers, RPA downstream primers and DNA (plasmid) to be tested (shown in SEQ ID NO.31 and 36) were synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd.; cas12a was produced by Suzhou Cree Gene Biotechnology Co., Ltd. (Sequence (As shown in SEQ ID NO.1); gRNA was synthesized by GenScrip; RPA enzyme premix and magnesium acetate were purchased from Jiangsu Qitian Gene Company (Cat. No.: B190122AA) and used according to the product instructions; NTC is nuclease-free water, purchased from Invitrogen Corporation. The concentration in Table 1 refers to the concentration of the original solution before each reagent is added to the reaction system.

The above 50μl reaction system was reacted at 37°C for 60 minutes, and the FAM fluorescence value was read every 2 minutes (Fluorescence Quantitative PCR Instrument, ABI 7500). Two repetitions are set for each concentration ratio. When the concentration of cas12a in the system is 250nM, the concentration of gRNA is 250nM, and the concentration of DNA to be tested is 1E6 copies/μl, the RPA enzyme premix and magnesium acetate are used in accordance with the product instructions. The experimental results are shown in Figure 3 and Figure 4. In Figures 3 and 4, F:R-1:1 indicates that the concentration of primer-F is 400nM, and the concentration of primer-R is 400nM; F:R-10:1 indicates that the concentration of primer-F is 400nM, and the concentration of primer-R is 400nM. The concentration of F:R-20:1 indicates that the concentration of primer-F is 400nM and the concentration of primer-R is 20nM; F:R-40:1 indicates that the concentration of primer-F is 400nM and the concentration of primer-R It is 10nM. F1R1-1:1 indicates that the concentration of primer-F1 is 400nM, the concentration of primer-R1 is 400nM; F1R1-1:10 indicates that the concentration of primer-F1 is 40nM, and the concentration of primer-R1 is 400nM; F1R1-1:20 indicates The concentration of primer-F1 is 20nM, the concentration of primer-R1 is 400nM; F1R1-1:40 means that the concentration of primer-F1 is 10nM, and the concentration of primer-R1 is 400nM. According to the results, the primer ratios of F:R-20:1 and F1:R1-1:20 were selected for downstream verification. The single-stranded product obtained by primer-F is shown in SEQ ID NO.41; the single-stranded product obtained by primer-R is shown in SEQ ID NO.42; the single-stranded product obtained by primer-F1 is shown in SEQ ID NO.43 Show; the single-stranded product obtained by primer-R1 is shown in SEQ ID NO.44.

Example 4. Asymmetric RPA and symmetric RPA combined with Cas12a one-step method for lower detection limit verification

Select gRNA4 and gRNA6, select the primer concentration ratio F:R-1:1, F:R-20:1, F1:R1-1:1, F1:R1-1:20 to verify the detectable target amount and target amount Set to 1E6, 1E5, 1E4, 1E3, 1E2. Prepare a 50μl reaction system according to the method of Example 3, react at 37°C for 80min, and read the FAM fluorescence value every 2min (fluorescence quantitative PCR instrument, ABI 7500). The results are shown in Figure 5-10. The detection effect of asymmetric RPA is better than that of symmetric RPA. In addition, from the results of Figure 7 and Figure 9, it can be seen that the primer F:R ratio is 20:1, and the target is as low as 1E2 copies. The fluorescent signal can be detected at 40 minutes, and it is significantly different from the negative control. Test results.

Example 5. Asymmetric RPA and symmetric RPA combined with Cas12a one-step detection and DETECTR system comparison

Select gRNA6, prepare a 50μl reaction system according to the method of Example 3, the primer concentration ratios are F1:R1-1:1, F1:R1-1:20, and the target amount is set to 1E6, 1E5, 1E4, 1E3, 1E2. At the same time, prepare the DETECTR system as follows, where the gRNA9 sequence: UAAUUUCUACUAAGUGUAGAUAAGACCUGCUUUCAGCAGUA (SEQ ID NO.45), the primer concentration ratios are F1:R1-1:1, F1:R1-1:20, and the target scalar is set to 1E6, 1E5 , 1E4, 1E3, 1E2.

table 3

成分Element	体积volume	浓度concentration
RPA上游引物RPA upstream primer	2μl2μl	0.1μM-10μM0.1μM-10μM
RPA下游引物RPA downstream primer	2μl2μl	0.1μM-10μM0.1μM-10μM
cas12acas12a	2.5μl2.5μl	5uM-10uM5uM-10uM
gRNA9gRNA9	2.5μl2.5μl	5uM-10uM5uM-10uM
RPA酶预混液RPA enzyme master mix	33.5μl33.5μl	n/an/a
乙酸镁Magnesium acetate	2.5μl2.5μl	n/an/a
待测DNA(质粒)DNA to be tested (plasmid)	1μl1μl	1E2-1E6拷贝/μl1E2-1E6 copies/μl
无核酸酶水Nuclease-free water	补充至50μlSupplement to 50μl	To

Set 37°C to react for 80 minutes, and read the FAM fluorescence value every 2 minutes (Fluorescence quantitative PCR instrument, ABI 7500). The results show that when the detection system in this application is an asymmetric reaction, the fluorescent signal can be detected within 40 minutes when the target is as low as 1E2 copies, and it is significantly different from the negative control, and the detection result is obtained.

In the DETECTR reaction system, there is no fluorescence signal within 60 minutes when the target concentration is 1E6 copies, and it cannot be detected. Please refer to Table 4 for specific fluorescence values:

Table 4

Example 6. Other asymmetric isothermal amplification combined with one-step detection of Cas12a

Select gRNA4 and gRNA6, select the primer concentration ratio F:R-20:1, F1:R1-1:20 to verify the detectable target amount, and the target amount is set to 1E6, 1E5, 1E4, 1E3, 1E2. Prepare a 50μl reaction system according to the following table, in which EMA (Normal Temperature Nucleic Acid Amplification) was purchased from Suzhou Dianjing Biotechnology Co., Ltd., reacted at 37°C for 80 minutes, and read the FAM fluorescence value every 2 minutes (Fluorescence Quantitative PCR Instrument, ABI 7500).

table 5

成分Element	体积volume	浓度concentration
上游引物Upstream primer	2μl2μl	0.1μM-10μM0.1μM-10μM
下游引物Downstream primer	2μl2μl	0.1μM-10μM0.1μM-10μM
cas12acas12a	2.5μl2.5μl	5uM-10uM5uM-10uM
gRNAgRNA	2.5μl2.5μl	5uM-10uM5uM-10uM
EMA酶预混液EMA enzyme premix	33.5μl33.5μl	n/an/a
乙酸镁Magnesium acetate	2.5μl2.5μl	n/an/a
待测DNA(质粒)DNA to be tested (plasmid)	1μl1μl	1E2-1E6拷贝/μl1E2-1E6 copies/μl
无核酸酶水Nuclease-free water	补充至50μlSupplement to 50μl	To

The results show that the detection effect of asymmetric EMA+Cas12 is the same as that of asymmetric RPA+Cas12: under the condition of as low as 1E2 copies of the target, the fluorescence signal can be detected within 40 minutes, and the detection result can be obtained. The specific fluorescence values are shown in Table 6.

Table 6

The foregoing detailed description is provided by way of explanation and examples, and is not intended to limit the scope of the appended claims. Various changes of the embodiments listed in the present application are obvious to those of ordinary skill in the art, and are reserved within the scope of the appended claims and their equivalents.

Claims

A method for detecting target nucleic acid in a sample, which includes:

a) Amplifying the target nucleic acid in the sample to obtain a single-stranded amplification product;

b) contacting the single-stranded amplification product with i) a type V CRISPR/Cas effector protein, ii) gRNA, and iii) an indicator nucleic acid, wherein the gRNA includes a region that binds to the type V CRISPR/Cas effector protein and A guide sequence that hybridizes with the target sequence in the target nucleic acid, the sequence adjacent to the 5'end or 3'end of the target sequence in the target nucleic acid does not contain a PAM sequence, and the indicator nucleic acid is a single-stranded nucleic acid molecule and does not interact with The guide sequence of the gRNA hybridizes; and

c) Measure the detectable signal generated by the indicator nucleic acid after being cleaved by the V-type CRISPR/Cas effector protein, so as to detect the target nucleic acid.
The method according to claim 1, wherein said a) and said b) are carried out in the same reaction system.
The method according to any one of claims 1-2, wherein the V-type CRISPR/Cas effector protein, the gRNA and the indicator nucleic acid are added to the reaction system containing the single-stranded amplification product.
The method of any one of claims 1-3, wherein the amplification comprises asymmetric amplification.
The method according to any one of claims 1 to 4, wherein the target nucleic acid comprises double-stranded DNA and/or single-stranded DNA.
The method according to any one of claims 1 to 5, wherein a forward amplification primer and a reverse amplification primer are used in the amplification, and the forward amplification primer and the reverse amplification primer The concentration ratio is 1:10-1:40.
The method according to any one of claims 1 to 6, wherein a forward amplification primer and a reverse amplification primer are used in the amplification, and the reverse amplification primer and the forward amplification primer The concentration ratio is 1:10-1:40.
The method of any one of claims 1-7, wherein the amplification comprises isothermal amplification.
8. The method of any one of claims 1-8, wherein the amplification comprises recombinase polymerase amplification (RPA).
The method according to any one of claims 1-9, wherein the sample contains a target nucleic acid derived from one or more of the following: cells of an organism, body fluids of an organism, and/or nucleic acid of an organism molecular.
The method according to claim 10, wherein the sample contains a target nucleic acid derived from a virus, and the virus is selected from the group consisting of African swine fever virus, HCV and HIV.
The method of any one of claims 1-11, wherein the contacting occurs under in vivo, in vitro, or ex vivo conditions.
The method of any one of claims 1-12, wherein the type V CRISPR/Cas effector protein comprises Cas12 protease.
The method according to claim 13, wherein the Cas12 protease is selected from the group consisting of Cas12a protease and Cas12b protease.
The method according to any one of claims 1-14, wherein the type V CRISPR/Cas effector protein comprises the amino acid sequence set forth in any one of SEQ ID NO. 1-11.
The method of any one of claims 1-15, wherein the indicator nucleic acid comprises a detectable label.
The method of any one of claims 1-16, wherein the detectable label comprises a fluorescent label.
The method of any one of claims 1-17, wherein the detectable signal comprises a fluorescent signal.
The method according to any one of claims 1-18, wherein the concentration of the target nucleic acid is at least 1*1E2 copies or more.
A kit for detecting a target nucleic acid in a sample, comprising i) a type V CRISPR/Cas effector protein, ii) gRNA and iii) an indicator nucleic acid; wherein the gRNA includes a region that binds to the type V CRISPR/Cas effector protein And a guide sequence that hybridizes with the target sequence in the target nucleic acid; and the guide sequence is designed such that the sequence at the 5'end or 3'end of the target sequence to which it hybridizes does not contain a PAM sequence; the indicator nucleic acid It is a single-stranded nucleic acid molecule and does not hybridize with the guide sequence of the gRNA.
The kit according to claim 20, further comprising a reagent for obtaining a single-stranded amplification product containing the target nucleic acid.
The kit according to claim 21, wherein said reagents for obtaining single-stranded amplification products comprise reagents required for asymmetric amplification.
The kit according to claim 22, wherein the reagent for asymmetric amplification comprises an upstream primer for amplifying the target nucleic acid and/or a downstream primer for amplifying the target nucleic acid.
The kit according to any one of claims 21-23, wherein the reagents for obtaining single-stranded amplification products comprise reagents required for isothermal amplification.
The kit according to claim 24, wherein the reagents required for the isothermal amplification include RPA recombinase, RPA polymerase and/or RPA buffer.
The kit according to any one of claims 21-25, wherein the reagent for the single-stranded amplification product of the target nucleic acid and the V-type CRISPR/Cas effector protein and the gRNA are located in the same container.
The kit according to any one of claims 20-26, wherein the type V CRISPR/Cas effector protein comprises Cas12 protease.
The kit according to claim 27, wherein the Cas12 protease is selected from the group consisting of Cas12a protease and Cas12b protease.
The kit according to any one of claims 27-28, wherein the type V CRISPR/Cas effector protein comprises the amino acid sequence of any one of SEQ ID NO. 1-11.
The kit according to any one of claims 20-29, wherein the indicator nucleic acid comprises a detectable label.
The kit according to any one of claims 30, wherein the detectable label comprises a fluorescent label.