LAMP-CRISPR Kit for Detecting HPV16 and Its Application

Abstract

The present invention discloses a LAMP-CRISPR kit for detecting HPV16 and its application, which is used for detecting the high-risk human papillomavirus HPV16, and includes: Step 1, using the DNA of the sample to be tested as a template for loop-mediated isothermal amplification (LAMP), and at the same time carrying out the CRISPR system reaction. The CRISPR-LAMP reaction system includes a specific primer pair containing components for amplifying HPV16, Buffer, Cas12b protein, sgRNA, probe, additives, etc.; Step 2, detecting the change in the fluorescence intensity of the CRISPR reaction, and judging whether HPV16 exists in the sample to be tested according to the Cas curve. By using the CRISPR detection method of the present invention, the nucleic acid amplification and target detection steps are integrated into one system, simplifying the operation procedure and avoiding cross-contamination, and improving the specificity and sensitivity of HPV16 detection.

Description

Technical Field

[0001] The present invention belongs to the technical fields of molecular biology technology CRISPR and nucleic acid detection technology, and relates to a detection technology based on the combination of LAMP and CRISPR / Cas12. Specifically, it relates to a primer set and a kit of LAMP-CRISPR for detecting high-risk human papillomavirus HPV16. Background Art

[0002] Nucleic acid detection of human papillomavirus (HPV) is the preferred method recommended by the WHO for primary screening of cervical cancer, but there is no guiding document in China for its application in cervical cancer screening practice. Therefore, multidisciplinary experts in public health, obstetrics and gynecology, laboratory medicine, pathology, etc. have formed an expert consensus by combining domestic and foreign guidelines, medical evidence-based evidence and actual situations, recommending HPV nucleic acid detection as the main method for cervical cancer screening, and putting forward scientific suggestions for aspects such as pre-evaluation and preparation before its application in cervical cancer screening practice, specific uses and applicable populations, laboratory operation procedures, and post-implementation monitoring and evaluation, so as to promote the comprehensive, in-depth, standardized and orderly high-quality development of HPV nucleic acid detection and provide a strong guarantee for achieving the goal of cervical cancer elimination. Among them, HPV16 is the most common (59.5%) high-risk human papillomavirus. During the continuous infection of HPV, the E6 and E7 genes of the virus may integrate with the host DNA, leading to cervical intraepithelial neoplasia (CIN) or even cervical cancer. Therefore, typing detection of HPV and clarifying whether there is persistent infection have important clinical significance.

[0003] Isothermal amplification technology, with its characteristics of rapidity, accuracy, specificity and independence from sophisticated instruments, has shown good application prospects in clinical and rapid diagnosis. Common ones include recombinase polymerase amplification (RPA), loop-mediated isothermal amplification (LAMP), strand displacement amplification (SDA), rolling circle amplification (RCA), etc. Loop-mediated isothermal amplification technology (LAMP) is an isothermal nucleic acid amplification technology reported by Notomi et al. in 1998. This method relies on 4 primers (2 outer primers 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, with simple operation and high amplification efficiency, and can amplify 10 9 ~10 10 times in 30 - 60 minutes, with relatively high specificity. However, compared with the mature qPCR technology, LAMP has problems such as insufficient detection sensitivity, difficult primer design, high false positives, easy contamination, and difficulty in multiplex implementation.

[0004] CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is an acquired immune method for most bacteria and archaea to resist virus invasion. The discovery of the CRISPR / Cas system can be traced back to 1987 when Nakata et al. first discovered tandemly repeated spacer sequences in the genome of Escherichia coli. In 2002, Jansen et al. officially named this peculiar repetitive spacer sequence as Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR).

[0005] In recent years, CRISPR technology has shown great application value in the field of genome editing.

[0006] The CRISPR detection technology introduced in 2017 is known as the "next-generation molecular detection technology" and features "rapid, sensitive, highly specific, simple, and low-cost". This technology can be widely applied in pathogen detection, cancer mutation detection, single nucleotide polymorphism detection, etc., and has great application potential.

[0007] As a type II CRISPR / Cas system, such as Cas12a and Cas13a, after the cis-cleavage of the formed Cas / crRNA / target DNA ternary complex, it has trans-cleavage activity and can non-specifically cleave nearby single-stranded DNA or RNA. The action process of CRISPR / Cas12 mainly consists of two stages. In the first stage, the exogenously introduced sequence, namely sgRNA, binds to the Cas12 protein. In the second stage, the transcribed crRNA forms pre-crRNA, which guides the Cas12 protein to cleave the target region on the genome that is the same as the sgRNA, thus achieving the purpose of gene editing. Cas12b is an RNP complex that can perform DNA cleavage according to the guidance of crRNA. Under the guidance of the target DNA, its nuclease domain will be activated and perform DNA endonuclease cleavage, realizing the efficient recognition and amplification of the target DNA.

[0008] Utilizing this property in combination with the FRET (Fluorescence Resonance Energy Transfer) technology enables nucleic acid detection. The specific principle is to design an oligonucleotide sequence with a fluorescent group and a fluorescence quenching group added to its ends, called a reporter probe. After the cis-cleavage of the CRISPR / Cas system, a fluorescent signal is generated through trans-cleavage of the reporter, and the content of the target nucleic acid sequence can be inferred by detecting the fluorescence signal intensity, achieving the effect of nucleic acid detection.

[0009] Combining the high amplification efficiency of isothermal amplification with the high specificity of CRISPR detection, researchers have developed a series of isothermal CRISPR amplification technologies. Due to problems such as template competition, system conflicts, and inconsistent reaction temperatures between the LAMP amplification reaction and the CRISPR detection reaction, the currently more widely used detection method is a two-step method that separates LAMP amplification and CRISPR detection. However, the two-step method has problems such as open-cap contamination and complex operations.

[0010] Therefore, subsequent researchers have successively developed "one-pot" reaction systems such as the HOLMES v2 system and the STOPCovid.v2 system, in which the LAMP amplification and CRISPR detection reactions are carried out synchronously in one system, with simple operation, avoiding contamination, and reducing the detection time.

[0011] However, in the "one-pot" system, the Bst polymerase of the LAMP amplification reaction and the Cas-sgRNA complex will inevitably compete with the DNA template for binding, and the cis-cleavage caused by the latter after binding to the DNA template will lead to the failure of LAMP amplification under the condition of extremely low-concentration templates, resulting in negative results, which also means that the sensitivity of the "one-pot" detection system is relatively low.

[0012] To solve this problem, several optimization strategies have been proposed by researchers, including reducing the Cas protein cleavage efficiency, modifying the sgRNA to delay triggering, and optimizing the reaction system. However, the design of specific primers and the detection process in the early stage require professional and skilled technicians, which further limits the wide application of the above methods. Summary of the Invention

[0013] The technical problems to be solved by the present invention are as follows: LAMP has insufficient detection sensitivity, high false positives, and is prone to contamination. Based on the integrated LAMP-CRISPR detection system, through the design and screening of specific primers, the binding of circular primers, and the optimization of the PAM site, reaction system, and reaction temperature of the Cas enzyme, the amplification sensitivity and specificity of the reaction system can be significantly improved, and non-specific amplification can be reduced.

[0014] The first object of the present invention is to provide a primer set, sgRNA in a LAMP-CRISPR reaction system for detecting high-risk human papillomavirus HPV16, and a LAMP-CRISPR kit containing the foregoing primer set and sgRNA.

[0015] The second object of the present invention is to develop a nucleic acid analysis method that is convenient, efficient, low-cost, avoids contamination, and does not require complex instrument equipment. An integrated system of LAMP, CRISPR, and Cas12b protein is established to detect HPV16, improve the reaction sensitivity and specificity, and reduce the risks of cross-contamination and non-specific amplification.

[0016] The third object of the present invention is to provide the application of the LAMP-CRISPR kit in the detection of high-risk human papillomavirus HPV16.

[0017] Technical solution: The present invention provides a set of LAMP primers, in particular a set of primers used in the LAMP-CRISPR reaction system. The present invention designs primer sets 1-9, and the best primer set is selected therefrom:

[0018] Primer set 1: Comprising F3-1, B3-1, FIP-1, BIP-1, and their nucleotide sequences are shown in SEQ ID NO: 1-4 in sequence;

[0019] Primer set 2: Comprising F3-2, B3-2, FIP-2, BIP-2, and their nucleotide sequences are shown in SEQ ID NO: 7-10 in sequence;

[0020] Primer set 3: Comprising F3-3, B3-3, FIP-3, BIP-3, and their nucleotide sequences are shown in SEQ ID NO: 13-16 in sequence;

[0021] Primer set 4: Comprising F3-4, B3-4, FIP-4, BIP-4, and their nucleotide sequences are shown in SEQ ID NO: 19-22 in sequence;

[0022] Primer set 5: Comprising F3-5, B3-5, FIP-5, BIP-5, and their nucleotide sequences are shown in SEQ ID NO: 25-28 in sequence;

[0023] Primer set 6: Comprising F3-6, B3-6, FIP-6, BIP-6, and their nucleotide sequences are shown in SEQ ID NO: 31-34 in sequence;

[0024] Primer set 7: Comprising F3-7, B3-7, FIP-7, BIP-7, and their nucleotide sequences are shown in SEQ ID NO: 37-40 in sequence;

[0025] Primer set 8: Comprising F3-8, B3-8, FIP-8, BIP-8, and their nucleotide sequences are shown in SEQ ID NO: 43-46 in sequence;

[0026] Primer set 9: Comprising F3-9, B3-9, FIP-9, BIP-9, and their nucleotide sequences are shown in SEQ ID NO: 49-52 in sequence.

[0027] If the average GC content of the primer nucleotide sequences in the primer set is too low, it will affect the amplification efficiency of the Bst enzyme. If it is too high, it is easy to form a stable secondary structure due to self-complementary pairing, making it difficult for the primer to bind to the template, and serious non-specific amplification bands may appear.

[0028] Preferably, the LAMP primer set of the present invention is primer set 4.

[0029] Preferably, the average GC content of the primer nucleotide sequences in the primer set is 50%-65%, more preferably selected from 55%-60%; the GΔ of the primer nucleotide sequences in the primer set is less than or equal to -4.0 kcal / mol, and more preferably the GΔ is less than or equal to -3.0 kcal / mol.

[0030] As a further optimization of the present invention, the amplification efficiency can be significantly improved by adding 2 loop primers.

[0031] Specifically, the primer sets 1-9 further include the following loop primers:

[0032] The primer set 1 further includes LF-1 and LR-1, and their nucleotide sequences are shown in SEQ ID NO:5-6 respectively;

[0033] The primer set 2 further includes LF-2 and LR-2, and their nucleotide sequences are shown in SEQ ID NO:11-12 respectively;

[0034] The primer set 3 further includes LF-3 and LR-3, and their nucleotide sequences are shown in SEQ ID NO:17-18 respectively;

[0035] The primer set 4 further includes LF-4 and LR-4, and their nucleotide sequences are shown in SEQ ID NO:23-24 respectively;

[0036] The primer set 5 further includes LF-5 and LR-5, and their nucleotide sequences are shown in SEQ ID NO:29-30 respectively;

[0037] The primer set 6 further includes LF-6 and LR-6, and their nucleotide sequences are shown in SEQ ID NO:35-36 respectively;

[0038] The primer set 7 further includes LF-7 and LR-7, and their nucleotide sequences are shown in SEQ ID NO:41-42 respectively;

[0039] The primer set 8 further includes LF-8 and LR-8, and their nucleotide sequences are shown in SEQ ID NO:47-48 respectively;

[0040] The primer set 9 further includes LF-9 and LR-9, and their nucleotide sequences are shown in SEQ ID NO:53-54 respectively.

[0041] As a further preference, the optimal primer set 4 of the present invention further includes LF-4 and LR-4, and their nucleotide sequences are shown in SEQ ID NO:23-24 respectively.

[0042] When designing the loop primer, the primer length is preferably 30-35bp, the Tm value is 60-70°C, and primer dimer phenomena should be avoided.

[0043] The present invention provides an sgRNA, especially an sgRNA used in the LAMP-CRISPR reaction system and specifically binding to the Cas12b protein, which contains any one of the nucleotide sequences of SEQ ID No:61-66.

[0044] As a further optimization of the present invention, the PAM site corresponding to the Cas12b protein is a T-rich PAM sequence, and the PAM sequence is TTA or TTT. Correspondingly, the sgRNA is preferably SEQ ID No:62.

[0045] Furthermore, the present invention also provides a LAMP-CRISPR kit, which includes the aforementioned LAMP primer set, the aforementioned sgRNA, Cas12b protein, Bst enzyme, reporter probe and buffer.

[0046] Furthermore, the reporter probe is ssDNA, which includes the nucleotide sequence shown in SEQ ID NO:69.

[0047] As a further optimization of the present invention, the buffer includes: 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.

[0048] The present invention also provides the application of the aforementioned LAMP primer set in the preparation of a LAMP-CRISPR kit for detecting high-risk human papillomavirus HPV16.

[0049] In the present invention, the method for detecting high-risk human papillomavirus HPV16 using the LAMP-CRISPR kit includes the following steps:

[0050] Step 1: Using the DNA of the HPV16 sample to be tested as a template, perform LAMP amplification to obtain an amplification product while performing a CRISPR reaction. The reaction system for LAMP-CRISPR includes a specific primer pair containing components for amplifying HPV16, Cas12b protein, sgRNA, a reporter probe, and additives, etc.;

[0051] Step 2: Detect the change in the fluorescence intensity of the CRISPR reaction, and determine whether HPV16 exists in the sample to be tested according to the curve.

[0052] The reaction system for amplification is 25 μL, including: 10×Buffer 2 - 4 μL, with an optimized amount of 2.5 μL. The components are 100 - 200 mM Tris-HCl, 100 - 200 mM NaCl, 200 - 600 mM (NH 4 ) 2 SO 4 4, 500 - 1000 mM KCL, 0.1% - 5% tween20, pH 8.5 - 8.8. Preferably 200 mM Tris-HCl, 100 mM NaCl, 500 mM (NH 4 ) 2 SO 4 4, 600 mM KCL, 0.1% tween20, pH 8.8.

[0053] Furthermore, the LAMP primer mix is 1 - 5 μM, and more optimally, the amount used is 2 μM.

[0054] Furthermore, MgCl 2 is 1 - 4 μM, and more optimally, the amount used is 1.2 μM.

[0055] Furthermore, the dNTP mix is 0.4 - 1 μM, and more optimally, the amount used is 0.6 μM.

[0056] Furthermore, the amount of Bst enzyme used is 8 - 20 U, and more optimally, the amount used is 15 U.

[0057] Furthermore, the Cas12b protein is 0.1 - 1 μM, and more optimally, the amount used is 0.2 μM.

[0058] Furthermore, an RNase inhibitor is added to inhibit the activities of RNaseA, RNaseB, and RNaseC in the reaction, thereby reducing RNase contamination. The RNase inhibitor is 10 - 50 U, and more optimally, the amount used is 20 U.

[0059] Furthermore, the sgRNA is 0.1 - 1 μM, and more optimally, the amount used is 0.2 μM.

[0060] Furthermore, the concentration of the probe is 0.1 - 1 μM, and more preferably, the dosage is 0.4 μM.

[0061] Furthermore, the concentration of DMSO is 1 - 5%, and more preferably, the dosage is 4%.

[0062] Furthermore, formamide is added to the amplification reaction system. It can bind to the major and minor grooves of DNA, thereby reducing the stability of the DNA double helix and decreasing the DNA melting temperature, thus improving the amplification efficiency. The concentration of formamide is 1 - 5%, and more preferably, the dosage is 5%.

[0063] Furthermore, the dosage of betaine is 0.1 - 0.5 M, and more preferably, the dosage is 0.2 M.

[0064] Furthermore, the dosage of propanesulfonic acid is 0.2 - 2 M, and more preferably, the dosage is 0.5 M. The balance is nuclease - free water.

[0065] The reaction procedure of LAMP - CRISPR amplification is as follows: react at 60 - 65 °C for 1 min, with 60 - 70 cycles, preferably react at 61 °C for 1 min, with 30 cycles; inactivate at 85 - 95 °C for 5 - 10 min, preferably inactivate at 85 °C for 5 min.

[0066] Compared with the prior art, the present invention has the following beneficial effects:

[0067] 1) The kit of the present invention has high detection specificity. The LAMP amplification combined with the highly specific detection of CRISPR based on the characteristic sequence greatly enhances the specificity of the molecular detection method, and the integrated detection reduces the false positive rate.

[0068] 2) The present invention has high detection sensitivity and can ultrasensitively detect templates with an abundance as low as 1 copy.

[0069] 3) The present invention has low equipment cost and simple operation. After LAMP - CRISPR amplification, electrophoresis is not required, and the results can be directly interpreted.

[0070] 4) The present invention has a short detection time. The fluorescence signal and data can be directly read by a fluorescence instrument, and the detection process can be completed within 30 min.

[0071] 5) The present invention is an innovative technical exploration and application in the field of nucleic acid detection of cervical cancer by the LAMP - CRISPR detection technology. It is the first innovative research and application of the combination technology of LAMP and CRISPR in cervical cancer screening and prevention and control, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] Figure 1 It is a screening comparison of the target primer and loop primer combinations by the LAMP - CRISPR method.

[0073] Figure 2 It is the amplification effect of screening additives in the LAMP-CRISPR system.

[0074] Figure 3 It is the screening and verification of the amplification temperature of the LAMP-CRISPR system.

[0075] Figure 4 It is the screening and verification of the sgRNA targeting the HPV16 target by the LAMP-CRISPR method.

[0076] Figure 5 It is the preferred primer combination, PAM site, and the positional relationship of the sgRNA in the LAMP-CRISPR method.

[0077] Figure 6 It is the screening and verification of the reporter probe in the LAMP-CRISPR system.

[0078] Figure 7 It is the screening and verification of the buffer in the LAMP-CRISPR system.

[0079] Figure 8 It is the detection specificity of the LAMP-CRISPR method targeting the HPV16 target.

[0080] Figure 9 It is the detection sensitivity of the LAMP-CRISPR method targeting the HPV16 target.

[0081] Figure 10 It is the false positive test of the LAMP-CRISPR method targeting the HPV16 target. Detailed implementation manner

[0082] In order to make the technical means, creative features, achieved purposes, and effects of the present invention easy to understand, the present invention will be further described below in conjunction with specific embodiments. However, the following embodiments are only the preferred embodiments of the present invention and not all of them. Based on the embodiments in the implementation manner, other embodiments obtained by those skilled in the art without creative efforts all belong to the protection scope of the present invention. The experimental methods in the following embodiments are all conventional methods unless otherwise specified. The materials, reagents, etc. used in the following embodiments can all be obtained from commercial channels unless otherwise specified.

[0083] Example 1

[0084] The detection process of the embodiment of the present invention: Using HPV16 nucleic acid as a template, sterile water as a blank control, and using HPV16-specific primers and templates for LAMP amplification and CRISPR reaction and detecting fluorescence.

[0085] The Cas12b protein of the present invention was purchased from Tolo Biotech., and the bst enzyme was purchased from Beijing TransGen Biotech Co., Ltd. (product number: LP311-01).

[0086] (1) Design of LAMP primers, CRISPR sgRNA and probes

[0087] In this example, candidate primers were selected through the ORF region of the HPV16 E1 target, which contains three intron regions (240bp, 70bp, 279bp). The complete DNA sequence was obtained from NCBI (National Center for Biotechnology Information). The online tool of the V5 version of the dedicated software PrimerExplore (http: / / primerexplorer.jp / e / ) for designing LAMP primers was used for sequence analysis and alignment, and four pairs of primers, namely FIP, BIP, F3, and B3, were designed: After loading the HPV16 target sequence into the system, PrimerExplorer will automatically judge the GC content and match different primer design parameters for the target sequence according to three types: AT-rich sequences (GC% < 45), general sequences (45 < GC% < 60), and GC-rich sequences (GC% > 60). The system can automatically check the terminal sequences of the designed primers and remove primer pairs with complementary sequences or multiple identical nucleotide sequences.

[0088] In the present invention, primer combinations were initially screened by comparing different GC contents. Too low a GC content will affect the amplification efficiency of the Bst enzyme, and too high a GC content is likely to form a stable secondary structure due to self-complementary pairing, making it difficult for the primer to bind to the template, and serious non-specific amplification bands may appear. The 3' ends of F2 and B2, and the 5' ends of F1c and B1c, which are the starting sites of amplification, are very important for their stability. In the present invention, different groups of primers were designed with a GC content of 50% - 65% and the key parameter GΔ representing stability less than or equal to -4.0 kcal / mol. To improve the amplification efficiency, 2 additional circular primers, namely the upstream circular primer (LF) and the downstream circular primer (LR), were added. The lengths of the LF and LR primers are 30 - 35bp, and the Tm values are 60 - 70°C. Primer dimer and other phenomena should be avoided, and the optimal circular primers were selected according to the screening principles of conventional primers.

[0089] When designing sgRNA, the F2 / B2 of the LAMP primer was used as the boundary, and the PAM site (T)TTG of Cas12b was searched within this interval. Each PAM site corresponds to an sgRNA, and the subsequent 20 bases were pulled as the spacer to design the sgRNA. Attention should be paid to avoiding the generation of primer dimers.

[0090] When designing the reporter probes, a series of polyA / polyT / polyC were designed as candidates according to the Cas12b cleavage function. They are random single-stranded DNA sequences, with Cy5 labeled at the 5'-end of the probe and BHQ2 labeled at the 3'-end. The designed primers, sgRNAs and probes were compared, and finally 10 sets of primers, 6 sgRNAs and 5 reporter probes were selected. The primer sequences are shown in Table 1, and the sgRNAs and reporter probes are shown in Table 2. All primers, probes and RNAs were synthesized by Shanghai Biolink Biotechnology Co., Ltd.

[0091] Table 1. Primer sequences

[0092]

[0093]

[0094]

[0095] Table 2. sgRNA / Probe sequences

[0096]

[0097]

[0098]

[0099]

[0100] Example 2

[0101] (1) Screening of LAMP-CRISPR HPV16 target primer combinations

[0102] Ten pairs of designed LAMP-CRISPR primer combinations were selected for screening, and their GC contents and ΔG were statistically analyzed respectively. And the effects of circular primers LF and LR on LAMP-CRISPR were tested. The template used was 100 copies of HPV16 nucleic acid, and the reaction was carried out according to the LAMP-CRISPR initial reaction system and procedure.

[0103] (2) The LAMP-CRISPR initial reaction system and reaction procedure are shown in Table 3 and Table 4.

[0104] Table 3. LAMP-CRISPR reaction system

[0105] Reagent Concentration primers mix 2μM dNTP 0.6μM 10×Buffer 2.5μL <![CDATA[MgCl 2 > 1.2μM ssDNA 0.4μM Bst 15U AacCas12b 0.2μM sgRNA 0.2μM Template 5μL ddH2O To 25μL

[0106] Table 4. LAMP-CRISPR initial reaction procedure

[0107] Step Temperature Time Number of cycles 1 62℃ 1 minute 60 2 85℃ 5 minutes 1

[0108] (3) Result determination

[0109] Judgment is made based on the fluorescence value of CRISPR at 60 minutes of reaction. If the peak value is significantly higher than the fluorescence value of the control nuclease-free water, it is determined as positive for CRISPR detection; otherwise, it is determined as negative for CRISPR detection.

[0110] The test results are shown in Figure 1 and Table 5.

[0111] Table 5. Screening results of primer combinations (comparing the detection time with and without loop primers)

[0112]

[0113] The results show that: compared with the CRISPR detection time and peak value after adding LF and LR loop primers, they are significantly increased. Therefore, adding loop primers can improve the amplification efficiency of the system. The preferred combination is primer 4 combination containing loop primers, with a GC content of 57% and ΔG of -2.2 kcal / mol. The detection time of its 100-copy nucleic acid template is 18 minutes, which is better than other combinations, indicating that the LAMP primers of this combination have better amplification effect in the CRISPR reaction system.

[0114] (2) Screening test of LAMP-CRISPR reaction system.

[0115] The present invention screens and tests the influence of 8 additives and RRI inhibitors (optimized dosage) on the amplification effect of the LAMP-CRISPR system. The template uses 100 copies of HPV16 nucleic acid, and the test results are shown in Figure 2 and Table 6.

[0116] Table 6. Amplification effect of additives

[0117] Serial number Additive name Optimized dosage Amplification effect 1 None None Control 2 DMSO 4％ - 3 Propane sulfonic acid 0.02M + 4 Glycine 0.1M - 5 Serine 0.5M - 6 Glutathione 0.1M - 7 Betaine 0.2M + 8 DTT 0.1％ - 9 Formamide 5％ + 10 RRI inhibitor 20U +

[0118] Note: “+” indicates a promoting effect compared with the control group, and “-” indicates no promoting or inhibiting effect compared with the control group. The results show that RRI inhibitor, propanesulfonic acid, Betaine, and formamide additives have a promoting effect on the amplification of the LAMP-CRISPR system.

[0119] (3) Screening test of amplification temperature of LAMP-CRISPR system

[0120] The preferred reaction activity of the Bst enzyme of the LAMP is 60 - 65°C, and 62°C is preferred in the preliminary test. The optimal activity of the Cas12b enzyme is at 58 - 63°C. Too low or too high a temperature will affect the Cas amplification efficiency. Therefore, the temperature is increased without affecting the Bst enzyme to test the Cas enzyme amplification effect. The present invention sets 8 groups of reaction temperatures from 58 - 65°C, uses 100 copies of the template, and screens for the most suitable LAMP-CRISPR amplification temperature for the Tm value of the preferred primer combination pair 4. The test results are as Figure 3 shown in Table 7.

[0121] Table 7. Screening results of amplification temperature

[0122]

[0123]

[0124] The results show that the optimal temperature for the LAMP-CRISPR reaction is 61°C.

[0125] (4) Screening test of LAMP-CRISPR for HPV16 target sgRNA

[0126] The two-step method is adopted, that is, first perform LAMP amplification to obtain the HPV16 product, and then perform CRISPR to detect the amplification effect of sgRNAs at 6 PAM sites. The reaction temperature is 61°C and the time is 30 minutes. The test results are as Figure 4 shown in Table 8.

[0127] Table 8. Cas-sgRNA screening results

[0128] Serial number PAM site sgRNA name Two-step screening effect 1 TTA sgRNA1 Poor 2 TTG sgRNA2 Optimal 3 TTC sgRNA3 Poor 4 TTT sgRNA4 Good 5 TTA sgRNA5 Good 6 TTC sgRNA6 Worst

[0129] The results show that: the preferred PAM site is TTG, and the corresponding preferred sgRNA is the sgRNA2 combination.

[0130] The preferred primer combination (including loop primers), PAM site, and sgRNA for the HPV16 target are shown in Figure 5 .

[0131] (5) Screening test of the ssDNA reporter probe for the LAMP-CRISPR system

[0132] Select the preferred primers and sgRNA for CRISPR probe screening. The template is 50 copies of nucleic acid of HPV16. The test results are as Figure 6 shown in Table 9. The reaction temperature is 61°C and the time is 30 minutes.

[0133] Table 9. Cas-ssDNA probe screening results

[0134]

[0135]

[0136] The results showed that the preferred reporting probe was ssDNA3, and the detection time was 21 minutes.

[0137] (6) Screening test of LAMP-CRISPR system Buffer

[0138] Six 10×Buffer formulations suitable for the CRISPR reaction system were selected for screening tests, namely

[0139] Buffer1: 300 mM Tris-HCl, 100 mM (NH 4 ) 2 SO 4 , 500 mM KCL, 20 mM MgSO 4 , 0.1% tween20, PH 8.8;

[0140] Buffer2: 100 mM Tris-HCl, 500 mM (NH 4 ) 2 SO 4 , 1000 mM KCL, 0.1 tween20, PH 8.5, 0.1% tween20, 0.2 M betaine, PH 8.8;

[0141] Buffer3: 100 mM Tris-HCl, 100 mM NaCl, 500 mM (NH 4 ) 2 SO 4 , 500 mM KCL, 0.1% tween20, 200 mM trehalose, PH 8.8;

[0142] Buffer4: 200 mM Tris-HCl, 800 mM (NH 4 ) 2 SO 4 , 500 mM KCL, 0.1% trixon-100, PH 8.5;

[0143] Buffer5: 400 mM Tris-HCl, 500 mM (NH 4 ) 2 SO 4 , 40 mM MgSO 4 , 800 mM KCL, 0.1% trixon-100, PH 8.8;

[0144] Buffer6: 200 mM Tris-HCl, 100 mM NaCl, 500 mM (NH 4 ) 2 SO 4 , 500 mM KCl, 0.1% tween20, pH 8.8. The template is 50 copies of HPV16 nucleic acid, the reaction temperature is 61 °C, and the time is 30 minutes. The test results are as shown in Figure 7 and Table 10.

[0145] Table 10. Screening results of 10×Buffer

[0146] Serial number 10×Buffer 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

[0147] The results show that Buffer6 has the best amplification effect, and the average detection time with a template concentration of 50 copies is 18 minutes, which is better than other Buffer combinations.

[0148] Example 3 Specific detection

[0149] Select 10 other common HPV nucleic acids as templates, with a template of 50 copies, and test by the LAMP-CRISPR method using HPV16 primers and probes. The reaction temperature is 61 °C and the time is 30 minutes. The results are as shown in Figure 8 and Table 11.

[0150] Table 11. Specificity experiment results

[0151] Serial number Template type Pathogen name LAMP-CRISPR result 1 Nucleic acid HPV18 Negative 2 Nucleic acid HPV31 Negative 3 Nucleic acid HPV33 Negative 4 Nucleic acid HPV35 Negative 5 Nucleic acid HPV39 Negative 6 Nucleic acid HPV45 Negative 7 Nucleic acid HPV51 Negative 8 Nucleic acid HPV59 Negative 9 Nucleic acid HPV68 Negative 10 Nucleic acid HPV16 Positive

[0152] The results show that except for HPV16, the LAMP-CRISPR detection results of other pathogen nucleic acids are all negative, indicating that this method performs well in terms of specificity.

[0153] Example 4 Detection limit test

[0154] The detection limit refers to the minimum concentration or minimum amount of the analyte that can be detected from a sample within a given reliability level. The HPV16 nucleic acid template was serially diluted to different concentrations with nuclease-free water, and 5 μL of the template was taken for LAMP-CRISPR testing respectively. The detection time was statistically analyzed. The results are as shown in Figure 9 and Table 12.

[0155] Table 12. Detection sensitivity of the LAMP-CRISPR method for the HPV16 target

[0156]

[0157]

[0158] The results showed that the amplification sensitivity of the LAMP-CRISPR method for detecting HPV16 could stably detect 1 copy without false positives.

[0159] Example 5 False Positive Test

[0160] In the present invention, combining LAMP and CRISPR and adopting an integrated system can significantly reduce false positives in isothermal reactions. Two positive controls and 48 negative controls were selected for verification, and the test results are as Figure 10 shown in Table 13.

[0161] Table 13. False Positive Test Results

[0162] Serial number Template concentration Average detection time 1-2 100 copies 15 minutes 3-50 0 0

[0163] The results showed that there were no false positives in the 48 negative control groups for detecting HPV16 by the LAMP-CRISPR method, avoiding contamination and non-specific amplification of conventional LAMP.

[0164] As mentioned above, only the preferred embodiments of the present invention are described, and there is no limitation in any form and substance to the present invention. It should be pointed out that for those of ordinary skill in the art in this technical field, without departing from the premise of the method of the present invention, several improvements and supplements can still be made, and these improvements and supplements should also be regarded as the protection scope of the present invention. Those skilled in the art, without departing from the spirit and scope of the present invention, when making some equivalent changes, modifications and evolutions using the technical content disclosed above, are all equivalent embodiments of the present invention; at the same time, any equivalent changes, modifications and evolutions made to the above embodiments based on the essential technology of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A LAMP-CRISPR kit, characterized in that: It includes a LAMP primer set, sgRNA, Cas12b protein, Bst enzyme, a reporter probe and a buffer; the LAMP primer set contains F3-4, B3-4, FIP-4, BIP-4, LF-4, LR-4, and their nucleotide sequences are shown in SEQ ID NO:19-24 respectively; the sgRNA specifically binds to the Cas12b protein, and the sgRNA is the sequence shown in SEQ ID No:62; the sequence of the reporter probe ssDNA is shown in SEQ ID NO:

69.

2. A LAMP-CRISPR kit according to claim 1, characterized in that: The buffer comprises: 100-200 mM Tris-HCl, 100-200 mM NaCl, 200-600 mM (NH4)2SO4, 500-1000 mM KCl, 0.1%-5% tween-20, pH 8.5-8.

8.

3. A LAMP-CRISPR kit according to claim 1, characterized in that: The reaction procedure is: 60-65°C for 1 minute, 30-60 cycles; 85-95°C for inactivation for 5-10 minutes.

4. Use of the LAMP-CRISPR kit according to any one of claims 1 to 3 in preparing a kit for detecting high-risk human papillomavirus HPV16.

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