CN111560482B - Detection method based on CRISPR/Cas and nucleic acid test paper and human papilloma virus detection kit - Google Patents

Detection method based on CRISPR/Cas and nucleic acid test paper and human papilloma virus detection kit Download PDF

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CN111560482B
CN111560482B CN202010530653.6A CN202010530653A CN111560482B CN 111560482 B CN111560482 B CN 111560482B CN 202010530653 A CN202010530653 A CN 202010530653A CN 111560482 B CN111560482 B CN 111560482B
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汤光辉
龚姣姣
薛良
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Decipher Bioscience Shenzhen Co ltd
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Abstract

The invention relates to the technical field of gene detection, in particular to a CRISPR/Cas (CRISPR/Cas)/nucleic acid test paper-based nucleic acid detection method and a human papilloma virus rapid detection kit. The human papillomavirus detection kit comprises crRNA, cas12, an RPA upstream primer, an RPA downstream primer, a buffer system and a single-stranded DNA reporter molecule; the reporter molecule has a sequence of 5'-NNN … -3' and a length of 3-30 base, FAM, DIG and Biotin are marked, and the nucleic acid molecule is modified to prevent degradation of DNase or cas12 after activation. The CRISPR/Cas and nucleic acid test paper-based nucleic acid detection method and the human papilloma virus rapid detection kit have the following advantages: the novel report molecule design and modification are adopted, and the corresponding test strips are combined, so that the nonspecific color development is completely stopped, and the method has remarkable significance for clinical practical application.

Description

Detection method based on CRISPR/Cas and nucleic acid test paper and human papilloma virus detection kit
Technical Field
The invention relates to the technical field of gene detection, in particular to a CRISPR/Cas (CRISPR/Cas)/nucleic acid test paper-based nucleic acid detection method and a human papilloma virus rapid detection kit.
Background
The diagnosis industry is an important component of the biopharmaceutical industry, and the Chinese diagnosis industry is currently in the initial stage of product research and development and production, and the growth period is not yet reached. The current diagnostic market in China is less than 1/10 of the global market. In general, the technical level of the main manufacturers in China basically reaches the international advanced level on the projects (such as hepatitis, venereal disease and pregnancy test series in immune reagents, enzyme series, lipid series, liver function series, blood sugar series, urine test series and the like in clinical biochemistry) with wider clinical application and wide market prospect at present; the PCR technology series in gene detection has reached the international advanced level, and the gene chip and cancer series are rapidly pursuing the international level. However, due to the limitation of market factors and technical factors, the current domestic molecular rapid detection level, namely POCT products and the international advanced level, still have a great gap.
Global POCT market size in 2018 is about $280 billion, and 2021 is expected to exceed $350 billion. Wherein in the IVD subdivision field, the POCT market size is about 28%, and the top is the top. In recent years, the POCT market in China develops very rapidly, and the industry speed-up is kept at 10% -20% which is far higher than the speed-up of 6% -7% worldwide. With the aging aggravation and the high incidence of chronic diseases in China and the gradual landing of the grading diagnosis policy, the POCT market in the future still keeps developing at a high speed. The POCT market scale in 2018 is over 70 hundred million yuan, which accounts for about 10.9% of the IVD industry, and the 2022 industry scale is expected to reach 200 hundred million yuan. Therefore, the POCT in China has huge market space and has not been developed yet.
Molecular diagnostic reagent products still dominate the diagnostic field because of their higher specificity and sensitivity. At present, most laboratories adopt a PCR method to detect pathogens, and the sensitivity and specificity are good, but the time is long, the instrument and the equipment are expensive, and the method is difficult to popularize in basic-level inspection institutions. Compared with the temperature-changing nucleic acid detection technologies such as PCR and the like, the isothermal amplification technology does not need temperature-rising circulation, so that the requirement on amplification equipment is low, and the nucleic acid POCT detection is facilitated.
The emerging recombinase-dependent polymerase amplification technique (Recombinase polymerase amplification, RPA) is capable of shortening the detection time to 15min-30min, and is called a nucleic acid detection technique that can replace PCR. However, this technique does not substantially improve the detection sensitivity compared to the real-time quantitative PCR (Q-PCR) method currently on the market. There is therefore a need to establish a detection technique that is applicable on-site, simple, fast, and highly sensitive, and a CRISPR/Cas detection technique developed in recent years is an ideal candidate.
CRISPR (Clustered regularly interspaced short palindromic repeats) is an adaptive immune modality against viral invasion in most bacteria and archaea. Upon viral invasion, the bacteria produce a corresponding crRNA that recognizes the viral genome, which directs the recognition and cleavage of the viral target sequence by Cas protein with endonuclease activity (CRISPR-associated proteins). CRISPR systems are sequence-paired from their own RNA and target DNA, and direct cas proteins to specifically cleave targets. For double stranded DNA, the CRISPR system needs to recognize one Protospacer Adjacent Motif (PAM) sequence on the target DNA. This feature allows us to conveniently use the CRISPR system to identify different nucleic acid sequences. For example, using guide RNA (referred to as sgRNA or crRNA) of different sequences, double-stranded DNA with PAM sequence that is specifically recognized and matched to the guide RNA sequence. S. pyogenes Cas9 (SpCas 9, abbreviated as Cas 9) is most widely used because of its simple PAM sequence and high intracellular gene editing efficiency.
The group Zhang Feng subject found a CRISPR effector protein Cas13a, 6 months 2016. The protein is an endonuclease that binds to and degrades the target RNA under the direction of crRNA (Makarova et al 2011). An endonuclease called Leptotrichia buccalis Cas a (LbuCas 13 a) was found by Doudna et al (East-Seletsky et al 2016) at 10 months of the year to have not only cleavage activity for target RNAs but also activity for cleavage of non-target RNAs, which is called accessory cleavage. In 2017, zhang Feng, in cooperation with Collins, combined Cas13a with a recombinase polymerase isothermal amplification technique (Recombinase polymerase amplification, RPA), developed a highly Specific and sensitive nucleic acid detection system (specificity High-Sensitivity Enzymatic Reporter UnLOCKing, SHERLOCK) (Gootenberg et al, 2017). The introduction of RPA obviously improves the sensitivity of the system, and the detection limit of SHERLOCK on the target is as low as 2X10 3 The detection of single molecules was achieved by copies/mL (3.2 amol/L). This breakthrough research effort again demonstrates the value of CRISPR/Cas systems in the field of nucleic acid diagnostics, with the hope of having a significant impact in the public health field.
In 2015, zhang Feng et al found a class ii v CRISPR effector protein Cas12 (Zetsche et al, 2015). Cas12 can bind to target double-stranded DNA and cleave genomic DNA under the guidance of crRNA. In 2018, doudna et al found that Cas12 has activity of non-specifically cleaving single-stranded DNA (ssDNA) after specifically binding and cleaving target dsDNA, cas12 cleaves single-stranded DNA extremely rapidly, reaching more than 1000 molecules per second, and can last for more than several hours. Researchers have exploited this activity of Cas12 to develop a nucleic acid detection system designated DETECTR (DNA Endonuclease Targeted CRISPR Trans Reporter) (Chen et al, 2018). The system introduces a single-stranded reporter DNA molecule marked by a fluorescent group and a quenching group into the system, and when activated cas12 cuts single-stranded DNA, the quenching group and the fluorescent group are separated, so that the fluorescent light molecule emits light, and a light signal is detected. The detection combines the RPA isothermal amplification technique with Cas12, the amplification product activates the accessory cleavage activity of Cas12, the cleavage substrate generates a fluorescent signal, and single molecule level sensitivity is achieved. The system has wide application potential in the aspects of single nucleotide polymorphism analysis, cancer screening, bacterial and virus infection detection, drug resistance screening and the like.
Although these CRISPR/Cas-based techniques can achieve rapid and sensitive detection of nucleic acid molecules, there is still a need for the aid of a fluorescence detector and the result cannot be interpreted visually, which makes the technique difficult to popularize in a basic-level inspection mechanism.
Currently, there are embodiments of interpreting results using nucleic acid strips, but the use of strips that produce severe non-specific color development, which makes interpretation of results extremely difficult.
Disclosure of Invention
The invention aims at: provides a nucleic acid detection method and a corresponding rapid detection kit for human papilloma virus, which can be used for a CRISPR/Cas system and can completely eradicate a non-specific chromogenic nucleic acid test strip and a reporter molecule construction mode.
The technical scheme provided by the invention is as follows: the detection method based on CRISPR/Cas12 and nucleic acid test paper comprises the following steps:
step 1) designing and synthesizing a single-stranded DNA reporter molecule; the single-stranded DNA reporter molecule has a sequence of 5'-NNN … -3' and a length of 3-30 base, is marked by FAM, DIG and Biotin, and is modified to prevent degradation of DNase or cas12 enzyme after activation;
step 2) designing and synthesizing RPA upstream and downstream primers for amplifying target genes;
step 3) designing specific crRNA (complementary ribonucleic acid) aiming at target genes, wherein the length of the crRNA is 17-25 nucleotides, and a PAM sequence exists at the near 5' end of the crRNA: TTTN, the PAM sequence is used for complex recognition consisting of crRNA and cas 12;
step 4) mixing freeze-dried RPA reaction particles, RPA hydration buffer, RPA upstream and downstream primers, target nucleic acid to be detected and magnesium acetate, and reacting the obtained mixture at a constant temperature of 37-42 ℃ for 10-30min to obtain RPA reaction liquid;
step 5) mixing components comprising RPA reaction solution, crRNA molecules, cas12, single-stranded DNA reporter molecules, RNase inhibitors, magnesium ions and buffer solution to obtain a reaction mixture, and incubating the reaction mixture at 37 ℃ for 5-30min;
and 6) adding the reaction mixture obtained in the step 5) into a reaction diluent for dilution, inserting a test strip into the diluted liquid, and carrying out chromatography for 2-5min, so as to judge a detection result by naked eyes.
Preferably, in the above detection method based on CRISPR/Cas12 and nucleic acid test paper, the modification is: phosphothioate, 2'-O-methyl or 2' -O-Methoxyethyl (MOE) nucleotides.
Preferably, in the above detection method based on CRISPR/Cas12 and nucleic acid test paper, the Cas12 is one of FnCas12a, asCas12a, lbCas12a, lb5Cas12a, hkCas12a, osCas12a, tsCas12a, bbCas12a, boCas12a and Lb4Cas12 a.
The other technical scheme provided by the invention is as follows: the detection method based on CRISPR/Cas13 and nucleic acid test paper comprises the following steps:
step 1) designing and synthesizing a single-stranded RNA reporter molecule; the sequence of the single-stranded RNA reporter molecule is 5'-rNrNrN … -3', the length is 3-30 base, the single-stranded RNA reporter molecule is marked by FAM, DIG and Biotin, and the single-stranded RNA reporter molecule is modified to prevent degradation of RNase or cas13 enzyme after activation;
step 2) designing and synthesizing RPA upstream and downstream primers which are used for amplifying target genes and provided with T7 transcription promoter sequences;
step 3) designing specific crrnas for the target gene;
step 4) mixing freeze-dried RPA reaction particles, RPA hydration buffer, RPA upstream and downstream primers, target nucleic acid to be detected and magnesium acetate, and reacting the obtained mixture at a constant temperature of 37-42 ℃ for 10-30min to obtain RPA reaction liquid;
step 5) mixing the components comprising RPA reaction solution, crRNA molecules, cas13, single-stranded RNA reporter RNA rnase inhibitor, T7 transcriptase, magnesium ions and buffer solution to obtain a reaction mixture, and incubating the reaction mixture at 37 ℃ for 5-30min;
and 6) adding the reaction mixture obtained in the step 5) into a reaction diluent for dilution, inserting a test strip into the diluted liquid, and carrying out chromatography for 2-5min, so as to judge a detection result by naked eyes.
Preferably, in the above detection method based on CRISPR/Cas13 and nucleic acid test paper, the modification is: phosphothioate, 2'-O-methyl or 2' -O-Methoxyethyl (MOE) nucleotides.
Preferably, in the above detection method based on CRISPR/Cas13 and nucleic acid test paper, the Cas13 is one of psucas 13b, lwaCas13a and Cca13b
The invention also provides a human papillomavirus detection kit, which comprises crRNA, cas12, RPA upstream and downstream primers, a buffer system and a single-stranded DNA reporter molecule;
the sequence of the reporter molecule is 5'-NNN … -3', the length is 3-30 base, the reporter molecule is marked by FAM, DIG and Biotin, and the nucleic acid molecule is modified to prevent degradation of DNase or cas12 after activation;
the crRNA is specifically designed aiming at target genes, the length of the crRNA is 17-25 nucleotides, and a sequence is present near the 5' end of the crRNA: TTTN, the PAM sequence is used for complex recognition consisting of crRNA and cas 12.
Preferably, in the above novel coronavirus detection kit, the modification is: phosphothioate, 2'-O-methyl or 2' -O-Methoxyethyl (MOE) nucleotides.
Preferably, in the above novel coronavirus detection kit, the sequence of the primer upstream and downstream of RPA is:
HPV16-F：SEQ ID NO：4
HPV16-R：SEQ ID NO：5
the crRNA sequence is as follows:
crRNA-HPV16：SEQ ID NO：6。
preferably, in the above novel coronavirus detection kit, the Cas12 is one of FnCas12a, asCas12a, lbCas12a, lb5Cas12a, hkCas12a, osCas12a, tsCas12a, bbCas12a, boCas12a and Lb4Cas12 a.
The invention has the beneficial effects that: the CRISPR/Cas12 and nucleic acid test paper-based nucleic acid detection method and the human papilloma virus rapid detection kit have the following advantages:
1. compared with the existing PCR-based nucleic acid detection technology on the market, the method has the following advantages:
1) And (3) quick: the amplification efficiency and crRNA cleavage efficiency only need 15-40min from sample to detection result according to the template to be detected, wherein the sample is processed for 5-10min, and the amplification detection reaction is carried out for 10-30min.
2) The accuracy is that: the invention has extremely high sensitivity and can detect single nucleic acid molecules; meanwhile, since crRNA is sensitive to single base mismatch, the invention has excellent specificity and can detect mutation of single nucleotide in a sample.
3) Convenient: the invention is carried out at the constant temperature of 37 ℃, so the requirement on equipment is extremely low, and the reaction can be carried out by simple constant temperature equipment even by using the body temperature, thereby being beneficial to popularization in basic-level inspection institutions.
2. Compared with the existing CRISPR/Cas 12-based nucleic acid detection technology, the method has the following advantages:
1) The operation is simpler and more convenient: because the prior art mostly adopts fluorescence detection, the fluorescence detection equipment is needed, and the nucleic acid test strip is adopted for detection, so that the operation is more convenient, and the method has great significance in areas lacking instruments and equipment.
2) The specificity is good: the detection method of the nucleic acid test strip in the prior art can generate serious nonspecific color development, and brings difficulty to interpretation of results.
Drawings
FIG. 1 is a schematic diagram of a combination of novel reporter and test strip structures of example 1 in accordance with embodiments of the present invention;
FIG. 2 is a schematic diagram showing a combination of a conventional reporter and a test strip according to example 1 of the present invention;
FIG. 3 is a graph showing a control of the detection of positive and negative samples using conventional test strips and reporter molecules according to example 1 of the present invention;
FIG. 4 is a graph showing the comparison of positive and negative samples detected using the test strips and reporter molecules of example 1 in accordance with an embodiment of the present invention;
FIG. 5 is a schematic diagram showing a combination of novel reporter and dipstick structures of example 2 in accordance with embodiments of the invention;
FIG. 6 is a schematic diagram showing a combination of a conventional reporter and a test strip according to example 2 of the present invention;
FIG. 7 is a control chart of example 2 of the present invention for detecting positive and negative samples using a conventional test strip and a single-stranded DNA reporter;
FIG. 8 is a control chart of example 2 of the present invention for detecting positive and negative samples using the test strip and single-stranded DNA reporter of the present invention.
Detailed Description
In order to describe the technical content, the constructional features, the achieved objects and effects of the present invention in detail, the following description is made in connection with the embodiments and the accompanying drawings.
The CRISPR/Cas12 system has the characteristics of accuracy, rapidness and convenience, and the nucleic acid diagnosis technology developed by combining the CRISPR/Cas12 system with the RPA gets rid of the dependence on a precise PCR instrument, so the CRISPR/Cas12 system is an important direction of the development of nucleic acid diagnosis. The current mainstream method is to use fluorescent signals to interpret the results of the system. Some researchers use test strips to interpret results, but the test strips adopted at present all generate serious nonspecific strips, and the invention aims to provide a probe and test strip combination mode and a construction method which can be applied to CRISPR/Ca12 and CRISPR/Cas13 systems and can completely stop nonspecific color development.
The invention is realized by the following technical routes:
1. CRISPR/Cas12 systems technology route:
1) The reporter molecule with the designed sequence of 5'-NNN … -3' can be 3-30 base in length, FAM, DIG and Biotin are labeled on the reporter molecule, and modification (including but not limited to phosphothioate, 2'-O-methyl or 2' -O-Methoxyethyl (MOE) nucleotides) is carried out on the nucleic acid molecule to prevent degradation of the DNase or cas12 after activation.
2) The RPA upstream and downstream primers for amplifying the target gene are designed and synthesized, and the length of the primers is 30-35 nucleotides.
3) Specific crrnas were designed for target genes, 17-25 nucleotides in length, and TTTN sequences (PAM sequences for recognition of complexes consisting of crrnas and cas 12) should be present near the 5' end of the crrnas. Directly chemically synthesizing or constructing a crRNA in-vitro transcription vector according to the designed crRNA sequence, and carrying out in-vitro transcription and purification by using a T7 transcription kit;
4) And mixing the freeze-dried RPA reaction particles, RPA hydration buffer, RPA upstream and downstream primers, target nucleic acid to be detected and magnesium acetate according to a proper proportion. The reaction mixture was allowed to react at a constant temperature of 37-42℃for 10-30min (depending on the amount of template to be detected, amplification efficiency).
5) Mixing the RPA reaction solution with crRNA molecule, cas12, single-stranded DNA reporter molecule, RNase inhibitor, magnesium ion and buffer solution according to proper proportion, and incubating the reaction mixture at 37 ℃ for 5-30min.
6) Adding a proper amount of reaction diluent (Tris-HCl, PH=7-8) into the reaction mixture, inserting a test strip into the diluted liquid, performing chromatography for 2-5min at room temperature, judging the result by naked eyes and photographing.
2. CRISPR/Cas13 systems technology route:
1) The reporter molecule with the designed sequence of 5'-rUrU … -3' can be 3-30 bases in length, FAM, DIG and Biotin are labeled on the reporter molecule, and modification (including but not limited to phosphothioate, 2'-O-methyl or 2' -O-Methoxyethyl (MOE) nucleotides) is performed on the nucleic acid molecule to prevent degradation of RNase or cas13 after activation.
2) The RPA upstream and downstream primers were designed to synthesize the sequence for amplification of the target gene and with the T7 transcriptional promoter.
3) Specific crRNA is designed aiming at target genes, crRNA in vitro transcription vectors are directly synthesized chemically or constructed according to the designed crRNA sequence, and in vitro transcription and purification are carried out by using a T7 transcription kit;
4) And mixing the freeze-dried RPA reaction particles, RPA hydration buffer, RPA upstream and downstream primers, target nucleic acid to be detected and magnesium acetate according to a proper proportion. The reaction mixture was allowed to react at a constant temperature of 37-42℃for 10-30min (depending on the amount of template to be detected, amplification efficiency).
5) Mixing the RPA reaction solution with crRNA molecule, cas13, single-stranded RNA reporter molecule, RNase inhibitor, T7 transcriptase, magnesium ion and buffer solution according to proper proportion, and incubating the reaction mixture at 37 ℃ for 5-30min.
6) And adding a proper amount of reaction diluent (Tris-HCl, pH=7-8) into the reaction mixture, inserting a test strip into the diluted liquid, performing chromatography for 2-5min at room temperature, and judging and photographing the result by naked eyes.
Example 1
A detection method based on CRISPR/Cas12 and nucleic acid test paper, the following operation steps of the method are described by taking detection of human beta Actin (ACTB) as an example:
1) Searching a 5'-TTTN-3' sequence in a human beta actin region to design and synthesize crRNA; primer sequences were designed upstream and downstream of the crRNA, and both the crRNA and the primer used in the present invention were synthesized by Shanghai. The structure and the combination mode of the reporter molecule and the test strip adopted in the invention are shown in figure 1 and figure 2.
The primers, crrnas, and reporter sequences selected in this example are shown in table 1 below:
TABLE 1
2) Healthy volunteer samples were taken with throat swabs and added to quick lysates formulated as follows in Table 2, and lysed at 80℃for 5 minutes.
TABLE 2
Reagent(s) Concentration of
Guanidine hydrochloride 800mM
Tween 20 0.5％
Polyethylene glycol octyl phenyl ether 1％
DEPC water -
3) 2 mu L of the lysate in the step 1) is added into RPA reaction particles, and an appropriate proportion of RPA hydration buffer solution, RPA upstream and downstream primers, pure water and magnesium acetate are added. The reaction mixture was allowed to react at 42℃for 30min. The invention adopts an Anpu future isothermal amplification kit (DNA basic type).
The RPA reaction mixture ratio in the present invention is shown in Table 3 below.
TABLE 3 Table 3
4) 10. Mu.L of RPA was added to 40. Mu.L of crRNA, cas12a and single-stranded DNA reporter reaction assay, and reacted at 37℃for 20 minutes.
The ratios of cas12a detection solutions in the present invention are shown in Table 4 below.
TABLE 4 Table 4
5) 10. Mu.L of the cleavage product was diluted to 100. Mu.L with a diluent, and the dilution was added dropwise to a test strip, and the result was visually observed and photographed. A positive sample using a conventional test strip and reporter combination showed a distinct band at the detection line (T), while a negative sample also showed a weak signal at the detection line (fig. 3); positive samples tested with the reporter and test strip combinations of the invention showed a distinct band at the test line, negative samples were not band at the test line, showing good specificity (fig. 4).
Example 2
A human papillomavirus detection kit, which comprises crRNA, cas12, RPA upstream and downstream primers, a buffer system and a single-stranded DNA reporter molecule; the specific design is as follows:
1) Searching a 5'-TTTN-3' sequence in a conserved L1 gene region of the human papillomavirus, and designing and synthesizing crRNA; primer sequences were designed upstream and downstream of the crRNA, and both the crRNA and the primer used in the present invention were synthesized by Shanghai. The reporter molecule is synthesized and modified, and a nucleic acid test strip is prepared, and the structure and the combination mode of the reporter molecule and the test strip adopted in the invention are shown in fig. 5 and 6.
In this example, primers, crrnas, and reporter sequences are shown in table 5 below.
TABLE 5
2) And (3) taking RPA reaction particles, and adding an RPA hydration buffer solution, an RPA upstream primer, an RPA downstream primer, nucleic acid to be detected, pure water and magnesium acetate in a proper proportion. The reaction mixture was allowed to react at a constant temperature of 37℃for 30min. The invention adopts an Anpu future isothermal amplification kit (DNA basic type). The RPA reaction solution in the invention is prepared as shown in Table 3.
3) 10. Mu.L of RPA was added to 40. Mu.L of crRNA, cas12a and the reporter reaction test solution, and reacted at 37℃for 20 minutes. The ratios of cas12a detection solutions in the present invention are shown in Table 4.
5) 10. Mu.L of the cleavage product was diluted to 100. Mu.L with a diluent, and the dilution was added dropwise to a test strip, and the result was visually observed and photographed. Positive samples using a conventional test strip and reporter combination showed a distinct band at the test line (T) while negative samples also showed a weak signal at the test line (fig. 7); positive samples tested with the reporter and test strip combinations of the invention showed a distinct band at the test line, negative samples were not band at the test line, showing good specificity (fig. 8).
The foregoing description is only illustrative of the present invention and is not intended to limit the scope of the invention, and all equivalent structures or equivalent processes or direct or indirect application in other related technical fields are included in the scope of the present invention.
SEQUENCE LISTING
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Claims (4)

1. The detection method based on CRISPR/Cas and nucleic acid test paper is characterized by comprising the following steps:
step 1) designing and synthesizing a single-stranded DNA reporter molecule; the sequence of the single-stranded DNA reporter molecule is 5'-T TTATT-3', the single-stranded DNA reporter molecule is marked by FAM, DIG and Biotin, and the single-stranded DNA reporter molecule is modified to prevent degradation of DNase or cas12 enzyme after activation; carrying out Biotin and DIG labeling on the 5 'end of the single-stranded DNA reporter molecule, and carrying out FAM labeling on the 3' end; modification to prevent degradation of dnase or cas12 enzyme after activation is performed between Biotin and DIG of single-stranded DNA reporter; the modification is thio modification of a phosphodiester bond between two T's at positions T' TTATT in the single-stranded DNA reporter;
step 2) designing and synthesizing RPA upstream and downstream primers for amplifying target genes; the target gene is a human actin gene sequence;
step 3) designing specific crrnas for target genes, wherein PAM sequences are present near the 5' end of the crrnas: TTTN, the PAM sequence is used for complex recognition consisting of crRNA and cas 12;
the sequence of the RPA upstream and downstream primer is as follows:
ACTB-F1：SEQ ID NO：1；
ACTB-R1：SEQ ID NO：2；
the crRNA sequence is as follows:
crRNA-ACTB：SEQ ID NO：3；
step 4) mixing freeze-dried RPA reaction particles, RPA hydration buffer, RPA upstream and downstream primers, target nucleic acid to be detected and magnesium acetate, and reacting the obtained mixture at a constant temperature of 37-42 ℃ for 10-30min to obtain RPA reaction liquid;
step 5) mixing components comprising RPA reaction solution, crRNA molecules, cas12, single-stranded DNA reporter molecules, RNase inhibitors, magnesium ions and buffer solution to obtain a reaction mixture, and incubating the reaction mixture at 37 ℃ for 5-30min;
and 6) adding the reaction mixture obtained in the step 5) into a reaction diluent for dilution, inserting a test strip into the diluted liquid, and carrying out chromatography for 2-5min, so as to judge a detection result by naked eyes.
2. The CRISPR/Cas and nucleic acid test paper based detection method according to claim 1, wherein the Cas12 is one of FnCas12a, asCas12a, lbCas12a, lb5Cas12a, hkCas12a, osCas12a, tsCas12a, bbCas12a, boCas12a and Lb4Cas12 a.
3. The human papilloma virus detection kit is characterized by comprising crRNA, cas12, RPA upstream and downstream primers, a buffer system and a single-stranded DNA reporter molecule;
the single-stranded DNA reporter molecule is 5'-T TTATT-3', and is marked by FAM, DIG and Biotin, and the nucleic acid molecule is modified to prevent degradation of DNase or cas12 after activation; carrying out Biotin and DIG labeling on the 5 'end of the single-stranded DNA reporter molecule, and carrying out FAM labeling on the 3' end; modification to prevent degradation of dnase or cas12 enzyme after activation is performed between Biotin and DIG of single-stranded DNA reporter; the modification is thio modification of a phosphodiester bond between two T's at positions T' TTATT in the single-stranded DNA reporter;
the crRNA is a crRNA with specificity designed for target genes, and a PAM sequence exists at the near 5' end of the crRNA: TTTN, the PAM sequence is used for complex recognition consisting of crRNA and cas 12;
the sequence of the RPA upstream and downstream primer is as follows:
HPV16-F：SEQ ID NO：4
HPV16-R：SEQ ID NO：5
the crRNA sequence is as follows:
crRNA-HPV16 ：SEQ ID NO：6。
4. the human papillomavirus detection kit of claim 3, wherein the Cas12 is one of FnCas12a, asCas12a, lbCas12a, lb5Cas12a, hkCas12a, osCas12a, tsCas12a, bbCas12a, boCas12a, and Lb4Cas12 a.
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