CN117844984A - LAMP-CRISPR kit for detecting HPV16 and application thereof - Google Patents
LAMP-CRISPR kit for detecting HPV16 and application thereof Download PDFInfo
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Abstract
The invention discloses an LAMP-CRISPR kit for detecting HPV16 and application thereof, which is used for detecting high-risk human papilloma virus HPV16 and comprises the following steps: step one, performing loop-mediated isothermal amplification (LAMP) by taking DNA of a sample to be detected as a template, and simultaneously performing a CRISPR system reaction, wherein the CRISPR-LAMP reaction system comprises a specific primer pair containing amplified HPV16 components, buffer, cas12b protein, sgRNA, a probe, an additive and the like; and step two, detecting CRISPR reaction fluorescence intensity change, and judging whether HPV16 exists in the sample to be detected according to the Cas curve. The CRISPR detection method integrates the nucleic acid amplification and target detection steps into a system, simplifies the operation procedure, avoids cross contamination and improves the HPV16 detection specificity and sensitivity.
Description
Technical Field
The invention belongs to the technical field of molecular biology technology CRISPR and nucleic acid detection, and relates to a detection technology based on LAMP combined with CRISPR/Cas 12. In particular to a primer group and a kit for LAMP-CRISPR of high-risk human papilloma virus HPV16.
Background
Human Papillomavirus (HPV) nucleic acid detection is the preferred method recommended by WHO for cervical cancer primary screening, but there is no guidance document in our country for cervical cancer screening practice. Therefore, multidisciplinary specialists such as public health, gynaecology and obstetrics, inspection medicine, pathology and the like form specialist consensus by combining domestic and foreign guidelines, medical evidence-based evidence and actual conditions, HPV nucleic acid detection is recommended to be used as a main method for screening cervical cancer, scientific suggestions are provided for the aspects of evaluation and preparation before cervical cancer screening practice, specific application and applicable crowd, laboratory operation flow, monitoring and evaluation after implementation and the like, so that comprehensive, deep, standard and orderly high-quality development of HPV nucleic acid detection is promoted, and powerful guarantee is provided for achieving a cervical cancer elimination target. Among them, HPV16 is the most common (59.5%) as high-risk human papillomavirus, and E6 and E7 genes of the virus may integrate with host DNA during the continuous infection of HPV, resulting in Cervical Intraepithelial Neoplasia (CIN) and even cervical cancer, so that it has important clinical significance to perform typing detection on HPV and to determine whether there is continuous infection.
The isothermal amplification technology has good application prospect in clinic and rapid diagnosis due to the characteristics of rapidness, accuracy, specificity and independence of precise instruments, and common techniques include Recombinase Polymerase Amplification (RPA), loop-mediated isothermal amplification (LAMP), chained amplification (SDA), rolling Circle Amplification (RCA) and the like. Loop-mediated isothermal amplification (LAMP) is an isothermal nucleic acid amplification reported by Notomi et al in 1998. The method relies on 4 primers (2 outer and 2 inner primers) that recognize 6 specific fragments of conserved sequence DNA and a strand displacement DNA polymerase (Bst DNA polymerase). The amplification of genes and the detection of products in the LAMP detection system can be completed in one step, the operation is simple, the amplification efficiency is high, and the amplification can be carried out for 10min to 60min 9 ～10 10 The specificity is higher. However, LAMP is sensitive to presence detection relative to the mature qPCR techniqueThe primer design is difficult, the false positive is high, the pollution is easy, the multiple realization is difficult, and the like.
CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is an adaptive immune modality against viral invasion in most bacteria and archaea. The discovery of CRISPR/Cas systems dates back to 1987, nakata et al first found tandem spacer repeats in the E.coli genome. In 2002, jansen et al formally named this unique repeat spacer sequence as tandem spacer short palindromic repeat (CRISPR).
In recent years, CRISPR technology has shown great application value in the field of genome editing.
The detection technology of CRISPR which is known as the molecular detection technology of the next generation in 2017 can achieve the characteristics of rapidness, sensitivity, high specificity, simplicity and low price, and the technology can be widely applied to the fields of pathogen detection, cancer mutation detection, single nucleotide polymorphism detection and the like and has great application potential.
As a class II CRISPR/Cas system, such as Cas12a and Cas13a, the formed Cas/crRNA/target DNA ternary complex has trans-cleavage activity after cis-cleavage, and can non-specifically cleave nearby single-stranded DNA or RNA. The CRISPR/Cas12 action process mainly comprises two stages, namely, capturing exogenously introduced sequences, namely, sgRNA and Cas12 protein binding; in the second stage, the transcribed crRNA forms pre-crRNA, and Cas12 protein is guided to cut the targeting region which is the same as the sgRNA on the genome, so that the aim of gene editing is fulfilled. Cas12b is RNP complex capable of performing DNA cutting according to crRNA guidance, and under the guidance of target DNA, the nuclease domain of the complex is activated and performs DNA inscription, so that efficient recognition and amplification of the target DNA are realized.
By utilizing this characteristic in combination with FRET (fluorescence energy resonance transfer) technology, nucleic acid detection can be performed. The specific principle is that an oligonucleotide sequence with a fluorescent group and a fluorescence quenching group added at the tail end is designed, the oligonucleotide sequence is called a report probe (reporter), after the CRISPR/Cas system carries out cis-cleavage, a fluorescent signal is generated by the trans-cleavage reporter, and the content of a target nucleic acid sequence is deduced by detecting the intensity of the fluorescent signal, so that the effect of nucleic acid detection is achieved.
Combining the high amplification efficiency of isothermal amplification with the high specificity of CRISPR detection, researchers have developed a range of isothermal CRISPR amplification techniques. Because of the problems of template competition, system conflict, inconsistent reaction temperature and the like in the LAMP amplification reaction and the CRISPR detection reaction, more two-step detection which separates LAMP amplification from CRISPR detection is currently used. However, the two-step detection has the problems of cover opening pollution, complex operation and the like.
Therefore, subsequent researchers successively develop a one-pot reaction system such as an HOLMES v2 system and a STOPCOV id.v2 system, and the like, and the LAMP amplification and the CRISPR detection reaction are synchronously carried out in one system, so that the operation is simple, the pollution is avoided, and the detection time is shortened.
However, in the "one-pot" system, the Bst polymerase and Cas-sgRNA complex of the LAMP amplification reaction must bind competitively to the DNA template, and the cis-cleavage induced after the latter binds to the DNA template will cause LAMP amplification failure under the condition of extremely low concentration of template, thus resulting in negative results, which also means that the sensitivity of the "one-pot" detection system is lower.
To address this issue, researchers have proposed several optimization strategies including reducing Cas protein cleavage efficiency, modifying sgRNA delay triggering, optimizing the reaction system, etc. However, the design and detection of pre-specific primers and the like require specialized and skilled technicians, which further limit the wide spread application of the above-described methods.
Disclosure of Invention
The technical problems to be solved by the invention are as follows: LAMP has the advantages of insufficient detection sensitivity, high false positive, easy pollution and the like, and the amplification sensitivity and specificity of the reaction system can be obviously improved finally and nonspecific amplification is reduced by designing and screening specific primers, combining annular primers and optimizing PAM sites, reaction systems and reaction temperatures of Cas enzymes based on a CRISPR integrated detection system of LAMP.
The first object of the invention is to provide a primer group, sgRNA and a LAMP-CRISPR kit comprising the primer group and the sgRNA, which are used in an LAMP-CRISPR reaction system for detecting high-risk human papilloma virus HPV16.
The second purpose of the invention is to develop a nucleic acid analysis method which is convenient, efficient, low in cost, free from pollution and free from complex instruments and equipment, and establish an integrated system for detecting HPV16 by LAMP, CRISPR and Cas12b proteins, thereby improving the reaction sensitivity and specificity and reducing the risks of cross-contamination and non-specific amplification.
The third object of the invention is to provide an application of the LAMP-CRISPR kit in high-risk human papilloma virus HPV16 detection.
The technical scheme is as follows: the invention provides an LAMP primer group, in particular to a primer group used in an LAMP-CRISPR reaction system, which designs a primer group 1-9, and screens out the primer group to obtain the optimal primer group:
primer set 1: comprises F3-1, B3-1, FIP-1 and BIP-1, and the nucleotide sequences of the F3-1, the B3-1, the FIP-1 and the BIP-1 are shown as SEQ ID NO. 1-4 in sequence;
primer set 2: comprises F3-2, B3-2, FIP-2 and BIP-2, and the nucleotide sequences of the F3-2, the B3-2, the FIP-2 and the BIP-2 are shown as SEQ ID NO 7-10 in sequence;
primer set 3: comprises F3-3, B3-3, FIP-3 and BIP-3, and the nucleotide sequences of the F3-3, the B3-3, the FIP-3 and the BIP-3 are shown as SEQ ID NO. 13-16 in sequence;
primer set 4: comprises F3-4, B3-4, FIP-4 and BIP-4, and the nucleotide sequences of the F3-4, the B3-4, the FIP-4 and the BIP-4 are shown as SEQ ID NO. 19-22 in sequence;
primer set 5: comprises F3-5, B3-5, FIP-5 and BIP-5, and the nucleotide sequences of the F3-5, the B3-5, the FIP-5 and the BIP-5 are shown as SEQ ID NO. 25-28 in sequence;
primer set 6: comprises F3-6, B3-6, FIP-6 and BIP-6, and the nucleotide sequences of the F3-6, the B3-6, the FIP-6 and the BIP-6 are sequentially shown as SEQ ID NO. 31-34;
primer set 7: comprises F3-7, B3-7, FIP-7 and BIP-7, and the nucleotide sequences of the F3-7, the B3-7, the FIP-7 and the BIP-7 are shown as SEQ ID NO. 37-40 in sequence;
primer set 8: comprises F3-8, B3-8, FIP-8 and BIP-8, and the nucleotide sequences of the F3-8, B3-8, FIP-8 and BIP-8 are shown as SEQ ID NO. 43-46 in sequence;
primer set 9: comprises F3-9, B3-9, FIP-9 and BIP-9, and the nucleotide sequences of the F3-9, B3-9, FIP-9 and BIP-9 are shown as SEQ ID NO. 49-52 in sequence.
The primer nucleotide sequence in the primer group, such as the average GC content, is too low, so that the amplification efficiency of Bst enzyme is affected, and the primer is difficult to bind to a template due to the fact that the primer is easy to form a stable secondary structure by self-complementary pairing, and serious nonspecific amplification bands can occur.
Preferably, the LAMP primer group of the present invention is primer group 4.
Preferably, the average GC content of the primer nucleotide sequences in the primer set is 50% -65%, more preferably selected from 55% -60%; the primer nucleotide sequence in the primer set has a G.DELTA.less than or equal to-4.0 kcal/mol, more preferably has a G.DELTA.less than or equal to-3.0 kcal/mol.
As a further optimization of the present invention, amplification efficiency can be significantly improved by adding 2 loop primers.
Specifically, the primer set 1-9 further comprises the following loop primers:
the primer group 1 further comprises LF-1 and LR-1, and the nucleotide sequences of the primer group are respectively shown as SEQ ID NO. 5-6;
the primer group 2 further comprises LF-2 and LR-2, and the nucleotide sequences of the primer group are respectively shown as SEQ ID NO. 11-12;
the primer group 3 further comprises LF-3 and LR-3, and the nucleotide sequences of the primer group are respectively shown as SEQ ID NO. 17-18;
the primer group 4 further comprises LF-4 and LR-4, and the nucleotide sequences of the primer group are respectively shown as SEQ ID NO. 23-24;
the primer group 5 further comprises LF-5 and LR-5, and the nucleotide sequences of the primer group are shown as SEQ ID NO. 29-30 respectively;
the primer group 6 further comprises LF-6 and LR-6, and the nucleotide sequences of the primer group are shown as SEQ ID NO. 35-36 respectively;
the primer group 7 further comprises LF-7 and LR-7, and the nucleotide sequences of the primer group are respectively shown as SEQ ID NO. 41-42;
the primer group 8 further comprises LF-8 and LR-8, and the nucleotide sequences of the primer group are respectively shown as SEQ ID NO. 47-48;
the primer group 9 further comprises LF-9 and LR-9, and the nucleotide sequences of the primer group are respectively shown as SEQ ID NO. 53-54.
As a further preferred aspect, the optimal primer set 4 of the present invention further comprises LF-4 and LR-4, the nucleotide sequences of which are shown in SEQ ID NOS.23-24, respectively.
The loop primer is preferably 30-35bp in primer length and 60-70 ℃ in Tm value, and the phenomenon of primer dimer and the like is avoided.
The invention provides an sgRNA, in particular to an sgRNA which is specifically combined with Cas12b protein in a LAMP-CRISPR reaction system and comprises any one nucleotide sequence of SEQ ID No. 61-66.
As a further optimization of the invention, the PAM site corresponding to the Cas12b protein is a T-rich PAM sequence, which is TTA or TTT. Correspondingly, the sgRNA is preferably SEQ ID No. 62.
Further, the invention also provides a LAMP-CRISPR kit, which comprises the LAMP primer group, the sgRNA, the Cas12b protein, the Bst enzyme, the report probe and the buffer solution.
Further, the reporter probe is ssDNA, which includes the nucleotide sequence shown as SEQ ID NO. 69.
As a further optimization of the present invention, the buffer comprises: 100-200mM Tris-HCl, 100-200mM NaCl,200-600mM (NH) 4 ) 2 SO 4 、500-1000mM KCl、0.1％-5％tween-20，pH 8.5-8.8。
The invention also provides application of the LAMP primer group in preparation of an LAMP-CRISPR kit for detecting high-risk human papilloma virus HPV16.
In the invention, the LAMP-CRISPR kit is used for detecting the high-risk human papilloma virus HPV16, and comprises the following steps:
step one, performing LAMP amplification by taking DNA of HPV16 to be detected as a template to obtain an amplification product, and performing CRISPR reaction. The LAMP-CRISPR reaction system comprises a specific primer pair containing amplified HPV16 components, cas12b protein, sgRNA, a reporter probe, additives and the like;
and step two, detecting CRISPR reaction fluorescence intensity change, and judging whether HPV16 exists in the sample to be detected according to the curve.
The reaction system for amplification was 25. Mu.L, including: buffer 10 XBuffer 2-4. Mu.L, optimized 2.5. Mu.L. The components are 100-200mM Tris-HCl, 100-200mM NaCl,200-600mM (NH) 4 ) 2 SO 4 500-1000mM KCL, 0.1% -5% tween20, pH 8.5-8.8. Preferably 200mM Tris-HCl, 100mM NaCl,500mM (NH) 4 ) 2 SO 4 、600mM KCL、0.1％tween20、PH 8.8。
Further, the LAMP primer mix is 1-5. Mu.M, more preferably, 2. Mu.M.
Further, mgCl 2 1-4. Mu.M, more preferably 1.2. Mu.M.
Further, dNTP mix is 0.4 to 1. Mu.M, more preferably, 0.6. Mu.M.
Further, bst enzyme is used in an amount of 8-20U, more preferably 15U.
Further, the Cas12b protein is 0.1-1 μm, more optimally in an amount of 0.2 μm.
Further, RNase inhibitor was added to inhibit the activities of RNaseA, RNaseB and RNaseC in the reaction, thereby reducing RNase contamination. The RNase inhibitor is 10 to 50U, more preferably 20U.
Further, the sgRNA is 0.1-1. Mu.M, more preferably, 0.2. Mu.M.
Further, the probe is used in an amount of 0.1 to 1. Mu.M, more preferably, 0.4. Mu.M.
Further, DMSO is present in an amount of 1-5%, more preferably 4%.
Furthermore, formamide is added in the amplification reaction system and can be combined with major grooves and minor grooves in DNA, so that the stability of DNA double helix is reduced, the melting temperature of DNA is reduced, and the amplification efficiency is improved. The concentration of formamide is 1-5%, more preferably 5%.
Further, betaine is used in an amount of 0.1 to 0.5M, more preferably in an amount of 0.2M.
Further, the amount of propane sulfonic acid is 0.2-2M, more preferably 0.5M. The balance being water without nuclease.
The reaction procedure for LAMP-CRISPR amplification was: 1min at 60-65℃for 60-70 cycles, preferably 1min at 61℃for 30 cycles; inactivating at 85-95deg.C for 5-10min, preferably 85 deg.C for 5 min.
Compared with the prior art, the invention has the following beneficial effects:
1) The kit has high detection specificity, combines LAMP amplification with high-specificity detection based on a characteristic sequence by CRISPR, greatly enhances the specificity of a molecular detection method, and reduces false positive by integrated detection.
2) The invention has high detection sensitivity and can ultra-sensitively detect templates with abundance as low as1 copy.
3) The invention has low equipment cost and simple operation, does not need electrophoresis after LAMP-CRISPR amplification, and can directly interpret results.
4) The invention has short detection time, the fluorescent signal and data are directly read by the fluorometer, and the detection process is completed within 30 minutes.
5) The invention is innovative technology development and application exploration of LAMP-CRISPR detection technology in the aspect of nucleic acid detection field of cervical cancer, is innovative research and application of LAMP and CRISPR combined technology in cervical cancer screening prevention and control for the first time, and has good application prospect.
Drawings
FIG. 1 is a screening comparison of LAMP-CRISPR method for target primer and loop primer combinations.
FIG. 2 shows the amplification effect of LAMP-CRISPR system screening additives.
FIG. 3 is a LAMP-CRISPR system amplification temperature screening and verification.
FIG. 4 is LAMP-CRISPR method screening and validation against HPV16 target sgRNA.
FIG. 5 is a preferred primer combination and PAM site, sgRNA positional relationship for LAMP-CRISPR method.
FIG. 6 is LAMP-CRISPR system reporter probe screening and validation.
FIG. 7 is LAMP-CRISPR system buffer screening and validation.
FIG. 8 is the detection specificity of the LAMP-CRISPR method for HPV16 targets.
FIG. 9 is the detection sensitivity of the LAMP-CRISPR method against HPV16 targets.
FIG. 10 is a false positive test of LAMP-CRISPR method against HPV16 target.
Detailed Description
In order to facilitate understanding of the technical means, the creation characteristics, the achievement of the objects and the effects achieved by the present invention, the present invention will be further described with reference to specific examples, but the following examples are only preferred examples of the present invention, and not all the examples are included. Based on the examples in the embodiments, those skilled in the art can obtain other examples without making any inventive effort, which fall within the scope of the invention. The experimental methods in the following examples are conventional methods unless otherwise specified, and materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.
Example 1
The detection process of the embodiment of the invention comprises the following steps: using HPV16 nucleic acid as a template, sterile water as a blank, LAMP amplification and CRISPR reactions were performed using HPV16 specific primers and template and fluorescence was detected.
The Cas12b protein of the invention was purchased from Tolo Biotech, inc., and the bst enzyme was purchased from Beijing Quantum gold Co., ltd. (cat.: LP 311-01).
(1) LAMP primer, CRISPR sgRNA and probe design
In this example, candidate primer selection was performed by using the ORF region of HPV 16E 1 target, comprising three intron regions (240 bp,70bp,279 bp). Complete DNA sequences were obtained from NCBI (National Center for Biotechnology Information ), sequence analysis and alignment were performed using the proprietary software PrimerExplore (http:// PrimerexExplorer. Jp/e /) version V5 on-line tool to design LAMP primers, four pairs of primers FIP, BIP, F3 and B3 were designed: after loading HPV16 target sequences into the system, primerExplorer will automatically judge GC content and match the target sequences to different primer design parameters according to AT-rich sequence (GC% < 45), general sequence (45 < GC% < 60) and GC-rich sequence (GC% > 60) types, respectively. The system can automatically check the designed primer terminal sequence to remove the primer pair with complementary sequence or multiple identical nucleotide sequences.
The primer combination is initially screened by comparing different GC contents, the amplification efficiency of Bst enzyme is influenced by excessively low GC content, and the primer is difficult to bind to a template due to the fact that the excessively high GC content is easy to form a stable secondary structure due to self-complementary pairing, so that serious nonspecific amplification bands can possibly appear. The 3 'end of F2, B2, the 5' end of F1c, B1c, which serve as the start sites for amplification, are very important for stability. The invention designs different groups of primers by the GC content of 50% -65% and the critical parameter Gdelta representing stability of less than or equal to-4.0 kcal/mol. In order to improve the amplification efficiency, 2 circular primers, namely an upstream circular primer (LF) and a downstream circular primer (LR), are added, the lengths of the LF and LR primers are 30-35bp, the Tm value is 60-70 ℃, the phenomenon of primer dimer and the like is avoided, and the optimal circular primer is selected according to the screening principle of the conventional primers.
F2/B2 of LAMP primer is used as limit when designing sgRNA, PAM locus (T) TTG of Cas12B is searched in the interval, each PAM locus corresponds to one sgRNA, and the subsequent 20 bases are pulled to be space for designing the sgRNA, and care should be taken to avoid primer dimer generation.
When the report probe is designed, a series of polyA/polyT/polyC is designed as candidates according to the cutting function of Cas12b, and is a random single-stranded DNA sequence, cy5 is marked at the 5 'end of the probe, and BHQ2 is marked at the 3' end of the probe. Comparing the designed primers, the sgRNAs and the probes, and finally screening 10 groups of primers, 6 sgRNAs and 5 reporting probes, wherein the sequences of the primers are shown in table 1, and the sequences of the sgRNAs and the reporting probes are shown in table 2. All primers, probes and RNA were synthesized by Shanghai Bai Ge biosystems.
TABLE 1 primer sequences
TABLE 2 sgRNA/Probe sequences
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Example 2
(1) LAMP-CRISPRHPV target index primer combination screening
10 pairs of LAMP-CRISPR primer combinations are selected and designed for screening, and the GC content and the delta G of the LAMP-CRISPR primer combinations are counted respectively. And the influence of loop primers LF and LR on LAMP-CRISPR is tested, and the template is performed according to an LAMP-CRISPR initial reaction system and program by using 100 copies of HPV16 nucleic acid.
(2) The LAMP-CRISPR initial reaction system and reaction procedure are shown in tables 3 and 4.
TABLE 3 LAMP-CRISPR reaction System
| Reagent(s) | Concentration of |
| primers mix | 2μM |
| dNTP | 0.6μM |
| 10×Buffer | 2.5μL |
| MgCl 2 | 1.2μM |
| ssDNA | 0.4μM |
| Bst | 15U |
| AacCas12b | 0.2μM |
| sgRNA | 0.2μM |
| Template | 5μL |
| ddH2O | To 25μL |
TABLE 4 LAMP-CRISPR initial reaction procedure
| Step (a) | Temperature (temperature) | Time | Cycle number |
| 1 | 62℃ | For 1 minute | 60 |
| 2 | 85℃ | For 5 minutes | 1 |
(3) Result determination
Judging according to the fluorescence value of CRISPR in 60 minutes of reaction, and judging that CRISPR detection is positive if the peak value is obviously higher than the fluorescence value of the control nuclease-free water; otherwise, it is determined that the CRISPR detection is negative.
The test results are shown in FIG. 1 and Table 5.
TABLE 5 primer combination screening results (vs. detection time for acyclic primers)
The results show that: compared with the CRISPR detection time and peak value which are increased by LF and LR loop primers, the increase of the loop primers can improve the amplification efficiency of the system. The preferred combination is a primer 4 combination containing loop primers, the GC content is 57%, the delta G is-2.2 kcal/mol, and the detection time of 100 copies of the nucleic acid template is longer than that of other combinations in 18 minutes, which shows that the LAMP primer of the combination has better amplification effect in a CRISPR reaction system.
(2) And screening and testing the LAMP-CRISPR reaction system.
The invention screens and tests the influence of 8 additives and RRI inhibitors (optimized dosage) on the amplification effect of the LAMP-CRISPR system, 100 copies of HPV16 nucleic acid are used as templates, and the test results are shown in FIG. 2 and Table 6.
TABLE 6 additive amplification Effect
| Sequence number | Additive name | Optimizing dosage | Amplification Effect |
| 1 | Without any means for | Without any means for | Control |
| 2 | DMSO | 4％ | - |
| 3 | Propane sulfonic acid | 0.02M | + |
| 4 | Glycine (Gly) | 0.1M | - |
| 5 | Serine (serine) | 0.5M | - |
| 6 | Glutathione | 0.1M | - |
| 7 | Betaine | 0.2M | + |
| 8 | DTT | 0.1％ | - |
| 9 | Formamide | 5％ | + |
| 10 | RRI inhibitors | 20U | + |
Note that: "+" indicates a promoting effect as compared to the control group, and "-" indicates no promoting or inhibiting effect as compared to the control group. The results show that RRI inhibitor, propane sulfonic acid, betaine and formamide additive have promotion effect on LAMP-CRISPR system amplification.
(3) LAMP-CRISPR system amplification temperature screening test
The Bst enzyme of LAMP preferably has a reactivity of 60-65deg.C, and the preliminary test is preferably carried out at 62 deg.C. The activity of the Cas12b enzyme is optimally between 58 and 63 ℃, and the Cas amplification efficiency is influenced by too low and too high, so that the temperature is increased on the premise of not influencing Bst enzyme so as to test the Cas enzyme amplification effect. In the invention, 8 groups of reaction temperatures are set at 58-65 ℃, 100 copies of templates are used, and the optimal LAMP-CRISPR amplification temperature is screened out according to the Tm value of the preferred primer combination pair 4. The test results are shown in fig. 3 and table 7.
TABLE 7 amplification temperature screening results
The results show that the LAMP-CRISPR reaction is at an optimal temperature of 61 ℃.
(4) LAMP-CRISPR screening test for HPV16 target sgRNA
The two-step method is adopted, namely, the LAMP amplification is firstly carried out to obtain HPV16 products, and then CRISPR is carried out to detect the sgRNA amplification effect of 6 PAM sites. The reaction temperature was 61℃for 30 minutes. The test results are shown in fig. 4 and table 8.
TABLE 8 Cas-sgRNA screening results
| Sequence number | PAM site | sgRNA name | Two-step screening effect |
| 1 | TTA | sgRNA1 | Poor quality |
| 2 | TTG | sgRNA2 | Optimum for the production of a product |
| 3 | TTC | sgRNA3 | Poor quality |
| 4 | TTT | sgRNA4 | Preferably, it is |
| 5 | TTA | sgRNA5 | Preferably, it is |
| 6 | TTC | sgRNA6 | Worst case of |
The results show that: preferably the PAM site TTG, the corresponding preferred sgrnas are the sgRNA2 combination.
Preferably the primer combination (loop containing primers), PAM site and sgRNA are found in HPV16 target in figure 5.
(5) LAMP-CRISPR system ssDNA report probe screening test
Preferred primers and sgrnas were selected for CRISPR probe screening with 50 copies of nucleic acid for HPV16 as templates and the test results are shown in fig. 6 and table 9. The reaction temperature was 61℃for 30 minutes.
TABLE 9 Cas-ssDNA Probe screening results
The results show that: preferably, the reporter probe is ssDNA3 and the detection time is 21 minutes.
(6) LAMP-CRISPR system Buffer screening test
6 10 XBuffer formulas suitable for CRISPR reaction systems are selected for screening test, respectively
Buffer1：300mM Tris-HCl、100mM(NH 4 ) 2 SO 4 、500mMKCL、20mM MgSO 4 、0.1％tween20、PH8.8；
Buffer2：100mM Tris-HCl、500mM(NH 4 ) 2 SO 4 、1000mM KCL、0.1tween20、PH 8.5、0.1％tween20、0.2M betaine、PH8.8；
Buffer3：100mM Tris-HCl、100mM NaCl，500mM(NH 4 ) 2 SO 4 500mM KCL, 0.1% tween20, 200mM trehalose, pH8.8;
Buffer4：200mM Tris-HCl、800mM(NH 4 ) 2 SO 4 、500mM KCL、0.1％trixon-100、PH8.5；
Buffer5：400mM Tris-HCl、500mM(NH 4 ) 2 SO 4 、40mM MgSO 4 、800mM KCL、0.1％trixon-100、PH8.8；
Buffer6：200mM Tris-HCl、100mM NaCl，500mM(NH 4 ) 2 SO 4 500mM KCL, 0.1% tween20, pH 8.8. The template is HPV16 nucleic acid 50 copies, the reaction temperature is 61 ℃ and the time is 30 minutes. The test results are shown in fig. 7 and table 10.
TABLE 10.10 XBuffer screening results
| Sequence number | 10 Xbuffer name | Average detection time |
| 1 | Buffer1 | 20 minutes |
| 2 | Buffer2 | 0 |
| 3 | Buffer3 | 25 minutes |
| 4 | Buffer4 | 22 minutes |
| 5 | Buffer5 | 28 minutes |
| 6 | Buffer6 | 18 minutes |
The result shows that the Buffer6 has optimal amplification effect, and the average detection time of 50 copies of template concentration is 18 minutes, which is superior to other Buffer combinations.
Example 3 specific assay
Other 10 common HPV nucleic acids are selected as templates, 50 copies of the templates are used, and the primers and probes of HPV16 are used for carrying out LAMP-CRISPR method test, wherein the reaction temperature is 61 ℃ and the reaction time is 30 minutes. The results are shown in FIG. 8 and Table 11.
TABLE 11 specificity test results
| Sequence number | Template type | Pathogen name | LAMP-CRISPR results |
| 1 | Nucleic acid | HPV18 | Negative of |
| 2 | Nucleic acid | HPV31 | Negative of |
| 3 | Nucleic acid | HPV33 | Negative of |
| 4 | Nucleic acid | HPV35 | Negative of |
| 5 | Nucleic acid | HPV39 | Negative of |
| 6 | Nucleic acid | HPV45 | Negative of |
| 7 | Nucleic acid | HPV51 | Negative of |
| 8 | Nucleic acid | HPV59 | Negative of |
| 9 | Nucleic acid | HPV68 | Negative of |
| 10 | Nucleic acid | HPV16 | Positive and negative |
The results show that the LAMP-CRISPR detection results of other pathogen nucleic acids except HPV16 are all negative, and the method is good in specificity.
Example 4 detection limit test
The detection limit refers to the minimum concentration or minimum amount of a substance to be detected from a sample within a given degree of reliability. Sequentially diluting HPV16 nucleic acid templates to different concentrations by nuclease-free water, respectively taking 5 mu L of templates for LAMP-CRISPR test, and counting detection time, wherein the results are shown in FIG. 9 and Table 12.
TABLE 12 detection sensitivity of LAMP-CRISPR method against HPV16 targets
The results show that the amplification sensitivity of the LAMP-CRISPR method for detecting HPV16 can stably detect 1 copy and has no false positive.
Example 5 false positive test
The invention combines LAMP and CRISPR and adopts an integrated system, thus being capable of obviously reducing false positive in constant temperature reaction, selecting 2 positive controls and 48 negative controls for verification, and the test results are shown in figure 10 and table 13.
TABLE 13 false positive test results
| Sequence number | Template concentration | Average detection time |
| 1-2 | 100 copies | 15 minutes |
| 3-50 | 0 | 0 |
The results show that the LAMP-CRISPR method detects no false positive in 48 negative control groups of HPV16, and avoids the pollution and non-specific amplification of conventional LAMP.
While the invention has been described with respect to preferred embodiments thereof, it will be understood by those skilled in the art that various modifications and additions may be made without departing from the scope of the invention. Equivalent embodiments of the present invention will be apparent to those skilled in the art having the benefit of the teachings disclosed herein, when considered in the light of the foregoing disclosure, and without departing from the spirit and scope of the invention; meanwhile, any equivalent changes, modifications and evolution of the above embodiments according to the essential technology of the present invention still fall within the scope of the technical solution of the present invention.
Claims (9)
1. A LAMP primer set, characterized in that the LAMP primer set comprises:
primer set 4: comprises F3-4, B3-4, FIP-4 and BIP-4, and the nucleotide sequences of the F3-4, the B3-4, the FIP-4 and the BIP-4 are shown as SEQ ID NO. 19-22 in sequence.
2. The LAMP primer set as claimed in claim 1, wherein the primer set 4 optionally comprises the following loop primers:
the primer group 4 further comprises LF-4 and LR-4, and the nucleotide sequences of the primer group are respectively shown as SEQ ID NO. 23-24.
3. An sgRNA that specifically binds to Cas12b protein and comprises any one of the nucleotide sequences of SEQ ID nos 61-66.
4. The sgRNA of claim 3, wherein the PAM site corresponding to the Cas12b protein is a T-rich PAM sequence, wherein the PAM sequence is TTA or TTT, and wherein the sgRNA is the sequence shown in SEQ ID No. 62.
5. A LAMP-CRISPR kit comprising the LAMP primer set of claim 1, the sgRNA of claim 3, a Cas12b protein, bst enzyme, a reporter probe, and a buffer.
6. The LAMP-CRISPR kit of claim 5 wherein the sequence of the reporter probe ssDNA is shown in SEQ ID NO: 69.
7. The LAMP-CRISPR kit of claim 5, wherein said buffer comprises: 100-200mM Tris-HCl, 100-200mM NaCl,200-600mM (NH) 4 ) 2 SO 4 、500-1000mM KCl、0.1％-5％tween-20，pH 8.5-8.8。
8. The LAMP-CRISPR kit of claim 5, wherein the reaction procedure is: reacting for 1 minute at 60-65 ℃ and 30-60 cycles; inactivating at 85-95deg.C for 5-10 min.
9. The use of the LAMP primer set of claim 1 in the preparation of LAMP-CRISPR kit for detecting high risk human papillomavirus HPV16.
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