CN115029416A - Nucleic acid detection reagent based on SENSR and CRISPR technology, kit and application - Google Patents

Abstract

The invention belongs to the technical field of gene detection, and in particular relates to a nucleic acid detection reagent, kit and application based on SENSR and CRISPR technology. Disclosed is a reagent for detecting nucleic acid; the reagent is based on SENSR and CRISPR technology to detect nucleic acid. By replacing the existing SENSR reporter probe's dye-bound RNA aptamer template sequence with a common sequence recognized by crRNA, Cas protein is in the Under the guidance of CrRNA, after the target sequence is recognized, the Cas protein with para-cleavage activity is stimulated, and the detection signal is generated by cleaving a specific reporter probe; and, compared with the SENSR method and the improved SENSR method, the blank or negative control after using this reagent has no effect. Significant amplification signal, while lower copy target nucleic acid can have significant signal after loading, which can achieve highly sensitive one-pot detection of target nucleic acid; at the same time, the reagent can be used for two or more target nucleic acids at the same time.

Description

A nucleic acid detection reagent, kit and application based on SENSR and CRISPR technology

technical field

The invention belongs to the technical field of gene detection, and in particular relates to a nucleic acid detection reagent, kit and application based on SENSR and CRISPR technology.

Background technique

Some dangerous infectious diseases often have signs and symptoms similar to cold or flu-like syndrome, so early diagnosis is crucial for disease treatment. For the rapid diagnosis of infectious diseases, nucleic acid-based diagnostics have become an alternative to traditional culture or immunoassay methods due to their rapidity and specificity. To improve sensitivity, current nucleic acid detection methods usually perform target amplification before the detection step. Traditional amplification methods are based on PCR, which requires a thermal cycler for precise temperature regulation. As an alternative based on PCR, isothermal amplification methods such as recombinant chain polymerase reaction (RPA) at constant temperature have complex active components. In order to maintain the long-term stability of the active components in the complex detection system, detection reagents need to be Freeze-drying increases production and testing costs.

The nucleic acid detection method based on the SplintR enzymatic ligation reaction is a sequence-specific method, which mainly relies on the ligation of two independent probes to hybridize to the adjacent sites of the RNA target sequence. Because its specificity depends on the method of ligation reaction, it is often used for the detection of genetic disease markers and pathogen detection. Based on this, Chang Ha Woo et al. developed a ligation method based on SplintR enzyme, which is complementary to connect two DNAs through an RNA scaffold, combined with signal amplification to achieve highly sensitive detection of nucleic acids, and developed a one-pot isothermal RNA detection reaction (SENSR). SENSR consists of two enzymatic reactions: ligation by SplintR ligase and subsequent transcription by T7 RNA polymerase. In SENSR, after adding pathogen-derived RNA samples, the process of detection, amplification and signal generation can be carried out through the reaction steps of ligation, transcription and dye aptamer binding, respectively. Chang Ha Woo et al. designed two single-stranded DNA probes, a promoter probe and a reporter probe, respectively. The promoter probe consists of an upstream hybridization sequence (UHS) and a stem-loop structure of the T7 promoter. UHS hybridizes to the 5-terminal half of the target RNA, and the T7 promoter part adopts a stem-loop structure to form an active duplex. The reporter probe consists of a downstream hybridization sequence (DHS) and a dye-conjugated RNA aptamer template sequence. DHS contains the complementary sequence of the other half of the target RNA region. After the UHS and DHS probes hybridize to the target RNA, SplintR ligase ligates the promoter and reporter probes. Subsequently, T7 RNA polymerase uses the full-length ligation probe as a DNA template to synthesize RNA aptamers, which bind to fluorescent dyes and emit fluorescence. The SENSR reaction has a two-fold amplification mechanism: (1) T7 RNA polymerase ligates the full-length probe to the multiplex transcription reaction; (1) The target RNA sequence (including the target RNA formed by multiple transcription) ligates the full-length probe to the ligation reaction . SENSR utilizes this mechanism to achieve highly sensitive detection of RNA.

However, the inventors found in the process of using the SENSR method to detect RNA, the pre-mixed SENSR components at room temperature had significant non-specific amplification before the RNA components were added, and necessary measures must be taken, such as RNA samples and The probes were pre-hybridized and then detected at 37°C to eliminate or reduce non-specific reactions. Although the improved SENSR method can detect positive samples, there is still a problem of high background detection signal during blank loading, and the real-time detection signal will be improved to a certain extent. The signal difference was not significant, affecting the sensitivity of the detection.

SUMMARY OF THE INVENTION

The object of the first aspect of the present invention is to provide a reagent for detecting nucleic acid.

The purpose of the second aspect of the present invention is to provide the application of the above reagents in the preparation of nucleic acid detection products.

The object of the third aspect of the present invention is to provide a kit comprising the reagent of the first aspect of the present invention.

The purpose of the fourth aspect of the present invention is to provide the application of the reagent of the first aspect of the present invention and/or the kit of the third aspect of the present invention in nucleic acid detection.

The purpose of the fifth aspect of the present invention is to provide a non-diagnostic nucleic acid detection method.

In order to achieve the above object, the technical scheme adopted by the present invention is:

A first aspect of the present invention provides a reagent for detecting nucleic acid, comprising: a probe pair and crRNA for detecting target nucleic acid;

The probe pair comprises a promoter probe and a reporter probe;

The promoter probe comprises an upstream hybridization sequence (UHS) and a T7 promoter sequence;

The reporter probe comprises a downstream hybridization sequence (DHS) and a universal sequence;

Described crRNA comprises anchor sequence and spacer sequence;

The upstream hybridization sequence is complementary to a partial sequence of the target nucleic acid;

the downstream hybridization sequence is complementary to the remainder of the target nucleic acid;

The universal sequence is the same as the spacer sequence, so that it can be recognized by crRNA;

The anchor sequence specifically recognizes the Cas protein.

Preferably, the 5' end of the promoter probe contains a phosphate group.

Preferably, the promoter probe includes an upstream hybridization sequence (UHS) and a T7 promoter sequence in sequence from the 5' to 3' end.

Preferably, the reporter probe includes a universal sequence and a downstream hybridization sequence (DHS) in order from the 5' to the 3' end.

Preferably, the crRNA includes an anchor sequence and a spacer sequence from the 5' to the 3' end in order; or a spacer sequence and an anchor sequence.

Preferably, the T7 promoter sequence is a stem-loop structure T7 promoter sequence.

Preferably, the T7 promoter sequence is shown in positions 25-86 of SEQ ID NO.2.

Preferably, the number of bases of the upstream hybridization sequence is 18-24; further, it is 19-24.

Preferably, the upstream hybridization sequence is complementary to the 5' end of the target nucleic acid, and the downstream hybridization sequence is complementary to the 3' end of the target nucleic acid; or

The upstream hybridization sequence is complementary to the 3' end of the target nucleic acid, and the downstream hybridization sequence is complementary to the 5' end of the target nucleic acid.

Preferably, the target nucleic acid is RNA.

Preferably, when the sequence of the target nucleic acid is shown in SEQ ID NO.1, the upstream hybridization sequence is shown in positions 1 to 24 of SEQ ID NO.2; the downstream hybridization sequence is shown in SEQ ID NO.4 shown in positions 31 to 48.

Preferably, when the sequence of the target nucleic acid is shown in SEQ ID NO.6, the upstream hybridization sequence is shown in positions 1 to 19 of SEQ ID NO.7; the downstream hybridization sequence is shown in SEQ ID NO.8 shown in positions 31 to 50.

Preferably, when the sequence of the target nucleic acid is shown in SEQ ID NO.10, the upstream hybridization sequence is shown in positions 1 to 20 of SEQ ID NO.11; the downstream hybridization sequence is shown in SEQ ID NO.13 shown in positions 31 to 50.

Preferably, the universal sequence is not homologous or complementary to the upstream hybridizing sequence and the downstream hybridizing sequence.

Preferably, the number of bases of the universal sequence is 26-34; furthermore, it is 26-30.

Preferably, the universal sequence is shown in positions 1-30 of SEQ ID NO.4, or shown in positions 1-30 of SEQ ID NO.13.

Preferably, when the sequence of the target nucleic acid is shown in SEQ ID NO.1 or SEQ ID NO.6, the general sequence is shown in positions 1-30 of SEQ ID NO.4.

Preferably, when the sequence of the target nucleic acid is shown in SEQ ID NO. 10, the general sequence is shown in positions 1-30 of SEQ ID NO. 13.

Preferably, the Cas protein is at least one of LwaCas13a, PsmCas13b, and CcaCas13b; further, it is at least one of LwaCas13a and PsmCas13b.

Preferably, when the Cas protein is LwaCas13a, the anchor sequence is shown in positions 1-35 of SEQ ID NO.5.

Preferably, when the Cas protein is PsmCas13b, the anchor sequence is shown in positions 31-66 of SEQ ID NO.14.

Preferably, when the sequence of the target nucleic acid is shown in SEQ ID NO.1 or SEQ ID NO.6, the anchor sequence is shown in positions 1-35 of SEQ ID NO.5.

Preferably, when the sequence of the target nucleic acid is shown in SEQ ID NO. 10, the anchor sequence is shown in positions 31-66 of SEQ ID NO. 14.

Preferably, the spacer sequence needs to replace T in the universal sequence with U.

Preferably, the type of the target nucleic acid is greater than or equal to 1.

Preferably, when the type of the target nucleic acid is greater than or equal to 2, the reagent comprises: N groups of probe pairs for detecting nucleic acid and crRNA, where N is equal to the type of the target nucleic acid.

Preferably, when the types of the target nucleic acid are greater than or equal to 2, the universal sequences in the reporter probes of the probe pairs of different target nucleic acids are different.

Preferably, when the types of the target nucleic acids are greater than or equal to 2, the anchor sequences of crRNAs of different target nucleic acids are different.

Preferably, when the target nucleic acid comprises: a nucleic acid whose sequence is shown in SEQ ID NO.6 and a nucleic acid whose sequence is shown in SEQ ID NO.10, the reagent comprises a target nucleic acid whose detection sequence is shown in SEQ ID NO.6 The probe pair and crRNA, and the probe pair and crRNA of the target nucleic acid whose detection sequence is shown in SEQ ID NO.10,

The probe pair of the target nucleic acid whose detection sequence is as shown in SEQ ID NO.6 comprises a promoter probe and a reporter probe of the target nucleic acid whose detection sequence is as shown in SEQ ID NO.6;

The detection sequence is shown in SEQ ID NO.6, and the upstream hybridization sequence of the target nucleic acid promoter probe is shown in positions 1 to 19 of SEQ ID NO.7; the detection sequence is shown in SEQ ID NO.6 The downstream hybridization sequence of the reporter probe of the target nucleic acid shown is shown in the 31st to 50th positions of SEQ ID NO.8; the detection sequence is shown as the general sequence of the reporter probe of the target nucleic acid shown in SEQ ID NO.6. The 1st to 30th positions of SEQ ID NO.4 are shown;

The detection sequence is shown in SEQ ID NO.6, and the anchor sequence of the crRNA of the target nucleic acid is shown in positions 1 to 35 of SEQ ID NO.5;

The probe pair of the target nucleic acid whose detection sequence is shown in SEQ ID NO.10 comprises the promoter probe and the reporter probe of the target nucleic acid whose detection sequence is shown in SEQ ID NO.10;

The detection sequence is shown in SEQ ID NO.10, and the upstream hybridization sequence of the target nucleic acid promoter probe is shown in positions 1 to 20 of SEQ ID NO.11; the detection sequence is shown in SEQ ID NO.10 The downstream hybridization sequence of the reporter probe of the target nucleic acid shown is shown in the 31st to 50th position of SEQ ID NO.13; the detection sequence is shown as the general sequence of the reporter probe of the target nucleic acid shown in SEQ ID NO.10. The 1st to 30th positions of SEQ ID NO.13 are shown;

The detection sequence is shown in SEQ ID NO. 10, and the anchor sequence of the crRNA of the target nucleic acid is shown in positions 31 to 66 of SEQ ID NO. 14.

Preferably, when the sequence of the target nucleic acid is shown in SEQ ID NO.1, the sequence of the promoter probe is shown in SEQ ID NO.2; the sequence of the reporter probe is shown in SEQ ID NO.4 The sequence of the CrRNA is shown in SEQ ID NO.5.

Preferably, when the sequence of the target nucleic acid is shown in SEQ ID NO.6, the sequence of the promoter probe is shown in SEQ ID NO.7; the sequence of the reporter probe is shown in SEQ ID NO.8 The sequence of the CrRNA is shown in SEQ ID NO.5.

Preferably, when the sequence of the target nucleic acid is shown in SEQ ID NO.10, the sequence of the promoter probe is shown in SEQ ID NO.11; the sequence of the reporter probe is shown in SEQ ID NO.13 The sequence of the CrRNA is shown in SEQ ID NO.14.

Preferably, when the target nucleic acid comprises: a nucleic acid whose sequence is shown in SEQ ID NO.6 and a nucleic acid whose sequence is shown in SEQ ID NO.10, the reagent comprises a target nucleic acid whose detection sequence is shown in SEQ ID NO.6 The probe pair and crRNA, and the probe pair and crRNA of the target nucleic acid whose detection sequence is shown in SEQ ID NO.10,

The probe pair of the target nucleic acid whose detection sequence is as shown in SEQ ID NO.6 comprises a promoter probe and a reporter probe of the target nucleic acid whose detection sequence is as shown in SEQ ID NO.6;

The detection sequence is shown in SEQ ID NO.6, and the sequence of the promoter probe of the target nucleic acid is shown in SEQ ID NO.7;

The detection sequence is shown in SEQ ID NO.6, and the sequence of the reporter probe of the target nucleic acid is shown in SEQ ID NO.8;

The detection sequence is as shown in SEQ ID NO.6, and the sequence of the crRNA of the target nucleic acid is as shown in SEQ ID NO.5;

The probe pair of the target nucleic acid whose detection sequence is shown in SEQ ID NO.10 comprises the promoter probe and the reporter probe of the target nucleic acid whose detection sequence is shown in SEQ ID NO.10;

The detection sequence is shown in SEQ ID NO.10, and the sequence of the promoter probe of the target nucleic acid is shown in SEQ ID NO.11;

The detection sequence is shown in SEQ ID NO.10, and the sequence of the reporter probe of the target nucleic acid is shown in SEQ ID NO.13;

The detection sequence is shown in SEQ ID NO.10, and the sequence of the crRNA of the target nucleic acid is shown in SEQ ID NO.14.

The second aspect of the present invention provides the use of the reagent of the first aspect of the present invention in preparing a product for detecting nucleic acid.

Preferably, the nucleic acid is RNA.

Preferably, the product is a reagent, a kit, a test paper or a chip.

A third aspect of the present invention provides a kit comprising the reagent of the first aspect.

Preferably, the kit further comprises a signal reporter probe, the Cas protein in the first aspect of the present invention.

Preferably, the signal reporter probe comprises a nucleic acid sequence, the 5' end of the nucleic acid sequence is labeled with a fluorescent reporter group, and the 3' end is labeled with a quencher group.

Preferably, the fluorescent reporter group is any one of FAM, HEX, ROX, CY5, VIC, JOE, TAMRA.

Preferably, the quenching group is at least one of BHQ1 and BHQ2.

Preferably, the nucleic acid sequence of the signal reporter probe is AAAAA or UUUUU.

Preferably, when the sequence of the target nucleic acid is shown in SEQ ID NO.1 or SEQ ID NO.6, the Cas protein is LwaCas13a.

Preferably, when the sequence of the target nucleic acid is shown in SEQ ID NO.1 or SEQ ID NO.6, the signal reporter probe is FAM-5'UUUUU-3'BHQ1.

Preferably, when the sequence of the target nucleic acid is shown in SEQ ID NO. 10, the Cas protein is PsmCas13b.

Preferably, when the sequence of the target nucleic acid is shown in SEQ ID NO. 10, the signal reporter probe is HEX-5'AAAAA-3'BHQ1.

Preferably, when the type of the target nucleic acid is greater than or equal to 2, the Cas protein includes N types of Cas proteins, the N types of Cas proteins are different from each other, and N is equal to the type of the target nucleic acid.

Preferably, when the types of the target nucleic acid are greater than or equal to 2, the signal reporter probes comprise N kinds of signal reporter probes, the sequences and fluorescent reporter groups of the N kinds of signal reporter probes are different from each other, and N is equal to the Kind of target nucleic acid.

Preferably, when the target nucleic acid comprises: a nucleic acid whose sequence is shown in SEQ ID NO. 6 and a nucleic acid whose sequence is shown in SEQ ID NO. 10, the Cas protein comprises LwaCas13a and PsmCas13b.

Preferably, when the target nucleic acid comprises: the nucleic acid whose sequence is shown in SEQ ID NO.6 and the nucleic acid whose sequence is shown in SEQ ID NO.10, the signal reporter probe comprises FAM-5'UUUUU-3'BHQ1 ( For detection of nucleic acid whose sequence is shown in SEQ ID NO. 6) and HEX-5'AAAAA-3'BHQ1 (for detection of nucleic acid whose sequence is shown in SEQ ID NO. 10).

Preferably, the kit further comprises SENSR buffer, ET-SSB, T7 RNA polymerase, RNase inhibitor, NTPs and SplintR ligase.

Preferably, the kit further comprises water.

Preferably, the water is RNase-free purified water.

A fourth aspect of the present invention provides the use of the reagent of the first aspect and/or the kit of the third aspect in nucleic acid detection for non-diagnostic purposes.

Preferably, the nucleic acid is RNA.

The fifth aspect of the present invention provides a non-diagnostic nucleic acid detection method, comprising the steps of using the reagent of the first aspect of the present invention and/or the kit of the third aspect of the present invention.

The nucleic acid detection method comprises the following steps:

Mixing the promoter probe, the reporter probe and the SENSR buffer in the kit of the third aspect of the present invention to obtain a hybridization solution;

Mix the water, NTPs, SENSR buffer, ET-SSB, T7RNA polymerase, RNase inhibitor, NTPs, SplintR ligase, Cas protein, signal reporter probe, CrRNA in the kit of the third aspect of the present invention to obtain detection fluid;

Mix the nucleic acid to be tested and the hybridization solution, incubate to obtain a mixed solution;

The mixed solution is mixed with the detection solution for reaction and detection.

Preferably, the incubation conditions are incubation at 50-60°C for 2-8 minutes; further, incubation at 55°C for 5 minutes.

Preferably, the temperature of the detection liquid before mixing with the mixed liquid is 35-39°C.

Preferably, the reaction conditions are 30-60 min at 35-39 °C.

The beneficial effects of the present invention are:

The present invention provides a reagent for detecting nucleic acid, comprising: a probe pair and crRNA for detecting target nucleic acid; the probe pair comprises a promoter probe and a reporter probe; the promoter probe comprises an upstream hybridization sequence (UHS ) and T7 promoter sequence; the reporter probe comprises a downstream hybridization sequence (DHS) and a universal sequence; the crRNA comprises an anchor sequence and a spacer sequence; the upstream hybridization sequence is complementary to a partial sequence of the target nucleic acid; the The downstream hybridization sequence is complementary to the remaining sequence of the target nucleic acid; the universal sequence is the same as the spacer sequence, so that it can be recognized by crRNA; the anchor sequence is specifically recognized by the Cas protein; the reagent is based on SENSR and CRISPR technology Detecting nucleic acid, by replacing the RNA aptamer template sequence of the dye-bound reporter probe in the existing SENSR with the universal sequence recognized by crRNA, the Cas protein recognizes the target sequence under the guidance of CrRNA (that is, it is transcribed into and universal). After the sequence template is complementary to the RNA sequence), the Cas protein with para-cleavage activity is stimulated, and the detection signal is generated by cleaving a specific reporter probe; and, compared with the SENSR method and the improved SENSR method, there is no significant difference in the blank or negative control after using this reagent. The amplification signal of the lower copy of the target nucleic acid can have a significant signal after loading the sample, which can realize the highly sensitive one-pot detection of the target nucleic acid, that is, the reagent greatly improves the sensitivity of nucleic acid detection; at the same time, the reagent can simultaneously Two or more target nucleic acids.

Detailed ways

The content of the present invention will be further described in detail below through specific embodiments.

The materials, reagents, etc. used in this example, unless otherwise specified, are reagents and materials obtained from commercial sources.

The malachite green reagent in the following examples is from SIGMA-ALDRICH company, and NTPs, Rnase inhibitor, single-stranded binding protein ET-SSB, SplintR ligase and T7 RNA polymerase are all from NEB company.

Example 1 Probe pair and CrRNA for synthesizing and detecting SARS-COV-2 RNA-dependent RNA polymerase (RdRp) gene

Entrusted Guangzhou Bolaisi Biotechnology Co., Ltd. to synthesize the upstream and downstream probe pairs (promoter probe and reporter probe) of SplintR ligase for detecting RdRp target gene fragments and the corresponding CrRNA for detection, wherein the promoter probe is a table MG-PP1 in 1, the reporter probe is G-RP1 in Table 1, CrRNA is CrRNA in Table 2.

Table 1 SplintR ligase upstream and downstream probe sequences for SARS-CoV-2 RdRp target sequence

*Note: The Tm value is calculated according to the online calculation tool provided by http://www.bio-toyobo.cn/file/tmcal.html ;

Document 1 is Sensitive fluorescence detection of SARS-CoV-2 RNA in clinical samples via one-pot isothermal ligation and transcription. NatBiomedEng.2020Dec;4(12):1168-1179.doi:10.1038/s41551-020-00617-5.

Table 2. R1fL (Reporter 1for LwaCas13a) CrRNA sequence identifying universal sequence 1 in reporter probes

Embodiment 2 Synthesis detects the probe pair and CrRNA of SARS-COV-2 ORF1ab gene

1. Sequence design:

Determine the conserved region (70nt length) of SARS-COV-2ORF1ab gene, and carry out UHS and DHS design. UHS of promoter probe and DHS of reporter probe were designed using Primer-BLAST software (https://www.ncbi.nlm.nih.gov/tools/primer-blast/):

a. PCR product length parameter: 40～60bps to find the best candidate sites (to find best candidate sites that ensure specific binding of both probes which are 20～30-nt long and adjacent to each other);

b. Primer pair specificity checking parameters: Primer pair specificity checking parameterDatabase: Refseq mRNA, Organization: Homo sapiens;

c. Other parameters are unchanged default values.

According to the above parameters, 10 pairs of candidate primers were screened, and the closest primer pair (the smallest space between primers or the smallest cross-overlapping fragment) was selected, and the fragment sequence was adjusted so that the corresponding UHS primer sequence and DHS primer sequence were adjacent (UHS The 5' end of the DHS sequence is adjacent to the 3' end of the DHS sequence), and the Tm value of the primers is between 58 and 66 °C and the Tm difference between the primers does not exceed 2.

According to the above steps, the UHS sequence of the promoter probe and the DHS sequence of the reporter probe designed for SARS-COV-2 ORF1ab were obtained. Entrusted Guangzhou Bolaisi Biotechnology Co., Ltd. to synthesize the SplintR ligase upstream and downstream probe pairs (promoter probe and reporter probe) for detecting ORF1ab target gene fragments and the corresponding detection CrRNA, wherein the promoter probe is the table UHS-O1 in 3, the reporter probe is DHS-O1 in Table 3, CrRNA is CrRNA in Table 2.

Table 3 SplintR ligase upstream and downstream probe sequences for SARS-CoV-2 ORF1ab target sequence

*Note: The Tm value is calculated according to the online calculation tool provided by http://www.bio-toyobo.cn/file/tmcal.html .

Example 3 Synthesis of probe pair and CrRNA for detection of influenza A virus (Influenza A) HA gene

Entrusted Guangzhou Bolaisi Biotechnology Co., Ltd. to synthesize the upstream and downstream probe pairs (promoter probe and reporter probe) of SplintR ligase for detecting HA target gene fragments and the corresponding CrRNA for detection, wherein the promoter probe is a table MG-PP in 4, the reporter probe is G-RP2 in Table 4, CrRNA is CrRNA in Table 5.

Table 4 SplintR ligase upstream and downstream probe sequences for Influenza A HA gene target sequence

*Note: The Tm value is calculated according to the online calculation tool provided by http://www.bio-toyobo.cn/file/tmcal.html .

Table 5 R2fP (Reporter 2for PsmCas13b) CrRNA sequence identifying the universal sequence 2 in the reporter probe

Comparative Example 1 Synthesis of probe pairs for the detection of SARS-COV-2 RNA-dependent RNA polymerase (RdRp) gene

According to the sequence provided in Document 1, Guangzhou Bolaisi Biotechnology Co., Ltd. was entrusted to synthesize the upstream and downstream probe pairs (promoter probe and reporter probe) of SplintR ligase for detecting the target gene fragment of RdRp, wherein the promoter probe is MG-PP1 in Table 1, and the reporter probe is MG-RP1 in Table 1.

Comparative Example 2 Synthesis of Probe Pairs for Detecting SARS-COV-2 ORF1ab Gene

Entrusted Guangzhou Bolaisi Biotechnology Co., Ltd. to synthesize the SplintR ligase upstream and downstream probe pairs (promoter probe and reporter probe) for detecting ORF1ab target gene fragments, wherein the promoter probe is UHS- O1, the reporter probe is DHS-O1MG in Table 3.

Comparative Example 3 Synthesis of Probe Pairs for Detecting Influenza A (Influenza A) HA Gene

Entrusted Guangzhou Bolaisi Biotechnology Co., Ltd. to synthesize the upstream and downstream probe pairs (promoter probe and reporter probe) of SplintR ligase for detecting HA target gene fragments, wherein the promoter probe is MG- PP, the reporter probe is MG-RP in Table 4.

Effect Example 1 Detection of SARS-COV-2 RNA-dependent RNA polymerase (RdRp) gene

1. Gene synthesis

委托广州艾基生物技术有限公司根据文献1的资料进行RdRp基因片段的合成并亚克隆至pGSI质粒SmaI位点中；其中，合成的RdRp基因片段如下：ATAGGGAAACATACAACGTGTTGTAGCTTGTCACACCGTTTCTATAGATTAGCTAATGAGTGTGCTCAAGTATTGAGTGAAATGGTCATGTGTGGCGGTTCACTATATGTTAAACCAGGTGGAACCTCATCAGGAGATGCCACAACTGCTTATGCTAATAGTGTTTTTAACATTTGTCAAGCTGTCACGGCCAATGTTAATGCACTTTTATCTACTGATGGTAACAAAATTGCCGATAGTATGTCCGCAATTTAC(SEQ ID NO.15)。

2. In vitro RNA transcription synthesis

A high-fidelity PCR reaction was performed using the synthesized plasmid as a template. The kit was 2×Hieff CanaceGold Plus Super-Fidelity PCR Master Mix from Shanghai Yisheng. The operation was carried out according to the instructions, and the reaction conditions were: 94°C for 5 mins; 55°C for 30s, 72°C for 30s, 40 cycles; 72°C for 5 mins. The primer sequences are as follows:

PCR upstream primer sequence: GAAATTAATACGACTCACTATAGGG ACGTGTTGTAGCTTGTCACAC (SEQ ID NO. 16) (the underlined part is the T7 promoter sequence);

PCR downstream primer sequence: GTAAATTGCGGACATACTATCGG (SEQ ID NO. 17).

Gel Extraction Kit琼脂糖凝胶回收试剂盒，按其说明操作将扩增的PCR目的条带纯化。纯化的目的条带用RNase-free H₂O溶解，定容至1μg/μL，作为T7 RNA聚合酶体外转录模板。use

Gel Extraction Kit agarose gel recovery kit, operate according to its instructions to purify the amplified PCR target band. The purified target band was dissolved with RNase-free H ₂ O, and the volume was adjusted to 1 μg/μL, which was used as a template for in vitro transcription of T7 RNA polymerase.

In vitro transcription was also carried out with Shanghai Yisheng's T7 in vitro transcription kit according to its instructions. The reaction system and reaction procedures are shown in Table 6.

Table 6 Transcription system and program

After the transcription reaction was completed, add 1 μL of DNase I (RNase-free) to each tube, mix well, centrifuge briefly, and incubate at 37°C for 30 mins to remove template DNA. RNA purification: RNA obtained by transcription (after removing template DNA) was purified with RNA Cleaner magnetic beads (Shanghai Yisheng Cat. No: 12602). The purified RNA is qualified by electrophoresis, quantified by UV detection, diluted with DEPC water to about 1000 copies/uL, and stored in aliquots at -80°C for later use.

3. Detection of SARS-COV-2 RNA-dependent RNA polymerase (RdRp) gene

Adopt the probe pair and CrRNA of embodiment 1 respectively, adopt the probe pair of comparative example 1 with SENSR method, adopt the probe pair of comparative example 1 to detect SARS-COV-2 RNA-dependent RNA polymerase with the improved SENSR method ( RdRp) gene, as follows:

Using the probe pair of Comparative Example 1 to detect the SARS-COV-2 RNA-dependent RNA polymerase (RdRp) gene by the SENSR method, the steps are as follows: Add the following components in sequence: 53.72 μL of RNase-free purified water, SENSR buffer (500 mM Tris -HCl, 100 mM MgCl ₂ , pH 7.4) 10 μL, NTPs (25uM per component) 10 μL, Malachite Green (320 μM) 5 μL, promoter probe MG-PP1 (10 μM) 2 μL, reporter probe MG-RP1 (10 μM) 2.2μL, Rnase inhibitor (40U/μL) 0.5μL, ET-SSB (extremely thermostable single-stranded binding protein, 500ng/μL) 0.8μL, SplintR ligase (25U/μL) 10μL, T7 RNA polymerase (50U/μL) μL) 5 μL, RNA synthesized in step 2 (0, 10, 100, 1000 copies/μL) 0.78 μL. The above components were mixed and incubated at 37°C for 1 h, and 20 μL were sampled at reaction zero, 30 mins, and 1 h after the reaction for fluorescence detection (ex (excitation wavelength): 616 nm/em (emission wavelength): 665 nm).

Using the probe pair of Comparative Example 1 to detect the SARS-COV-2 RNA-dependent RNA polymerase (RdRp) gene by the improved SENSR method, the steps are as follows: Mix the following components: SENSR buffer (500 mM Tris-HCl, 100 mM MgCl) ₂ , pH 7.4) 6 μL, promoter probe MG-PP1 (10 μM) 12 μL, reporter probe MG-RP1 (10 μM) 12 μL, to obtain hybridization solution, store at -20 °C for later use; mix the following components: RNA-free Enzyme Purified Water 53.72μL, NTPs (25uM per component) 10μL, Malachite Green (320μM) 5μL, Rnase Inhibitor (40U/μL) 0.5μL, ET-SSB (500ng/μL) 0.8μL, SplintR Ligase (25U) /μL) 10μL, T7 RNA polymerase (50U/μL) 5μL, SENSR buffer (500mM Tris-HCl, 100mM MgCl ₂ , pH 7.4) 9μL, to obtain the detection solution, and store it at -20°C for later use; take 15μL RNA samples (300 copy/μL, 30 copies/μL, 3 copies/μL and 0 copies/μL), add 30uL of hybridization solution and mix, the obtained mixture was incubated at 55°C for 5min, and 10uL of the mixture was added to pre-warmed 37°C The reaction was carried out in the detection liquid tube (94.02 μL); 20 μL were sampled at the zero point of the reaction, after 30 mins and 1 h of the reaction, and placed in a 384-well flat-bottom black microplate for detection with a TECAN SPARK fluorescence detector (excitation wavelength: 616nm/emission wavelength: 665nm).

The steps of using the probe pair and CrRNA of Example 1 to detect the SARS-COV-2 RNA-dependent RNA polymerase (RdRp) gene are as follows: mix the following components: SENSR buffer (500mM Tris-HCl, 100mM MgCl ₂ , pH7 .4) 6 μL, promoter probe MG-PP1 (10 μM) 12 μL, reporter probe G-RP1 (10 μM) 12 μL to obtain hybridization solution, and store at -20°C for later use; mix the following components: RNase-free purification Water 50.5μL, NTPs (25uM per component) 10μL, Rnase inhibitor (40U/μL) 0.5μL, ET-SSB (500ng/μL) 0.8μL, LwaCas13aReporter (FAM-5'-UUUUU-3'BHQ1, 10μM) 5μL, LwaCas13a (Swiss-Prot: U2PSH1.1, 2mg/mL) 2.5μL, CrRNA (1.25uM) 1.25μL, SplintR ligase (25U/μL) 10μL, T7 RNA polymerase (50U/μL) 5μL, SENSR buffer (500mM Tris-HCl, 100mM MgCl ₂ , pH 7.4) 9 μL to obtain the detection solution, stored at -20°C for later use; take 15 μL RNA samples (respectively 300 copies/μL, 30 copies/μL, 3 copies/μL and 0 copies/μL) μL) add 30uL of hybridization solution and mix, the obtained mixture is incubated at 55°C for 5min, add 10uL of the mixture into a pre-warmed 37°C detection solution tube (94.55μL), mix well, and place it in the detection FAM channel The reaction and detection were carried out at 37°C in a POCT facility (Guangzhou Double Helix, Delix).

Using the probe pair and CrRNA of Example 1, using the probe pair of Comparative Example 1 to detect SARS-COV-2 RNA-dependent RNA polymerase (RdRp The result of ) gene is as shown in table 7,8,9: as seen from table 7 result, adopt the probe of comparative example 1 to detect SARS-COV-2 RNA-dependent RNA polymerase (RdRp) gene with SENSR method, blank sample There is no significant difference between the fluorescence intensity changes over time between positive samples and the positive samples, indicating that the SENSR detection method of mixing the detection components including promoter probes and reporter probes at room temperature to form the detection solution in advance is not feasible. This method was no longer used for SENSR; from the results in Table 8, the probe pair of Comparative Example 1 was used to detect the SARS-COV-2 RNA-dependent RNA polymerase (RdRp) gene with the improved SENSR method compared with the unmodified SENSR The method has better effect. When 100 copies, the positive signal can be judged by ΔRn value within 30min; when 1000 copies, positive signal can be judged by ΔRn value or Rn value within 30min; but the blank Rn value and ΔRn value are still relatively large In addition, there was no difference in Rn value or ΔRn with blank at lower sample copy number (10 copies); while the probe pair and CrRNA of Example 1 were used to detect SARS-COV-2 RNA-dependent RNA polymerase ( RdRp) gene, the Rn value and ΔRn value of the blank sample have a low growth rate with time, and the detection results of different copy numbers basically show a dose-effect relationship. When 100 copies are used, it can be judged as positive by Rn or ΔRn within 30 minutes, and when 10 copies are used, it can be judged as positive by Rn or ΔRn It can be judged as positive by ΔRn within 1 h; it can be seen that the sensitivity of the probe pair and CrRNA detection of Example 1 is higher.

Table 7 uses the probe pair of Comparative Example 1 to detect SARS-CoV-2 RdRf gene detection results by SENSR method

Table 8 uses the probe pair of Comparative Example 1 to detect SARS-CoV-2 RdRf gene detection results by the improved SENSR method

Note: * indicates that the detection value of the sample is more than twice the detection value of the blank group in the same period, and it is judged as a positive signal.

Table 9 adopts the probe pair and CrRNA of Example 1 to detect SARS-CoV-2 RdRf gene detection results

Note: * indicates that the detection value of the sample is more than twice the detection value of the blank group in the same period, and it is judged as a positive signal.

Effect Example 2 Detection of SARS-COV-2 ORF1ab Gene

Adopt the probe pair of embodiment 2 and CrRNA respectively, adopt the probe pair of comparative example 2 to detect SARS-COV-2 ORF1ab gene with the improved SENSR method, be specific as follows:

The steps of using the probe pair of Comparative Example 2 to detect the SARS-COV-2 ORF1ab gene by the improved SENSR method are as follows: take a pseudovirus sample (MS phage pseudovirus sample (containing 2019-nCoV ORF1ab+E+N gene positive strand RNA, MS bacteriophage pseudovirus samples were obtained from Guangzhou Bondsun Company, with the genome copy number information per milliliter of the sample solution attached, Cat. No.: BDS-IQC-276) Virus preservation solution (Universal Transport Medium, UTM) was purchased from Shandong Ampu Future Biotechnology Co., Ltd.) diluted to the genome copy numbers of 1 copy/μL, 5 copies/μL and 20 copies/μL, respectively, the blank sample was prepared by 9-fold UTM dilution in normal saline; the following components were mixed: SENSR buffer (500mM Tris-HCl, 100mM MgCl ₂ , pH 7.4) 6 μL, promoter probe UHS-O1 (10 μM) 12 μL, reporter probe DHS-O1MG (10 μM) 12 μL, to obtain hybridization solution, and store at -20°C for later use; Mixing of components: RNase-free purified water 53.72μL, NTPs (25uM per component) 10μL, malachite green (320μM) 5μL, RNase inhibitor (40U/μL) 0.5μL, ET-SSB (500ng/μL) 0.8 μL, SplintR ligase (25U/μL) 10μL, T7 RNA polymerase (50U/μL) 5μL, SENSR buffer (500mM Tris-HCl, 100mM MgCl ₂ , pH 7.4) 9μL to obtain detection solution, and store at -20°C for later use; Take 15 μL of pseudovirus samples with different copy numbers (1 copy/μL, 5 copies/μL and 20 copies/μL)/blank samples respectively, incubate at 95°C for 5mins; then mix with twice the volume of hybridization solution to obtain After the mixed solution was incubated at 55 °C for 5 min, 10 uL of the mixed solution was added to the detection tube (94.02 μL) pre-warmed at 37 °C for reaction; 20 uL were sampled at the zero point of the reaction, after 30 mins and 1 h of reaction, and placed in 384 wells. Detection was performed in a flat-bottom black microplate with a TECAN SPARK fluorescence detector (excitation wavelength: 616 nm/emission wavelength: 665 nm).

The steps of using the probe pair and CrRNA of Example 2 to detect the SARS-COV-2 ORF1ab gene are as follows: take a pseudovirus sample (MS bacteriophage pseudovirus sample (containing 2019-nCoV ORF1ab+E+N gene plus-strand RNA, MS bacteriophage pseudovirus) The sample was obtained from Guangzhou Bang Desheng Company, and the genome copy number information per milliliter of the sample solution has been attached. Item number: BDS-IQC-276) The virus preservation solution (Universal Transport Medium, UTM) was purchased from Shandong Anpu Future Biotechnology Co., Ltd. ) were diluted to the genome copy numbers of 1 copy/μL, 5 copies/μL and 20 copies/μL, respectively, and the blank sample was prepared by 9-fold UTM dilution in normal saline; the following components were mixed: SENSR buffer (500mM Tris- HCl, 100 mM MgCl ₂ , pH 7.4) 6 μL, promoter probe UHS-O1 (10 μM) 12 μL, reporter probe DHS-O1 (10 μM) 12 μL, to obtain a hybridization solution, which was stored at -20°C for later use; Mixing: RNase-free purified water 50.5μL, NTPs (25uM per component) 10μL, Rnase inhibitor (40U/μL) 0.5μL, ET-SSB (500ng/μL) 0.8μL, LwaCas13aReporter (FAM-5'UUUUU-3 'BHQ1, 10uM) 5μL, LwaCas13a (2mg/mL) 2.5μL, CrRNA (1.25uM) 1.25μL, SplintR ligase (25U/μL) 10μL, T7 RNA polymerase (50U/μL) 5μL, SENSR buffer (500mM Tris) -HCl, 100mM MgCl ₂ , pH 7.4) 9μL to obtain the detection solution, which was stored at -20°C for later use; 15μL of pseudovirus samples with different copy numbers (1 copy/μL, 5 copies/μL and 20 copies/μL)/blank were taken respectively. The samples were incubated at 95°C for 5 mins; then mixed with twice the volume of hybridization solution, and the resulting mixture was incubated at 55°C for 5 min, and 10uL of the mixture was added to a pre-warmed 37°C detection solution tube (94.55°C). μL), and then placed in a POCT device (Guangzhou Double Helix, Delix) for detecting FAM channels at 37°C for reaction and detection.

Using the probe pair and CrRNA of Example 2, using the probe pair of Comparative Example 2 to detect the SARS-COV-2 ORF1ab gene with the improved SENSR method, the results are shown in Tables 10 and 11: as can be seen from the results in Table 10, In the process of detecting the SARS-COV-2 ORF1ab gene using the probe pair and CrRNA of Example 2, the growth rate of the blank Rn value and ΔRn value with time was relatively low, and the detection results of different copy numbers basically showed a dose-effect relationship. The difference between the average Rn and ΔRn of the copy sample at 30min and the blank has exceeded 2 times, and it can be judged as positive; and the average ΔRn of the 5-copy sample at 60min is 2 times that of the blank sample, which can be judged as Positive; it shows that the probe pair and CrRNA of Example 2 can be used to detect low-copy positive samples (5 copies) in a relatively short period of time; and as can be seen from Table 11, the probe pair of Comparative Example 2 is used to improve the In the process of detecting the SARS-COV-2ORF1ab gene by the SENSR method, the detection signal of the blank sample increased to a certain extent with time, and the difference with the positive sample was not significant, and the ΔRn value of the high copy group (100 copies) reached 2 times that of the blank sample at 60min. Above; it can be seen that the sensitivity of the probe pair and CrRNA detection of Example 2 is higher.

Table 10 uses the probe pair and CrRNA of Example 2 to detect the results of SARS-CoV-2 ORF1ab

Note: * indicates that the detection value of the sample is more than twice the detection value of the blank group in the same period.

Table 11 Using the probe pair of Comparative Example 2 to detect SARS-CoV-2 ORF1ab by the improved SENSR method

Note: * indicates that the detection value of the sample is more than twice the detection value of the blank group in the same period.

Effect Example 3 Detection of Influenza A (Influenza A) HA Gene

1. Gene synthesis

Guangzhou Aike Biotechnology Co., Ltd. was entrusted to synthesize the HA gene fragment of influenza A virus according to reference 1 and subcloned it into the SmaI site of the pGSI plasmid. The synthesized HA gene fragment is as follows: AGCTATAGCAGGTTTTATAGAGGGAGGATGGCAGGGAATGGTAGATGGTTGGTATGGGTACCACCATAGCAATGAGCAGGGGAGTGGATACGCTGCAGACAAAGAATCCACTCAAAAGGCAATAGATGGAGTCACCAATAAGGTCAACTCGATCATTGACAAAATGAACACTCAGTTTGAGGCCGTTGGAAGGGAATTTAATAACTTGGAAAGGAGGATAGAGAATTTAAACAGAIDGAAGACGGA18.

2. In vitro RNA transcription synthesis

A high-fidelity PCR reaction was performed using the synthesized plasmid as a template. The kit was 2×Hieff CanaceGold Plus Super-Fidelity PCR Master Mix from Shanghai Yisheng. The operation was carried out according to the instructions, and the reaction conditions were: 94°C for 5 mins; 55°C for 30s, 72°C for 30s, 40 cycles; 72°C for 5 mins. The primer sequences are as follows:

PCR upstream primer sequence: GAAATTAATACGACTCACTATAGGG AGCTATAGCAGGTTTTATAGAGGGA (SEQ ID NO. 19) (the underlined part is the T7 promoter sequence);

PCR downstream primer sequence: AAGCAGATGGAAGACGGA (SEQ ID NO. 20).

Gel Extraction Kit琼脂糖凝胶回收试剂盒，按其说明操作将扩增的PCR目的条带纯化。纯化的目的条带用RNase-free H₂O溶解，定容至1μg/μL，作为T7 RNA聚合酶体外转录模板。use

Gel Extraction Kit agarose gel recovery kit, operate according to its instructions to purify the amplified PCR target band. The purified target band was dissolved with RNase-free H ₂ O, and the volume was adjusted to 1 μg/μL, which was used as a template for in vitro transcription of T7 RNA polymerase.

In vitro transcription was also carried out with Shanghai Yisheng's T7 in vitro transcription kit according to its instructions. The reaction system and reaction procedures are shown in Table 6. After the transcription reaction was completed, add 1 μL of DNase I (RNase-free) to each tube, mix well, centrifuge briefly, and incubate at 37°C for 30 mins to remove template DNA. RNA purification: RNA obtained by transcription (after removing template DNA) was purified with RNACleaner magnetic beads (Shanghai Yisheng Cat. No: 12602). The purified RNA is qualified by electrophoresis, quantified by UV detection, diluted with DEPC water to about 1000 copies/uL, and stored in aliquots at -80°C for later use.

3. Detection of HA gene of Influenza A virus

The probe pair and CrRNA of Example 3 and the probe pair of Comparative Example 3 were respectively used to detect the HA gene of influenza A virus (Influenza A) with the improved SENSR method, as follows:

Using the probe pair of Comparative Example 3 to detect the HA gene of influenza A virus (Influenza A) by the improved SENSR method, the steps are as follows: mix the following components: SENSR buffer (500mM Tris-HCl, 100mM MgCl ₂ , pH7 .4) 6 μL, promoter probe MG-PP (10 μM) 12 μL, reporter probe MG-RP (10 μM) 12 μL to obtain hybridization solution, and store at -20°C for later use; mix the following components: RNase-free purification Water 53.72μL, NTPs (25uM per component) 10μL, Malachite Green (320μM) 5μL, Rnase Inhibitor (40U/μL) 0.5μL, ET-SSB (500ng/μL) 0.8μL, SplintR Ligase (25U/μL) ) 10 μL, T7 RNA polymerase (50 U/μL) 5 μL, SENSR buffer (500 mM Tris-HCl, 100 mM MgCl ₂ , pH 7.4) 9 μL to obtain the detection solution, and store at -20 °C for later use; 15μL RNA samples of copies/μL, 30 copies/μL and 300 copies/μL were mixed with 30uL of hybridization solution. After the resulting mixture was incubated at 55°C for 5 minutes, 10uL of the mixture was added to the pre-warmed detection solution at 37°C. The reaction was carried out in a tube (94.02μL); 20uL were sampled at the zero point of the reaction, after 30mins and 1h of the reaction, and placed in a 384-well flat-bottom black microplate for detection with a TECAN SPARK fluorescence detector (excitation wavelength: 616nm/emission wavelength: 665nm) .

The steps of using the probe pair and CrRNA of Example 3 to detect the HA gene of influenza A virus (Influenza A) are as follows: mix the following components: SENSR buffer (500mM Tris-HCl, 100mM MgCl ₂ , pH 7.4) 6 μL, Promoter probe MG-PP (10 μM) 12 μL, reporter probe G-RP2 (10 μM) 12 μL, obtain hybridization solution, and store at -20°C for later use; mix the following components: RNase-free purified water 50.5 μL, NTPs (25uM per component) 10μL, Rnase inhibitor (40U/μL) 0.5μL, ET-SSB (500ng/μL) 0.8μL, PsmCas13b Reporter (HEX-5'-AAAAA3'-BHQ1, 10μM) 5μL, PsmCas13b (2mg) /mL) 2.5μL, CrRNA (1.25uM) 1.25μL, SplintR ligase (25U/μL) 10μL, T7 RNA polymerase (50U/μL) 5μL, SENSR buffer (500mM Tris-HCl, 100mM MgCl ₂ , pH 7.4) 9 μL of the detection solution was obtained, and stored at -20°C for later use; 15μL RNA samples of 0 copies, 3 copies/μL, 30 copies/μL and 300 copies/μL were added to 30uL of hybridization solution and mixed, and the obtained mixture was subjected to 55°C After incubation for 5 min, 10 uL of the mixture was added to the detection solution tube (94.55 μL) pre-warmed at 37 °C, and then placed in the BIORAD CFX96 Realtime PCR for HEX channel detection at 37 °C for reaction and detection.

Using the probe pair and CrRNA of Example 3 respectively, and using the probe pair of Comparative Example 3 to detect the HA gene of influenza A virus (Influenza A) by the improved SENSR method, the results are shown in Tables 12 and 13: from Table 13 As can be seen from the results, in the process of detecting the HA gene of influenza A virus (Influenza A) by using the probe pair and CrRNA of Example 3, the growth rate of the blank Rn value and the ΔRn value with time is relatively low, and the detection results of different copy numbers are basically the same. There is a dose-effect relationship. The difference between the average Rn and ΔRn of the 1000-copy sample at 30min and the blank has exceeded 2 times, and the difference between the average Rn and ΔRn of the 100-copy sample at 60min has been more than 2 times. The difference between the average value of ΔRn and the blank has exceeded 2 times when the copy sample is detected at 30min, and the difference between the average value of ΔRn and the blank has exceeded 2 times when the 10-copy sample is detected at 60min, and it can be judged as positive at this time; it shows that the probe of Example 3 is used Pair and CrRNA can detect low-copy positive samples (10 copies) in a relatively short period of time; and as can be seen from Table 12, the probe pair of Comparative Example 3 was used to detect influenza A virus (Influenza A virus) by the improved SENSR method. A) During the HA gene process, the detection signal of the blank sample increased with time, and the difference with the positive sample was not significant. The ΔRn value of the high-copy group (100 copies) reached more than 2 times that of the blank sample at 60 minutes, and it was higher at 60 minutes. The Rn value of the copy group (1000 copies) reached more than 2 times that of the blank sample; it can be seen that the sensitivity of the probe pair and CrRNA detection in Example 3 is higher.

Table 12 Using the probe pair of Comparative Example 3 to detect Influenza A HA gene detection results by the improved SENSR method

Note: * indicates that the detection value of the sample is more than twice the detection value of the blank group in the same period, and it is judged as a positive signal.

Table 13 adopts the probe pair of embodiment 3 and CrRNA to detect Influenza A HA gene detection result

Note: * indicates that the detection value of the sample is more than twice the detection value of the blank group in the same period.

Effect Example 4 Detection of SARS-COV-2 ORF1ab gene and influenza A virus (Influenza A) HA gene

1. RNA samples

In this effect example, the ORF1ab target RNA is derived from the pseudovirus of effect example 2. After the pseudovirus RNA is extracted and purified with a viral nucleic acid extraction kit, the volume of DEPC purified water is adjusted to about 200 copies/μL. The HA target RNA was derived from the synthetically purified sample in Effect Example 3, and the volume of DEPC purified water was adjusted to 200 copies/μL. The samples can be diluted with DEPC water according to the experimental requirements.

2. Detection of SARS-COV-2 ORF1ab gene and Influenza A HA gene

SARS-COV-2 ORF1ab was detected by the SENSR method using the probe pairs and CrRNA of Examples 2 and 3 respectively, using the probe pairs of Comparative Examples 2 and 3, and the improved SENSR method using the probe pairs of Comparative Examples 2 and 3. Gene and influenza A virus (Influenza A) HA gene, as follows:

Using the probe pairs of Comparative Examples 2 and 3 to detect the SARS-COV-2 ORF1ab gene and the HA gene of influenza A virus (Influenza A) by the SENSR method, the steps are as follows: Add the following components in turn: 29.52 μL of RNase-free purified water , SENSR buffer (500mM Tris-HCl, 100mM MgCl ₂ , pH 7.4) 10μL, NTPs (25uM per component) 10μL, Malachite Green (320μM) 5μL, DFHBI-1T (320μM) 20μL, promoter probe UHS-O1 (10 μM) 2 μL, reporter probe DHS-O1MG (10 μM) 2.2 μL, promoter probe MG-PP (10 μM) 2 μL, reporter probe BR-RP ( gtatgtgggagacggtcgggtccag atattcgtatctgtcgagtagagtgtgggctcccacatac TTTGTCTGCAGCGTATCCAC, SEQ ID NO.21, underlined in malachite green RNA aptamer sequence for fluorescence detection, 10μM) 2.2μL, Rnase inhibitor (40U/μL) 0.5μL, ET-SSB (500ng/μL) 0.8μL, SplintR ligase (25U/μL) 10μL, T7 RNA polymerase (50U/μL) 5μL, RNA from step 1 (200 copies/μL) 20-fold diluted sample or 2-fold diluted sample 0.78μL. After mixing the above components, they were incubated at 37°C for 1 h, and 20 μL were sampled at reaction zero, 30 mins, and 1 h for dual-channel fluorescence detection (ex: 616 nm/em: 665 nm for malachite green; ex: 460 nm). /em:520nm for DFHBI-1T).

Using the probe pairs of Comparative Examples 2 and 3 to detect the SARS-COV-2 ORF1ab gene and the HA gene of influenza A virus (Influenza A) by the improved SENSR method, the steps are as follows: Mix the following components: SENSR buffer (500mM Tris -HCl, 100 mM MgCl ₂ , pH 7.4) 6 μL, promoter probe UHS-O1 (20 μM) 6 μL, reporter probe MG-RP1 (20 μM) 6 μL, promoter probe MG-PP (20 μM) 6 μL, reporter probe BR-RP (20μM) 6μL to obtain hybridization solution, and store at -20°C for later use; mix the following components: 48.72μL of RNase-free purified water, 10μL of NTPs (25uM per component), and 5μL of malachite green (320μM) , DFHBI-1T (320μM) 5μL, Rnase inhibitor (40U/μL) 0.5μL, ET-SSB (500ng/μL) 0.8μL, SplintR ligase (25U/μL) 10μL, T7 RNA polymerase (50U/μL) 5μL, SENSR buffer (500mM Tris-HCl, 100mM MgCl ₂ , pH 7.4) 9μL to obtain detection solution, and store at -20°C for later use; take 15μL blank sample and 15μL with DEPC water to dilute to 3 copies/μL and 30 copies/μL respectively ORF1ab single-positive RNA samples, 15 μL of HA single-positive RNA samples diluted with DEPC water to 3 copies/μL and 30 copies/μL, and 30 μL of hybridization solution were added to mix, and the resulting mixture was incubated at 55°C for 5 min. 10uL of the mixture was added to a pre-warmed detection solution tube (94.02μL) at 37°C for the reaction; 20uL were sampled at the zero point of the reaction, 30mins and 1h after the reaction, and placed in a 384-well flat-bottom black microplate with a TECAN SPARK fluorescence detector. Two-channel fluorescence detection was performed (excitation wavelength: 616 nm/emission wavelength: 665 nm; excitation wavelength: 460 nm/emission wavelength: 520 nm).

The steps of using the probe pairs and CrRNA of Examples 2 and 3 to detect the SARS-COV-2 ORF1ab gene and the influenza A virus (Influenza A) HA gene are as follows: mix the components of the following contents: SENSR buffer (500mM Tris-HCl, 100mM MgCl ₂ , pH 7.4) 6 μL, promoter probe UHS-O1 (20 μM) 6 μL, reporter probe MG-RP1 (20 μM) 6 μL, promoter probe MG-PP (20 μM) 6 μL, reporter probe BR-RP (20μM) 6μL to obtain hybridization solution, and store at -20°C for later use; mix the following components: 43.75μL of RNase-free purified water, 10μL of NTPs (25uM per component), 0.5μL of RNase inhibitor (40U/μL), ET-SSB (500ng/μL) 0.8μL, LwaCas13aReporter (FAM-5'-UUUUU-3'BHQ1, 10μM) 5μL, LwaCas13a (2mg/mL) 1.5μL, PsmCas13bReporter (HEX-5'-AAAAA-3'-BHQ1 , 10μM) 5μL, PsmCas13b (2mg/mL) 1.5μL, CrRNA1 (1.25uM) 1.25μL, CrRNA2 (1.25uM) 1.25μL, SplintR ligase (25U/μL) 10μL, T7 RNA polymerase (50U/μL) 5μL , SENSR buffer (500mM Tris-HCl, 100mM MgCl ₂ , pH 7.4) 9 μL to obtain detection solution, and store at -20°C for later use; take 15 μL blank sample, 15 μL of ORF1ab diluted with DEPC water to 3 copies/μL and 30 copies/μL respectively Single-positive RNA samples, 15 μL of HA single-positive RNA samples diluted with DEPC water to 3 copies/μL and 30 copies/μL, add 30 μL of hybridization solution to mix, and the resulting mixture was incubated at 55°C for 5 min, and the mixture was taken. 10uL was added to the detection solution tube (94.55μL) that had been pre-warmed at 37°C, and then placed in the BIORAD CFX96 Realtime PCR for FAM/HE X dual-channel detection at 37°C for reaction and detection.

Using the probe pairs and CrRNA of Examples 2 and 3, using the probe pairs of Comparative Examples 2 and 3 to detect the SARS-COV-2 ORF1ab gene by the SENSR method, and using the probe pairs of Comparative Examples 2 and 3 to detect the SARS-COV-2 ORF1ab gene And the result of type A influenza virus (InfluenzaA) HA gene is as shown in table 14,15,16: as seen from table 14 result, adopt the probe pair of comparative example 2,3 to detect SARS-COV-2ORF1ab gene and with SENSR method. Influenza A virus (Influenza A) HA gene, there is no significant difference in fluorescence intensity changes over time between blank samples and positive samples, indicating that the detection components including promoter probes and reporter probes are mixed together at room temperature in advance. The SENSR detection method of the detection solution is not feasible; as seen from the results in Table 15, the probe pairs of Comparative Examples 2 and 3 are used to detect the SARS-COV-2 ORF1ab gene and the influenza A virus (Influenza A) HA gene with the improved SENSR method Compared with the unimproved SENSR method, it has a better effect. At 100 copies, the positive signal of the HA gene can be judged by the ΔRn value within 30 minutes; when the 100 copies are used, the positive signal of the ORF1ab gene can be judged by the ΔRn value within 60 minutes; but the blank Rn ΔRn value and ΔRn value still have a larger trend of growth; in addition, there is no difference between Rn value or ΔRn and blank at lower sample copy number (10 copies); while the probe pair and CrRNA of Examples 2 and 3 are used to detect SARS -COV-2ORF1ab gene and influenza A virus (Influenza A) HA gene, the Rn value and ΔRn value of the blank sample have a low growth rate with time, and the detection results of different copy numbers are basically in a dose-effect relationship. It can be judged as positive (ORF1ab gene, HA gene) by Rn or ΔRn within 30mins, and can be judged as positive (ORF1ab gene, HA gene) by ΔRn within 1h when 10 copies are used; it can be seen that the probe pairs of Examples 2 and 3 and CrRNA detection is more sensitive.

Table 14 Using the probe pairs of Comparative Examples 2 and 3 to detect ORF1ab/HA dual-channel detection results by SENSR method

Table 15 Using the probe pairs of Comparative Examples 2 and 3 to detect ORF1ab/HA dual-channel detection results by the improved SENSR method

Note: * indicates that the detection value of the sample is more than twice the detection value of the blank group in the same period.

Table 16 uses the probe pairs and CrRNA of Examples 2 and 3 to detect ORF1ab/HA dual-channel detection results

Note: * indicates that the detection value of the sample is more than twice the detection value of the blank group in the same period.

The above-mentioned embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above-mentioned embodiments, and any other changes, modifications, substitutions, combinations, The simplification should be equivalent replacement manners, which are all included in the protection scope of the present invention.

SEQUENCE LISTING

<110> Guangzhou Baiyunshan Baidi Biopharmaceutical Co., Ltd.

<120> A nucleic acid detection reagent, kit and application based on SENSR and CRISPR technology

<130>

<160> 21

<170> PatentIn version 3.5

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<211> 42

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<213> SARS-CoV-2

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acuauauguu aaaccaggug gaaccucauc aggagaugcc ac 42

<210> 2

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<213> Artificial sequences

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gttccacctg gtttaacata tagtccctat agtgagtcgt attaatttcg cgacaacacg 60

cgaaattaat acgactcact ataggg 86

<210> 3

<211> 56

<212> DNA

<213> Artificial sequences

<400> 3

ggatccattc gttacctggc tctcgccagt cgggatccgt ggcatctcct gatgag 56

<210> 4

<211> 48

<212> DNA

<213> Artificial sequences

<400> 4

ctaccaagta atccatattt ctagaggatc gtggcatctc ctgatgag 48

<210> 5

<211> 65

<212> DNA

<213> Artificial sequences

<400> 5

gauuuagacu accccaaaaa cgaaggggac uaaaacuacc aaguaaucca uauuucuaga 60

ggauc 65

<210> 6

<211> 39

<212> DNA

<213> SARS-CoV-2

<400> 6

ugugaucaac uccgcgaacc caugcuucag ucagcugau 39

<210> 7

<211> 81

<212> DNA

<213> Artificial sequences

<400> 7

gttcgcggag ttgatcacac cctatagtga gtcgtattaa tttcgcgaca acacgcgaaa 60

ttaatacgac tcactatagg g 81

<210> 8

<211> 50

<212> DNA

<213> Artificial sequences

<400> 8

ctaccaagta atccatattt ctagaggatc atcagctgac tgaagcatgg 50

<210> 9

<211> 58

<212> DNA

<213> Artificial sequences

<400> 9

ggatccattc gttacctggc tctcgccagt cgggatccat cagctgactg aagcatgg 58

<210> 10

<211> 40

<212> DNA

<213> Influenza A

<400> 10

ccauagcaau gagcagggga guggauacgc ugcagacaaa 40

<210> 11

<211> 82

<212> DNA

<213> Artificial sequences

<400> 11

tcccctgctc attgctatgg ccctatagtg agtcgtatta atttcgcgac aacacgcgaa 60

attaatacga ctcactatag gg 82

<210> 12

<211> 58

<212> DNA

<213> Artificial sequences

<400> 12

ggatccattc gttacctggc tctcgccagt cgggatcctt tgtctgcagc gtatccac 58

<210> 13

<211> 50

<212> DNA

<213> Artificial sequences

<400> 13

cctggttcat gagcttcctg ccactgccaa tttgtctgca gcgtatccac 50

<210> 14

<211> 66

<212> DNA

<213> Artificial sequences

<400> 14

ccugguucau gagcuuccug ccacugccaa guuguagaag cuuaucguuu ggauagguau 60

gacaac 66

<210> 15

<211> 255

<212> DNA

<213> SARS-COV-2

<400> 15

atagggaaac atacaacgtg ttgtagcttg tcacaccgtt tctatagatt agctaatgag 60

tgtgctcaag tattgagtga aatggtcatg tgtggcggtt cactatatgt taaaccaggt 120

ggaacctcat caggagatgc cacaactgct tatgctaata gtgtttttaa catttgtcaa 180

gctgtcacgg ccaatgttaa tgcactttta tctactgatg gtaacaaaat tgccgatagt 240

atgtccgcaa tttac 255

<210> 16

<211> 46

<212> DNA

<213> Artificial sequences

<400> 16

gaaattaata cgactcacta tagggacgtg ttgtagcttg tcacac 46

<210> 17

<211> 23

<212> DNA

<213> Artificial sequences

<400> 17

gtaaattgcg gacatactat cgg 23

<210> 18

<211> 250

<212> DNA

<213> Influenza A

<400> 18

agctatagca ggttttatag agggaggatg gcagggaatg gtagatggtt ggtatgggta 60

ccaccatagc aatgagcagg ggagtggata cgctgcagac aaagaatcca ctcaaaaggc 120

aatagatgga gtcaccaata aggtcaactc gatcattgac aaaatgaaca ctcagtttga 180

ggccgttgga agggaattta ataacttgga aaggaggata gagaatttaa acaagcagat 240

ggaagacgga 250

<210> 19

<211> 50

<212> DNA

<213> Artificial sequences

<400> 19

gaaattaata cgactcacta tagggagcta tagcaggttt tatagaggga 50

<210> 20

<211> 18

<212> DNA

<213> Artificial sequences

<400> 20

aagcagatgg aagacgga 18

<210> 21

<211> 85

<212> DNA

<213> Artificial sequences

<400> 21

gtatgtggga gacggtcggg tccagatatt cgtatctgtc gagtagagtg tgggctccca 60

catactttgt ctgcagcgta tccac 85

Claims (10)

1. A reagent for detecting a nucleic acid, comprising: detecting the crRNA and the pair of probes of the target nucleic acid;

the probe pair comprises a promoter probe and a reporter probe;

the promoter probe comprises an upstream hybridization sequence and a T7 promoter sequence;

the reporter probe comprises a downstream hybridization sequence and a universal sequence;

the crRNA comprises an anchor sequence and a spacer sequence;

the upstream hybridizing sequence is complementary to a partial sequence of the target nucleic acid;

the downstream hybridizing sequence is complementary to the remaining sequence of the target nucleic acid;

the universal sequence is identical to the spacer sequence;

the anchor sequence specifically recognizes the Cas protein.

2. The reagent according to claim 1, characterized in that:

the number of bases of the upstream hybridization sequence is 18-24;

preferably, the upstream hybridizing sequence is complementary to the 5 'end of the target nucleic acid and the downstream hybridizing sequence is complementary to the 3' end of the target nucleic acid; or

The upstream hybridizing sequence is complementary to the 3 'end of the target nucleic acid and the downstream hybridizing sequence is complementary to the 5' end of the target nucleic acid;

preferably, the T7 promoter sequence is shown in the 25 th to 86 th positions of SEQ ID NO. 2.

3. The reagent according to claim 1 or 2, characterized in that:

when the type of the target nucleic acid is 2or more, the reagent contains: n sets of probe pairs for detecting target nucleic acids and crRNA, N being equal to the species of the target nucleic acids;

preferably, when the type of the target nucleic acid is 2or more, the universal sequences of the reporter probes of the probe pairs of different target nucleic acids are different;

preferably, when the type of the target nucleic acid is 2or more, the anchor sequences of crrnas of different target nucleic acids are different.

4. The reagent according to claim 3, characterized in that:

when the sequence of the target nucleic acid is shown as SEQ ID NO.1, the upstream hybridization sequence is shown as1 st to 24 th sites of SEQ ID NO. 2; the downstream hybridization sequence is shown as 31 th to 48 th sites of SEQ ID NO. 4;

preferably, when the sequence of the target nucleic acid is shown as SEQ ID NO.6, the upstream hybridization sequence is shown as1 st to 19 th positions of SEQ ID NO. 7; the downstream hybridization sequence is shown as 31 th to 50 th sites of SEQ ID NO. 8;

preferably, when the sequence of the target nucleic acid is shown as SEQ ID NO.10, the upstream hybridization sequence is shown as 1-20 th position of SEQ ID NO. 11; the downstream hybridization sequence is shown as 31 th to 50 th sites of SEQ ID NO. 13;

preferably, when the sequence of the target nucleic acid is shown as SEQ ID NO.1 or SEQ ID NO.6, the universal sequence is shown as1 st to 30 th sites of SEQ ID NO. 4;

preferably, when the sequence of the target nucleic acid is shown as SEQ ID NO.10, the universal sequence is shown as1 st to 30 th positions of SEQ ID NO. 13;

preferably, when the sequence of the target nucleic acid is shown as SEQ ID NO.1 or SEQ ID NO.6, the anchoring sequence is shown as1 st to 35 th sites of SEQ ID NO. 5;

preferably, when the sequence of the target nucleic acid is shown as SEQ ID NO.10, the anchoring sequence is shown as 31-66 of SEQ ID NO. 14;

preferably, the target nucleic acid comprises: when the sequence of the nucleic acid is shown as SEQ ID NO.6 and the sequence of the nucleic acid is shown as SEQ ID NO.10, the reagent comprises a probe pair and crRNA for detecting the target nucleic acid shown as SEQ ID NO.6, and a probe pair and crRNA for detecting the target nucleic acid shown as SEQ ID NO.10,

the probe pair of the target nucleic acid with the detection sequence shown in SEQ ID NO.6 comprises a promoter probe and a report probe of the target nucleic acid with the detection sequence shown in SEQ ID NO. 6;

the upstream hybridization sequence of the promoter probe of the target nucleic acid with the detection sequence shown as SEQ ID NO.6 is shown as1 st to 19 th sites of SEQ ID NO. 7; the downstream hybridization sequence of the report probe of the target nucleic acid with the detection sequence shown as SEQ ID NO.6 is shown as 31-50 th site of SEQ ID NO. 8; the universal sequence of the report probe of the target nucleic acid with the detection sequence shown as SEQ ID NO.6 is shown as1 st to 30 th sites of SEQ ID NO. 4;

the anchoring sequence of the crRNA of the target nucleic acid with the detection sequence shown as SEQ ID NO.6 is shown as1 st to 35 th sites of SEQ ID NO. 5;

the probe pair of the target nucleic acid with the detection sequence shown as SEQ ID NO.10 comprises a promoter probe and a report probe of the target nucleic acid with the detection sequence shown as SEQ ID NO. 10;

the upstream hybridization sequence of the promoter probe of the target nucleic acid with the detection sequence shown as SEQ ID NO.10 is shown as1 st to 20 th sites of SEQ ID NO. 11; the downstream hybridization sequence of the report probe of the target nucleic acid with the detection sequence shown as SEQ ID NO.10 is shown as 31-50 th site of SEQ ID NO. 13; the universal sequence of the report probe of the target nucleic acid with the detection sequence shown as SEQ ID NO.10 is shown as1 st to 30 th sites of SEQ ID NO. 13;

the anchor sequence of the crRNA of the target nucleic acid with the detection sequence shown as SEQ ID NO.10 is shown as 31-66 of SEQ ID NO. 14.

5. Use of the reagent according to any one of claims 1 to 4 for the preparation of a product for detecting nucleic acids.

6. A kit, characterized in that: comprising the agent according to any one of claims 1 to 4.

7. The kit of claim 6, wherein: the kit further comprises a signaling reporter probe and a Cas protein of claims 1-4.

8. The kit of claim 7, wherein:

the signal report probe comprises a nucleic acid sequence, wherein the 5 'end of the nucleic acid sequence is marked with a fluorescent report group, and the 3' end of the nucleic acid sequence is marked with a quenching group;

preferably, the fluorescent reporter group comprises any one of FAM, HEX, ROX, CY5, VIC, JOE, TAMRA;

preferably, the quencher group comprises at least one of BHQ1, BHQ 2;

preferably, the nucleic acid sequence of the signal reporting probe is AAAAA or UUUUUUU;

preferably, when the species of the target nucleic acid is 2or more, the Cas protein comprises N Cas proteins, N Cas proteins are different from each other, and N is equal to the species of the target nucleic acid;

preferably, the Cas protein comprises at least one of LwaCas13a, PsmCas13b, CcaCas13 b;

preferably, when the type of the target nucleic acid is greater than or equal to 2, the signal reporting probe comprises N signal reporting probes, the sequences of the N signal reporting probes and the fluorescent reporting group are different from each other, and N is equal to the type of the target nucleic acid;

preferably, when the sequence of the target nucleic acid is shown as SEQ ID No.1 or SEQ ID No.6, the Cas protein is LwaCas13 a;

preferably, when the sequence of the target nucleic acid is shown as SEQ ID NO.1 or SEQ ID NO.6, the signal reporting probe is FAM-5 'UUUUUU-3' BHQ 1;

preferably, when the sequence of the target nucleic acid is shown as SEQ ID No.10, the Cas protein is PsmCas13 b;

preferably, when the sequence of the target nucleic acid is shown as SEQ ID NO.10, the signal reporting probe is HEX-5 'AAAAA-3' BHQ 1;

preferably, the target nucleic acid comprises: when the sequence is shown as SEQ ID NO.6 and the sequence is shown as SEQ ID NO.10, the Cas protein comprises LwaCas13a and PsmCas13 b;

preferably, the target nucleic acid comprises: when the sequence is the nucleic acid shown as SEQ ID NO.6 and the sequence is the nucleic acid shown as SEQ ID NO.10, the signal report probe comprises FAM-5 'UUUUUU-3' BHQ1 and HEX-5 'AAAAA-3' BHQ 1;

preferably, the kit further comprises SenSR buffer, ET-SSB, T7RNA polymerase, RNase inhibitor, NTPs and SplintR ligase.

9. Use of a reagent according to any one of claims 1 to 4 and/or a kit according to any one of claims 6 to 8 in the detection of nucleic acids for non-diagnostic purposes.

10. A method of detecting a nucleic acid for non-diagnostic purposes comprising the step of using a reagent according to any one of claims 1 to 4 and/or a kit according to any one of claims 6 to 8;

preferably, the nucleic acid detection method comprises the steps of:

mixing the promoter probe, the reporter probe and the SENSR buffer in the kit according to claim 8 to obtain a hybridization solution;

mixing water with NTPs, SENSSR buffer, ET-SSB, T7RNA polymerase, RNase inhibitor, SplintR ligase, Cas protein, signal reporter probe and CrRNA in the kit according to claim 8 to obtain a detection solution;

mixing the nucleic acid to be detected with the hybridization solution, and incubating to obtain a mixed solution;

and mixing the mixed solution with a detection solution, and carrying out reaction and detection.