CN112852921B - A nucleic acid detection method, detection probe and kit thereof based on instant detection test strips - Google Patents

Abstract

The invention relates to the technical field of nucleic acid detection, in particular to a nucleic acid detection method based on an instant detection test strip, a detection probe and a kit thereof. The nucleic acid detection method of the present invention is based on the combination of CRISPR/Cas12a system and early pregnancy test paper. The non-specific hCG detection probe of the present invention is combined with the crRNA sequence that specifically recognizes the sequence to be tested to detect nucleic acid, which greatly reduces the detection cost and has obvious advantages. The detection target is universal, with high sensitivity and good specificity. The detection sensitivity is comparable to the fluorescence endpoint characterization method, and the minimum number of copies of 2 molecules can be detected.

Description

A nucleic acid detection method based on instant detection test strips, detection probes and test strips thereof Kit

technical field

The invention relates to the technical field of nucleic acid detection, in particular to a nucleic acid detection method based on an instant detection test strip, a detection probe and a kit thereof.

Background technique

Sudden and a series of major acute and chronic infectious diseases have always been the most harmful to human health and caused the largest number of deaths. Especially for some infectious viruses with diverse genotypes and complex structural proteins, due to the long development cycle of vaccines, point-of-care testing (POCT) is an effective method for effective investigation and control while searching for new drugs. An important prerequisite for various major acute and chronic infectious pathogens. At present, there are mainly two detection methods for sudden viral infectious diseases on the market, one is the detection of viral nucleic acid, and the other is antigen antibody detection. Since the two methods have different window periods after infection detection, the window period of nucleic acid detection is significantly shorter than that of antigen antibody detection, so it is more conducive to early screening of infection. Therefore, the realization of sensitive, specific and portable nucleic acid detection of infectious diseases is still the goal of continuous improvement and development.

In recent years, with the vigorous development of the field of molecular diagnosis, in order to develop a portable diagnostic system, scientists have created a new sensing concept, that is, using commercial POCT products to detect non-original analytes; this method can not only reduce the cost of the instrument Development costs, but also can truly realize the portable detection of nucleic acids. This method has now been widely accepted as one of the most promising portable detection methods. So far, with the help of different recognition elements (nucleic acid aptamers and DNA enzymes, etc.), metal ions, small molecular compounds, trans-DNA, proteins, and even cancer cells can be transduced into existing POCT commercial devices . Among these innovative detection methods, portable detection methods based on test strips are rapidly developing in the qualitative or quantitative analysis of analytes due to their simple operation methods and intuitive detection results. At present, the existing commercial early pregnancy test strip detection technology (Angew.Chem.Int.Ed., 2017, 56, 992-996) needs to add a variety of nucleic acid probes, and finally use magnetic beads for separation. The experimental process is complicated and cannot be achieved. Higher signal ratio.

The CRISPR/Cas system is an immune system that exists in bacteria and archaea to resist the invasion of foreign substances. In recent years, the CRISPR/Cas system has gradually become an important tool in genetic engineering and is widely used in gene editing, gene expression regulation, and gene detection. Among them, the CRISPR/Cas12a system is a new type of CRISPR/Cas system, which has RNA-guided DNase cleavage activity, and can activate the non-specific single-stranded DNA (ssDNA) cleavage activity of Cas12a while specifically cutting double-stranded DNA (dsDNA). . Taking advantage of this feature, the crRNA (CRISPR-derived RNA) can be artificially designed for the target DNA to be complementary to the DNA near the PAM (protospacer-adjacent motif) region in the target DNA, so as to realize the direct detection of different target DNA. Introduction of tagged ssDNA by activating Cas12a non-specific DNA. Doudna's research group (Science 2018, 360, 436-439) used the CRISPR/Cas12a system to realize the detection of human papillomavirus (HPV); when the test DNA exists, crRNA can specifically recognize the test DNA and Cas12a protein to form a triplet The complex activates the non-specific cleavage activity of Cas12a, and realizes fluorescence detection by cutting single-stranded DNA with fluorescent and quenching groups at both ends. In addition to using fluorescent groups as the final output method, electrochemical signals (Angew.Chem.Int.Ed.2019, 58, 17399-17405) and test strips (NatBiotechnol.2020, 38, 870-874) can also be used as characterization.

The currently reported method of using early pregnancy test paper to carry out genetic diagnosis is limited to the loop-mediated isothermal amplification (LAMP) product of the nucleic acid to be tested, using a three-way nucleic acid probe (respectively P1, P2 and P3 for the convenience of illustration) ), where the 3' end of P1 is marked with the pre-expressed hCG protein, the 5' end of P2 is marked with a magnetic ball, and the 3' end is a foothold sequence. Three single-stranded nucleic acids P1, P2 and P3 form a three-way structure. When the LAMP product of the target to be detected exists, its single-stranded loop sequence LP1 can combine with the toehold sequence in P2, triggering a strand displacement reaction, and the single-stranded loop sequence LP3 can combine with P3, and the structure of the three-way probe is thus destroyed ;Finally, using the magnetic force of the magnet, the free hCG in the solution can be separated from the magnetic ball, and the early pregnancy test paper is positive. On the contrary, when there is no LAMP product of the target to be detected, the probe maintains a three-way structure, theoretically there is no free hCG protein in the solution, and the early pregnancy test paper is negative. The disadvantages of this method are: 1. The design of the probe sequence is complicated, and it is necessary to calculate and balance the binding enthalpy to ensure the smooth progress of the chain substitution reaction; 2. The three single-stranded DNAs that form the three-way probe need to optimize the system concentration to ensure the accuracy of the detection reaction. Low background; 3. Double-labeled probes increase the complexity of the experimental operation, and different batches of magnetic balls have different stability, and the conditions need to be re-optimized each time the magnetic balls are replaced; 4. The overall design of the experiment limits the amplification of the gene to be tested. The amplification method can only be used for loop-mediated isothermal amplification reactions. 5. This method can only be used for nucleic acid detection, not for detection of small molecules or proteins.

At present, the commonly used test strip method is used to characterize the nucleic acid detection based on the CRISPR/Cas12a system, which is mainly to label the FAM and Biotin groups at both ends of the detection probe; although the test strips used in this method can be commercialized, The ultimate portable test can also be realized, but the price of test strips is too high, which virtually increases the cost of the entire test, and does not use large quantities of samples for test.

Contents of the invention

In view of this, in order to overcome the defects and deficiencies of the existing nucleic acid detection technology, the present invention provides a nucleic acid detection method based on instant test strips, detection probes and kits thereof. The detection method has obvious target universality, can realize early nucleic acid screening of various infectious diseases, has good specificity, high sensitivity, and greatly reduces the detection cost.

The invention provides a nucleic acid detection method based on an instant detection test strip, comprising:

Dissolve the nucleic acid sample to be tested, hCG-labeled single-stranded DNA probe, crRNA and Cas12a protein in the buffer, and react to obtain the first reaction solution;

Adding a substrate substance to the first reaction solution for incubation to obtain a second reaction solution;

Add the second reaction solution dropwise to the sample pad of the early pregnancy test strip, wait for 5-8 minutes, observe the color development of the test line and the quality control line, and judge whether it contains the nucleic acid to be tested;

The crRNA is a crRNA sequence that specifically binds to the nucleic acid to be tested;

The base material is a macromolecular material capable of combining with hCG-labeled single-stranded DNA probes.

In the present invention, the nucleic acid sample to be tested is: the full length or fragment of DNA of the nucleic acid to be tested, the PCR product of the nucleic acid to be tested, or the constant temperature amplification product of the nucleic acid to be tested.

The detection method of the present invention can be applied to early nucleic acid screening of various infectious diseases without expensive instruments and equipment and professional standard laboratories, and is of great significance to the early monitoring, diagnosis, prevention and control of infectious diseases.

In some specific embodiments, using the detection method of the present invention to detect HPV16 and SARS-CoV-2 samples has high sensitivity, and the detection limit can reach 2 molecular copies.

In the present invention, the hCG-labeled single-stranded DNA probe is referred to as hCG probe for short. The hCG probe consists of two parts, one part is hCG protein, and the other part is single-stranded DNA probe sequence. In some embodiments, the amino acid sequence of the hCG protein is modified by NH2C6 at the 5' end of the single-stranded DNA probe. Specifically, the composition of the hCG-labeled single-stranded DNA probe from the 5' end to the 3' end is: hCG DNA fragment-NH2 C6-single-stranded DNA probe.

In some specific embodiments, the nucleotide sequence of the single-stranded DNA probe is shown in SEQ ID NO: 1:

SEQ ID NO: 1: 5-TTATTTTTATTTTATTCTCTCTGGATGATG-3'.

The 5-terminal NH2 C6 of the single-stranded DNA probe is connected to the HCG protein, the 1-15 nt of the 5'-end is the Cas12a non-specific cleavage region, and the last 14 nt (16-29 nt) is the LAMP substrate binding region.

In some embodiments, the base material is a large molecular weight single-stranded DNA of any sequence that binds to the single-stranded DNA probe through base complementarity, or is a macromolecular substance that binds to the single-stranded DNA probe through a chemical bond.

In some embodiments, the substrate material is the substrate of all products after LAMP amplification of any nucleic acid sequence.

In some embodiments, the buffer comprises 50-200 mM NaCl, 10-40 mM Tris-HCl, 10-40 mM MgCl ₂ and 100-400 μg/mL BSA.

In some specific embodiments, the buffer comprises 200 mM NaCl, 40 mM Tris-HCl, 40 mM MgCl ₂ and 400 μg/mL BSA.

In some embodiments, the reaction is specifically reacting at 25° C. to 37° C. for 30 to 60 minutes; the incubation is 25° C. for 20-40 minutes.

In some specific embodiments, the reaction is specifically 30 min at 37°C or 40-60 min at 25°C; and the incubation is specifically 40 min at 25°C.

The present invention also provides a hCG signal-labeled single-stranded DNA probe for nucleic acid detection, the nucleotide sequence of which is shown in SEQ ID NO:1.

The present invention also provides a nucleic acid detection kit, comprising the hCG-labeled single-stranded DNA probe of the present invention.

The nucleic acid detection kit of the present invention also includes at least one of crRNA, Cas12a protein, buffer, RNase inhibitor, LAMP product substrate, and early pregnancy test strips.

Wherein, the buffer solution includes 50-200 mM NaCl, 10-40 mM Tris-HCl, 10-40 mM MgCl ₂ and 100-400 μg/mL BSA.

In some specific embodiments, the buffer comprises 200 mM NaCl, 40 mM Tris-HCl, 40 mM MgCl ₂ and 400 μg/mL BSA.

The nucleic acid detection method of the present invention is based on the combination of the CRISPR/Cas12a system and the early pregnancy test paper. The nucleic acid is detected by the non-specific hCG detection probe and the crRNA sequence that specifically recognizes the sequence to be tested, which greatly reduces the detection cost and can not only ensure the specificity of the detection performance, while increasing the portability of the entire assay. The detection principle is shown in Figure 1: when the nucleic acid to be tested exists, it can form a ternary complex with Cas12a protein and crRNA, and the RuvC domain of Cas12a in the complex performs DNase activity to cut the dsDNA of the nucleic acid to be tested. The activated Cas12a also has non-specific ssDNA cutting activity, cutting single-stranded DNA probes labeled with human chorionic gonadotropin (hCG) signal. The uncleaved single-stranded DNA probe specifically binds to the single-stranded loop of a substrate with a complex structure and a large volume (such as a LAMP product). Because the substrate is too large, hCG cannot pass through the early pregnancy test paper by chromatography; on the contrary , the cleaved single-stranded DNA probe, because hCG has been completely dissociated in the solution, even if the substrate exists, it can also use chromatography to pass through the early pregnancy test paper. According to the color development of the early pregnancy test paper, it is judged whether the target nucleic acid is contained in the sample. The specific judgment method is:

Compared with the color development of the negative sample, if the red band in the detection area of the sample to be tested is darker than the red band in the detection area of the negative sample, the test result is positive, indicating that the sample to be tested contains the target nucleic acid; If the color of the detection area of the negative sample is the same or the color is lighter, the test result is negative, indicating that the sample does not contain the nucleic acid to be tested.

Compared with the traditional test strip method for the characterization of the CRISPR/Cas12a system nucleic acid detection system, the detection method of the present invention uses cheap commercialized early pregnancy test strips in the market instead of expensive detection test strips, which greatly reduces the cost of detection. Cost; at the same time, it has obvious universality of detection targets, high sensitivity and good specificity, and the detection sensitivity is comparable to that of fluorescence end-point characterization method, and can detect as low as 2 molecular copies. In addition, the hCG probes and LAMP substrates used in this system can be processed into dry powder, which is convenient for storage and transportation.

Description of drawings

Fig. 1 shows the detection principle diagram of nucleic acid detection method of the present invention;

Fig. 2 shows the detection result of the detection method of the present invention to the HPV-16-L1 simulated double-stranded DNA of different concentrations;

Figure 3 shows a schematic diagram of the non-specific ssDNA shearing process triggered by the amplified product activating the CRISPR/Cas12a system after the gene to be tested is subjected to a constant temperature amplification reaction (here, RPA reaction is taken as an example);

Fig. 4 shows the detection result of embodiment 2 early pregnancy test paper to HPV-16-L1;

Fig. 5 shows the detection result of the new coronavirus by the early pregnancy test paper of embodiment 3.

Detailed ways

The invention provides a nucleic acid detection method based on an instant detection test strip, a detection probe and a kit thereof. Those skilled in the art can refer to the content of this article to appropriately improve the process parameters to achieve. In particular, it should be pointed out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously make changes or appropriate changes and combinations to the method and application herein without departing from the content, spirit and scope of the present invention to realize and apply the present invention Invent technology.

The test materials used in the present invention are all common commercially available products, which can be purchased in the market.

Below in conjunction with embodiment, further set forth the present invention:

Embodiment 1 Utilizes the detection method of the present invention to detect the HPV gene

The HPV-16-L1 simulated DNA fragment sequence is tested with test strips, and the output method of the fluorescent signal is compared at the same time. The simulated DNA sequence is as follows:

ATTGGTTACAACGAGCACAGGGCCACAATAATGGCATTTGTTGGGGTAACCAACTATTTGTTACTGTTGTTGATACTACACGCAGTACAAATATGTCATTATGTGCTGCCATATCTACTTCAGAAACTACATAAAAATACTTAACTTTAAGGAGTACCTACGACATGGGGAGGAATATGATTTACAGTTTATTTTTCAACTGTGCAAAATAACCTTAACTGC AGACGTTATGACATACATACATTCTATGAATTCCAC.

The crRNA that specifically binds to the above-mentioned simulated DNA sequence has a nucleotide sequence of: 5'-UAAUUUCUACUAAGUGUAGAUUGAAGUAGAUAUGGCAGCAC-3' (SEQ ID NO: 2)

The reaction system is as follows: 1μM Cas12a, 2μM crRNA, 20U RNase Inhibitor (Murine), 14nM hCG probe, 5nM/1nM/500pM/100pM/50pM/10pM/5pM/0pM HPV-16-L1 simulated DNA double strand, a total of 10μL mixture . Dissolve the above mixture in the buffer system of 200mM NaCl, 40mM Tris-HCl, 40mM MgCl ₂ , 400μg/mL BSA solution, react at 37°C for 30min, add 30μL of LAMP product substrate and incubate at 25°C for 40min, take all 40μL of the reaction The liquid drops on the bottom of the early pregnancy test strip, and observe after standing still for 5 minutes.

It can be seen from the detection results shown in Figure 2B that after Cas12a cleavage was triggered by the addition of different concentrations of nucleic acid analytes, the test strip detection line bands were significantly deepened; compared with the lowest detection concentration of the fluorescent label output mode in Figure 2A , Early pregnancy test strips can also detect 5pM of the gene to be tested, and at the same time, as the concentration of the nucleic acid to be tested deepens, the detection line deepens.

Example 2 Utilize the detection method of the present invention to detect the trace HPV gene

First, using constant temperature amplification reaction (such as RPA or LAMP amplification), taking RPA amplification as an example, amplify the 16-L1 gene of the artificially synthesized HPV virus, and add the amplified product to the above detection system, as shown in the figure 3.

The sequences of the RPA amplification primers used are as follows:

HPV-16-L1 forward primer: TTGTTGGGGGTAACCAACTATTTGTTACTGTT (SEQ ID NO: 3).

HPV-16-L1 reverse primer: CCTCCCCATGTCGTAGGTACTCCTTAAAG (SEQ ID NO: 4).

The RPA amplification is combined with the CRISPR/Cas12a system, and the reaction system is as follows: 1 μM Cas12a, 2 μM crRNA, 20 URNase Inhibitor (Murine), 14 nM hCG probe, 2 μL of RPA amplification products of different copy number templates, a total of 10 μL of the mixture. Dissolve the above mixed solution in the buffer system of 200mM NaCl, 40mM Tris-HCl, 40mM MgCl ₂ , 400μg/mLBSA solution, react at 37℃ for 30min, add 30μL of LAMP product substrate and incubate at 25℃ for 40min, take all 40μL of the reaction solution Drop it on the bottom of the early pregnancy test strip, and observe it after standing still for 5 minutes.

Figure 4 is the non-specific ssDNA cleavage triggered by the RPA product activation of the CRISPR/Cas12a system, and the color development of the pregnancy test strip. Among them, the negative control is that the template concentration in the RPA reaction is 0 molecular copy number, and the positive control is that the template concentration is respectively 2, 20, 200, 2,000, and 20,000 molecular copy numbers. It can be seen from the results that the present invention can detect at least 2 Molecular copy number.

Example 3 Using the detection method of the present invention to detect traces of new coronavirus

In order to prove the universality of the detection method of the present invention, the detection method of the present invention is used to detect trace amounts of novel coronavirus (SAR-Cov-2) reverse-transcribed dsDNA.

First, the RPA amplification method was used to amplify the ORF1ab gene in the new coronavirus at a constant temperature, and the amplified product was directly triggered by the CRISPR/Cas12a system.

The sequence of the novel coronavirus ORF1ab gene amplification probe is as follows:

ORF1ab-F: CTTGAAATTCCACGTAGGAATGTGGCAACTTTAC (SEQ ID NO: 5);

ORF1ab-R: GTATGCCAGGTATGTCAACACATAAACCTTCAG (SEQ ID NO: 6).

Figure 5 is the non-specific ssDNA shearing triggered by the RPA product of ORF1ab after activating the CRISPR/Cas12a system, and the color development of the pregnancy test strip. Among them, the negative control is that the template concentration in the RPA reaction is 0 molecular copy number, and the positive control is that the template concentration is 2, 20, 200, 2,000, and 20,000 molecular copy number respectively.

The above results show that the detection method of the present invention has obvious detection target universality, and the end point detection of the test strip can detect as low as 2 molecular copy numbers. At the same time, the RPA amplification product of the HPV-16-L1 gene was added for control, and it was found that the system can specifically detect the ORF1ab target gene, and will not produce false positives and misdiagnosis even under the interference of a large number of other gene RPA products.

The above are only preferred embodiments of the present invention, and it should be pointed out that for those of ordinary skill in the art, some improvements and modifications can also be made without departing from the principle of the present invention, and these improvements and modifications should also be considered Be the protection scope of the present invention.

sequence listing

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Claims (11)

1. A method for detecting nucleic acid based on a test strip for non-disease diagnosis, comprising:

dissolving a nucleic acid sample to be detected, an hCG marked single-stranded DNA probe, crRNA and Cas12a protein in a buffer solution, and reacting to obtain a first reaction solution;

adding a substrate material into the first reaction solution for incubation to obtain a second reaction solution;

dripping the second reaction liquid onto a sample pad of the early pregnancy test strip, waiting for 5-8min, observing the color development conditions of the detection line and the quality control line, and judging whether the sample pad contains nucleic acid to be detected;

the crRNA is a crRNA sequence which specifically binds to nucleic acid to be detected;

the substrate material is a macromolecular material capable of being combined with the hCG marked single-chain DNA probe;

the nucleotide sequence of the single-stranded DNA probe is shown as SEQ ID NO: 1.

2. The method according to claim 1, wherein the nucleic acid sample to be tested is: the whole length or fragment of the DNA of the nucleic acid to be tested, the PCR product of the nucleic acid to be tested or the isothermal amplification product.

3. The method for detecting nucleic acid according to claim 1, wherein in the hCG-labeled single-stranded DNA probe, the amino acid sequence of the hCG protein is modified at the 5' end of the single-stranded DNA probe by NH2C 6.

4. The method for detecting nucleic acid according to claim 1, wherein the substrate material is a large molecular weight single-stranded DNA of an arbitrary sequence bound to a single-stranded DNA probe by base complementation,

or a macromolecular substance that is bound to the single-stranded DNA probe by a chemical bond.

5. The method of claim 4, wherein the substrate material is a total product substrate after LAMP amplification of any nucleic acid sequence.

6. The method for detecting nucleic acid according to claim 1, wherein the buffer comprises 50-200mM NaCl, 10-40mM Tris-HCl, 10-40mM MgCl ₂ And 100-400 μg/mLBSA.

7. The method for detecting nucleic acid according to claim 1, wherein the reaction is specifically carried out at 25℃to 37℃for 30 to 60 minutes; the incubation is carried out at 25 ℃ for 20-40min.

8. An hCG signal marked single-chain DNA probe for nucleic acid detection, which has a nucleotide sequence shown in SEQ ID NO: 1.

9. A nucleic acid detection kit comprising the hCG-labeled single-stranded DNA probe according to claim 8.

10. The nucleic acid detection kit of claim 9, further comprising at least one of crRNA, cas12a protein, buffer, rnase inhibitor, LAMP product substrate, early pregnancy test strip.

11. The nucleic acid detection kit according to claim 10, wherein the buffer comprises 200mM NaCl,40mM Tris-HCl,40mM MgCl ₂ And 400 μg/mLBSA.

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