Kit of enterovirus and EV-A71 thereof based on HNB-LAMP coupled CRISPR / Cas12a and use method

Abstract

The invention discloses an enterovirus and EV-A71 typing kit based on an HNB-LAMP (Hepatitis B Narrow Band Loop-Mediated Isothermal Amplification) coupled CRISPR / Cas12a technology and a use method of the kit. According to the invention, the difficulty of simultaneous detection of enterovirus and EV-A71 typing by a traditional detection technology is overcome, an HNB-LAMP coupled CRISPR / Cas12a technology is successfully developed on the premise of not reducing the sensitivity and accuracy of the detection method, simultaneous detection of two targets of enterovirus and EV-A71 typing can be completed within 50 min, the sensitivity of enterovirus is 101 copies / [mu] L, the detection sensitivity of EV-A71 is 100 copies / [mu] L, the detection sensitivity of EV-A71 is 100 copies / [mu] L, the detection sensitivity of EV-A71 is 100 copies / [mu] L, the detection sensitivity of EV-A71 is 100 copies / [mu] L, and the detection sensitivity of EV-A71 is 100 copies / [mu] L. The comprehensive accuracy is 95.7%, and the method has the characteristics of rapidness, high sensitivity and high accuracy.

Description

Technical Field

[0001] The present invention relates to a detection technology, and in particular to a detection method and kit for enterovirus and EV-A71 thereof based on HNB-LAMP coupled with CRISPR / Cas12a, belonging to the field of microbial testing technology. Background Art

[0002] Enterovirus belongs to the genus Enterovirus of the Picornaviridae family. Many enteroviruses can cause human diseases, including hand, foot and mouth disease (HMFD), acute flaccid paralysis, acute hemorrhagic conjunctivitis, aseptic meningitis and myocarditis. Enterovirus is transmitted through the respiratory and digestive tracts, and has the potential to cause large-scale epidemics. Enterovirus infection can occur at any age, and the infection rate in infants under 1 year old is 7 times higher than that in children and adults. Summer and autumn are the main epidemic periods of enterovirus. During the epidemic period of enterovirus infection, the infection rate of newborns is as high as 13%. And it is easy to cause an outbreak in the neonatal ward.

[0003] The International Committee on Taxonomy of Viruses divides enteroviruses into Enterovirus Group A, Group B, Group C, and Group D based on their biological and genetic characteristics. Among them, most severe and fatal cases of enterovirus infection are caused by Enterovirus 71 (EV-A71). EV-A71 is a highly neurotropic virus that can cause serious central nervous system complications in patients. EV-A71 infection has become an important public health issue worldwide and is a key monitoring target for enterovirus-related infections.

[0004] For the detection and identification of enterovirus infection, it is necessary not only to determine whether it is enterovirus infection, but also to focus on whether it is EV-A71. A rapid, sensitive and feasible diagnostic test method that can simultaneously detect and distinguish EVs and EV-A71 is an ideal auxiliary tool for EVs monitoring, which will help to respond to the epidemic in terms of predicting the possible severity of the epidemic, greatly improving the timeliness of early detection and treatment of the disease, and thus initiating appropriate intervention measures.

[0005] Cell culture virus isolation diagnosis is a conventional method for enterovirus detection, but it is time-consuming and cumbersome to operate, far from meeting the needs of rapid diagnosis. Molecular detection methods, such as reverse transcription real-time fluorescent polymerase chain reaction (RT-qPCR), have been developed for clinical detection of enterovirus, but the required thermal cycle equipment is complex, the probe is expensive, nucleic acid extraction is required, and the requirements for operators are high, and it is not suitable for clinical rapid diagnosis.

[0006] Loop-mediated isothermal amplification (LAMP) technology is considered a potential alternative to RT-qPCR. LAMP is more tolerant to inhibitors, and the Bst DNA polymerase used can complete extraction-free nucleic acid amplification under isothermal conditions. In addition, LAMP can be well combined with colorimetric indicators. For example, in the presence of hydroxynaphthol blue (HNB), the positive amplification of LAMP turns sky blue, and the negative amplification turns violet, which is conducive to the bedside visual evaluation of the test results. In recent years, especially with the development of CRISPR / Cas molecular diagnosis, its coupling with LAMP (LAMP-CRISPR / Cas12) has further enhanced the sensitivity and specificity of the test, and is expected to become an ideal solution for POC diagnosis. Under the guidance of CRISPR RNA (crRNA), CRISPR / Cas12 can recognize a large number of LAMP amplification products and trigger their trans-cleavage of the surrounding modified reporter fluorescent group ssDNA to produce a visible fluorescent signal. Nevertheless, the limitation of LAMP-CRISPR / Cas12 is that one tube can only detect one target and cannot screen enterovirus and EV-A71 subtypes at the same time. Summary of the invention

[0007] The purpose of the present invention is to overcome the shortcomings and deficiencies in the prior art. The present invention provides a method and a kit for simultaneous detection of enterovirus and its EV-A71 typing based on HNB-LAMP coupled CRISPR / Cas12a technology, which can realize rapid detection of enterovirus and its EV-A71 typing, and has the characteristics of good specificity, high sensitivity and low detection cost.

[0008] To achieve the above-mentioned purpose and other related purposes, the technical solution provided by the present invention is: a kit for simultaneous detection of enterovirus and EV-A71 typing thereof based on HNB-LAMP coupled with CRISPR / Cas12a technology, the kit comprising:

[0009] Two sets of LAMP amplification primers.

[0010] 5′-UTR-F3 5′-ACGGGACGCTAGTTGTGA-3

[0011] 5′-UTR-B3 5′-ATTGTCACCATAAGCAGCCA-3′

[0012] 5′-UTR-FIP 5′-ATTAGCCGCATTCAGGGGCCACAGGGTGTGAAGAGCTAT-3′

[0013] 5′-UTR-BIP 5′-TGTCGTAACGCGCAAGTCCGGAAACACGGACACCCAAAGT-3′

[0014] 5′-UTR-LF 5′-GGATTCTTATGTAGCCTC-3′

[0015] 5′-UTR-LB 5′-TGGCGGAACCGACT-3′ 。

[0016] VP1-F3 5′-GCGGAGTTCACTTTTGTTGC-3

[0017] VP1-B3 5′-CGCAGGTGACATGAATGGTA-3′

[0018] VP1-FIP 5′-GGCTCCAGGTGGCACAAACATCACACC CACAGGGGAAGT-3′

[0019] VP1-BIP 5′-AGCCAGATTCCAGGGAATCCCTAGGGT CTGACAGCTTGACAA-3′

[0020] VP1-LB 5′- GTATTGGAGCAATTGTGGGACA-3′

[0021] VP1-LF 5′- CATGGCAAACCGCCACCAA-3′

[0022] Design crRNA against EV-A71 VP1 sequence:

[0023] VP1-crRNA: 5′-AATTTCTACTCTTGTAGATTGCCACCTGGAGCCCCTAAGCCA-3′

[0024] ssDNA reporter molecule: FAM-TTATT-BHQ1

[0025] The kit also includes LAMP reagent components: 1×WarmStart LAMP mixture, 1.6μM inner primers 5′-UTR-FIP, VP1-FIP and 5′-UTR-BIP, VP1-BIP, 0.2μM outer primers 5′-UTR-F3, VP1-F3 and 5′-UTR-B3, VP1-B3, 0.16μM loop primers 5′-UTR-LF, VP1-LF and 5′-UTR-LB, VP1-LB, 120μM HNB, CRISPR reagent components: 2×rCutSmart buffer, 100nM AsCas12a, 200nM crRNA, 1000nM DNA reporter molecule.

[0026] The detection method comprises the following steps:

[0027] Step 1: Sample preparation and configuration of the detection system. After sample collection, place the sample in the release preservation solution;

[0028] Step 2: Using the enterovirus nucleic acid sample in the sample to be tested as a template, incubate at 65°C for 40 minutes, and use loop-mediated isothermal amplification (LAMP) technology and specific primers to amplify the target fragment to obtain an amplified product;

[0029] Step 3: Determine the test result of enterovirus by colorimetry under natural light. If it is sky blue, enterovirus is present; if it is violet, enterovirus is not present.

[0030] Step 4: Mix the amplified product obtained in step 1 with the CRISPR / Cas12a reagent, incubate at 37°C for 10 minutes, and perform a specific cleavage reaction mediated by crRNA;

[0031] Step 5: Use blue light to excite the reaction system after the reaction in step 3, observe the fluorescence color development, and determine whether EV-A71 is contained. If it is red fluorescence, EV-A71 is not present, and if it is green fluorescence, EV-A71 is present.

[0032] Beneficial effects:

[0033] The detection objects of the present invention include: common enteroviruses (EVs) and enterovirus type 71 (EV-A71). First, LAMP primers are designed based on the conservative sequence of enterovirus 5′-UTR for enterovirus detection, LAMP primers, CRISPR crRNA sequences and CRISPR reporter molecules are designed based on the VP1 sequence of enterovirus type 71 (EV-A71) for EV-A71 typing detection, and CRISPR reporter molecules are designed. At the same time, a detection system for enterovirus and its 71 typing is constructed based on this design. The detection method includes configuring the detection system, LAMP reaction and result interpretation, CRISPR reaction and result interpretation.

[0034] The present invention uses HNB as an indicator to develop an RT-LAMP-CRISPR / Cas12a detection method for simultaneously detecting EVs and EV-A71. The present invention combines EV-A71, EVs genomes with RT-LAMP amplification, interprets the results, then adds CRISPR / Cas12a reagents for trans-cleavage determination, and finally performs colorimetric analysis on a blue light transilluminator.

[0035] (1) Dual identification of target viruses: The present invention can use a single sample in a single tube to simultaneously complete the efficient identification of two targets, enterovirus and its EV-A71 type, breaking the current limitations of single detection and single target, and has great application value.

[0036] (2) High sensitivity: For EVs detection, HNB-RT-LAMP assay was performed using serially diluted EVs 5′-UTR genes, and the colorimetric Lod under natural light could reach 10 1 copies / μL( Figure 4 ), which is consistent with the sensitivity of conventional LAMP assay. For the detection of EV-A71, the HNB-CRISPR assay was performed on the serially diluted EV-A71 VP1 gene. As the RNA concentration decreased, the product transitioned from green to red under blue light, and the Lod could reach 10 0 The 6.75 copies / μL of SHERLOCK, the classic CRISPR / Cas diagnostic system, is more than six times higher.

[0037] (3) High precision: The LAMP primers for EVs in the present invention are designed based on the highly conserved 5'-UTR sequence of EVs. The VP1 gene of EV-A71 (GenBank accession number: GQ855293.1) was used to design the LAMP-specific primers for EV-A71 using Primer Explorer V5. The crRNA was designed based on the VP1 gene fragment amplified by the 6 primers designed independently and the PAM site requirements of CRISPRCRISPR / Cas12a. While realizing the detection of EVs by LAMP technology, its detection product can be directly used for the detection of EV-A71. At the same time, the present invention develops a "One-tube" strategy to achieve simultaneous detection of EVs and EV-A71, avoid aerosol contamination caused by opening the lid in the middle, reduce the false positive rate of EV-A71 detection, and greatly improve the accuracy and specificity of the detection method.

[0038] (4) Rapid identification of target viruses: Dual identification of enterovirus and its EV-A71 type can be completed within 50 minutes, which is much faster than the 1-2 hours required by traditional molecular detection technology PCR, greatly satisfying the rapid screening before clinical precision medication.

[0039] (5) Easy to use: The test results are fluorescently visible, the technical threshold is low, and no complex machines are required. Users can complete high-precision testing at home, which is convenient for screening and prevention during the virus pandemic. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 This is a schematic diagram of the HNB-LAMP coupled CRISPR / Cas12a detection principle in the present invention;

[0041] Figure 2 This is the design diagram of EV-A71 LAMP primer and crRNA in the present invention (a: schematic diagram b: specific sequence diagram)

[0042] Figure 3 The results of the LAMP primer and crRNA test in the present invention (a: EVs LAMP primer test diagram b: EVs + EV-A71 LAMP primer test diagram c: crRNA test)

[0043] Figure 4 This is the test result diagram of the system in the present invention (a: HNB-RT-LAMP system test diagram b: HNB-CRISPR system test diagram)

[0044] Figure 5 This is the sensitivity test result diagram of the present invention (a: HNB-RT-LAMP sensitivity test diagram b: HNB-CRISPR sensitivity test diagram)

[0045] Figure 6The specificity test result diagram of the present invention (a: EVs LAMP primer test result diagram b: EV-A71 LAMP primer test result diagram c: crRNA test result diagram)

[0046] Figure 7 This is the clinical sample test result diagram of the present invention DETAILED DESCRIPTION

[0047] Example 1, Design and Synthesis of Primers, crRNA and Reporter Molecules

[0048] EVs primers were designed based on the highly conserved 5′-UTR sequences of enteroviruses used in previous studies. The VP1 gene of EV-A71 (GenBank accession No: GQ855293.1) was used to design six LAMP-specific primers for amplifying EV-A71 using the free software PrimerExplorer V5 ( http: / / primerexplorer.jp / lampv5e / index.html ), and crRNA was designed based on the VP1 gene fragment amplified by the six primers designed independently and the PAM site requirements of CRISPRCRISPR / Cas12a (see Figure 1 ), while realizing the detection of EVs by LAMP technology, the amplified product can be directly used for the detection of EV-A71.

[0049] Table 1 shows the primers, crRNA and reporter sequences:

[0050]

[0051] Example 2 Reaction system construction

[0052] The HNB-CRISPR detection system consists of an equal volume of the HNB-RT-LAMP detection system and the CRISPR / Cas12a trans-cleavage system. The LAMP reagent components include: 1×WarmStart LAMP mixture, 1.6μM inner primers 5′-UTR-FIP, VP1-FIP and 5′-UTR-BIP, VP1-BIP, 0.2μM outer primers 5′-UTR-F3, VP1-F3 and 5′-UTR-B3, VP1-B3, 0.16μM loop primers 5′-UTR-LF, VP1-LF and 5′-UTR-LB, VP1-LB, 120μM HNB,

[0053] CRISPR reagent components: 2×rCutSmart buffer, 100nM AsCas12a, 200nM crRNA, 1000nM DNA reporter molecule.

[0054] WarmStart LAMP mixture and rCutSmart buffer were purchased from New England Biolabs (NEB). HNB, TAE, and agarose were purchased from Sigma-Aldrich (Shanghai), and AsCas12a was purchased from Shanghai Tolo Harbor Biotech Co., Ltd. DEPC water and DNA marker were purchased from Nanjing Novozyme Medical Technology Co., Ltd. All sequences, including RNA templates, primers, crRNA, and ssDNA reporter molecules, were synthesized by Beijing Qingke Biotechnology Co., Ltd. Sample release preservation solution was purchased from Zhenshi Biotechnology.

[0055] Specific steps for using the kit

[0056] (1) Sample preparation and configuration of detection system

[0057] (2) LAMP reaction

[0058] (3) Interpretation of EVs results

[0059] (3) CRISPR response

[0060] (4) Interpretation of EV-A71 results

[0061] (1) The sample preparation and configuration system is as follows:

[0062] Sample preparation: Sample collection is done by inserting a swab into the nostril parallel to the palate, leaving the swab for a few seconds to absorb the secretion sample, and placing the sample in a release preservation solution for lysis and release.

[0063] Prepare 10μL LAMP system: 1×WarmStart LAMP mixture, 1.6μM inner primers 5′-UTR-FIP, VP1-FIP and 5′-UTR-BIP, VP1-BIP, 0.2μM outer primers 5′-UTR-F3, VP1-F3 and 5′-UTR-B3, VP1-B3, 0.16μM loop primers 5′-UTR-LF, VP1-LF and 5′-UTR-LB, VP1-LB, 120μM HNB, 2μL sample;

[0064] Prepare 10μL CRISPR system: 2×rCutSmart buffer, 100nM AsCas12a, 200nM crRNA, 1000nM DNA reporter molecule, and place the LAMP system at the bottom of the ep tube and the CRISPR system at the top of the ep tube.

[0065] (2) LAMP reaction: Place the prepared LAMP system in a constant temperature incubator at 65°C for 40 min.

[0066] (3) Interpretation of EVs results: Observe the LAMP reaction product under natural light. If it is sky blue, enterovirus is present; if it is violet, enterovirus is not detected.

[0067] (4) CRISPR reaction: Invert the CRISPR reaction tube to mix the LAMP reaction product and the CRISPR system, and place it in a constant temperature incubator at 37°C for 10 minutes.

[0068] (5) The EV-A71 result was interpreted as follows: the CRISPR reaction product was observed under blue light. If it was green fluorescence, EV-A71 was detected; if it was red fluorescence, EV-A71 was not detected.

[0069] Example 3 Feasibility Test

[0070] (1) LAMP primer feasibility test

[0071] To test the usability of LAMP primers, the EV-A71 primer set was used alone to perform HNB-RT-LAMP reaction. The tube containing the EV-A71 genome appeared sky blue under natural light, distinguishing it from other EVs and the negative control (NTC). The LAMP amplification band of EV-A71 was also observed by agarose gel electrophoresis (see Figure 3 ), indicating that the EV-A71 primer set can successfully amplify the VP1 gene.

[0072] The EV-A71 primer set and the EVs universal primer set were mixed and multiple HNB-RT-LAMP reactions were performed. Positive signals were observed for EV-A71 and other EVs (see Figure 3 ).

[0073] The mixed primer set of these two pairs of primers will be used in the subsequent HNB-CRISPR system.

[0074] (2) CRISPR crRNA feasibility test

[0075] Next, to test the feasibility of crRNA for CRISPR / Cas12a assay of EV-A71, the genome of EV-A71, EVs was first amplified by RT-LAMP, and then CRISPR / Cas12a reagent was added for trans-cleavage assay.

[0076] The results showed that the assay using RT-LAMP products was very rapid, and the fluorescent signal of the EV-A71 group could be observed within 10 minutes, which could be clearly distinguished from EVs (see Figure 4 ). The above results confirmed the feasibility of the primer set and crRNA.

[0077] (3) Feasibility test of HNB-CRISPR system

[0078] The present invention develops a "One-tube" strategy to achieve simultaneous detection of EVs and EV-A71, avoiding aerosol contamination caused by opening the lid in the middle. 2+ Produce pyrophosphatase (Mg2P2O7) precipitation. EVs can be detected by colorimetric analysis under natural light.

[0079] After mixing the HNB-RT-LAMP and CRISPR / Cas12a systems, the amplified products of EV-A71 will be detected by the CRISPR / Cas12a system, generating fluorescent signals under blue light excitation to detect EV-A71.

[0080] To test the feasibility of using EV-A71 and other EVs to test HNB-CRISPR, after RT-LAMP amplification, EV-A71 and other EVs appeared sky blue, which was distinguished from the violet color of NTC. At the same time, under blue light excitation, HNB can be excited to red, but the red fluorescence of positive amplification is weaker than that of NTC (see Figure 4 ).

[0081] After 10 minutes of CRISPR / Cas12a trans-cleavage assay, EV-A71 was excited to emit green fluorescence under blue light excitation, while other EVs still appeared red, thus distinguishing EV-A71 (see Figure 4 This demonstrates the feasibility of HNB-CRISPR for the determination of EVs and EV-A71 in one tube.

[0082] Example 4 Sensitivity Test

[0083] To test the sensitivity of the HNB-CRISPR system, for EVs detection, serially diluted EVs 5′-UTR genes were used to perform HNB-RT-LAMP assay using the kit in Example 2. The colorimetric Lod under natural light could reach 10 1 copies / μL (see Figure 5 a), which is consistent with the sensitivity of conventional LAMP assay.

[0084] For the detection of EV-A71, the kit in Example 2 was used to perform HNB-CRISPR assay on the serially diluted EV-A71 VP1 gene. As the RNA concentration decreased, the product changed from green to yellow under blue light, and the Lod could reach 10 0 copies / μL (see Figure 5 b).

[0085] Example 5 Specificity Test

[0086] At the same time, we used the kit in Example 2 to test the specificity of EVs and EV-A71 detection. We synthesized five viral genes, including respiratory syncytial virus (RSV), influenza A virus (Influenza A), influenza B virus (Influenza A), human metapneumovirus (HMPV), and human rhinovirus (HRV) to test the specificity of LAMP primers for EVs and VP genes. The detection results of the five viruses were all violet and undetectable (see Figure 6 a), The results showed that the LAMP primer set for EVs and EV-A71 had excellent specificity.

[0087] We also selected 5 different EV-A71 virus fragments other than the VP1 gene to test the specificity of crRNA. The detection results of the 5 fragments under blue light irradiation were all red and undetectable (see Figure 6 b) The results showed that the crRNA designed for the VP1 gene fragment has excellent specificity.

[0088] Example 6 Clinical sample testing

[0089] The Qinhuai District Center for Disease Control and Prevention provided 94 nasopharyngeal swab samples from children under 12 years old suspected of HFMD. EVs and EV-A71 were tested using HNB-CRISPR. Standard RT-qPCR tests were also performed.

[0090] 94 clinical samples were used for HNB-CRISPR performance testing. These samples were nasopharyngeal swab samples from children under 12 years old suspected of having hand, foot and mouth disease provided by the CDC. Overall, 79 samples that were originally tested positive for EVs after HNB-RT-LAMP were further tested for CRISPR / Cas12a, and finally 3 samples were tested positive for EV-A71 (see Figure 7 HNB-CRISPR detected 84.0% EVs positivity and 3.8% EV-A71 positivity in children suspected of HZMD (see Figure 7 ). The accuracy of HNB-CRISPR for EVs and EV-A71 typing was 95.7% and 100%, respectively, indicating that HNB-CRISPR has good detection performance.

[0091] The above is only a preferred embodiment of the present invention and does not limit the present invention in any form. Although the present invention has been disclosed as above in the preferred embodiment, it is not used to limit the present invention. Any technician familiar with the art can make many possible changes and modifications to the technical solution of the present invention by using the methods and technical contents disclosed above without departing from the spirit and technical solution of the present invention, or modify it into an equivalent embodiment of equivalent changes. Therefore, any simple modification, equivalent replacement, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still falls within the scope of protection of the technical solution of the present invention.

Claims

1. A kit for detecting enterovirus and EV-A71 typing thereof based on HNB-LAMP coupled with CRISPR / Cas12a technology, characterized in that: It includes an HNB-CRISPR system suitable for the detection of enterovirus and its 71 typing, wherein the HNB-CRISPR system includes a LAMP primer set 1 designed for the enterovirus 5′-UTR conservative sequence, a LAMP primer set 2 designed for the EV-A71 VP1 sequence, and a crRNA and ssDNA reporter molecule designed for the EV-A71 VP1 sequence; The LAMP primer set 1 designed for the enterovirus 5′-UTR conservative sequence comprises primers with nucleotide sequences as shown in SEQ ID NO.1-6: The nucleotide sequence of 5′-UTR-F3 is shown in SEQ ID NO.1; The nucleotide sequence of 5′-UTR-B3 is shown in SEQ ID NO.2; The nucleotide sequence of 5′-UTR-FIP is shown in SEQ ID NO.3; The nucleotide sequence of 5′-UTR-BIP is shown in SEQ ID NO.4; The nucleotide sequence of 5′-UTR-LF is shown in SEQ ID NO.5; The nucleotide sequence of 5′-UTR-LB is shown in SEQ ID NO.6; The LAMP primer set 2 designed for the EV-A71 VP1 sequence comprises nucleotide sequences such as SEQ ID NO.7-12 as follows: The nucleotide sequence of VP1-F3 is shown in SEQ ID NO.7; The nucleotide sequence of VP1-B3 is shown in SEQ ID NO.8; The nucleotide sequence of VP1-FIP is shown in SEQ ID NO.9; The nucleotide sequence of VP1-BIP is shown in SEQ ID NO.10; The nucleotide sequence of VP1-LB is shown in SEQ ID NO.11; The nucleotide sequence of VP1-LF is shown in SEQ ID NO.12; The VP1-crRNA nucleotide sequence designed for the EV-A71 VP1 sequence is shown in SEQ ID NO.13; ssDNA reporter, FAM-TTATT-BHQ1.

2. The enterovirus and EV-A71 typing detection kit based on HNB-LAMP coupled CRISPR / Cas12a technology according to claim 1, characterized in that: The LAMP reaction reagent components include the following: 1×WarmStart LAMPmixture, 1.6 μM inner primers 5′-UTR-FIP and 5′-UTR-BIP as shown in SEQ ID NO.3 and SEQ ID NO.4, 0.2 μM outer primers 5′-UTR-FIP F3 and 5′-UTR-FIP B3 as shown in SEQ ID NO.1 and SEQ ID NO.2, 0.16 μM loop primers 5′-UTR- LF and 5′-UTR-LB as shown in SEQ ID NO.5 and SEQ ID NO.6, 1.6 μM inner primers VP1-FIP and VP1-BIP as shown in SEQ ID NO.9 and SEQ ID NO.10, 0.2 μM outer primers VP1-F3 and VP1-B3 as shown in SEQ ID NO.7 and SEQ ID NO.8, 0.16 μM loop primers VP1-LF and VP1-LB as shown in SEQ ID NO.11 and SEQ ID NO. As shown in NO.12, 120μM HNB (hydroxynaphthol blue).

3. The enterovirus and EV-A71 typing detection kit based on HNB-LAMP coupled CRISPR / Cas12a technology according to claim 2, characterized in that: CRISPR reaction reagent components: 100nM crRNA as shown in SEQ ID NO.13, 50nM AsCas12a protein, 1×rCutSmart buffer, 500nM DNA reporter.

4. The method for using the enterovirus and EV-A71 typing kit based on HNB-LAMP coupled with CRISPR / Cas12a according to claim 1, characterized in that: The steps include: (1) Configure the detection system, including the HNB-LAMP system for enterovirus detection and the CRISPR system for EV-A71 detection. (2) LAMP reaction; (3) Interpretation of enterovirus results; (3) CRISPR response; (4) Interpretation of EV-A71 results.

5. The method for using the enterovirus and EV-A71 typing kit based on HNB-LAMP coupled with CRISPR / Cas12a according to claim 4, characterized in that: (1) Prepare 10μL LAMP system: 1×WarmStart LAMP mixture, 1.6μM inner primers 5′-UTR-FIP, VP1-FIP and 5′-UTR-BIP, VP1-BIP, 0.2μM outer primers 5′-UTR-F3, VP1-F3 and 5′-UTR-B3, VP1-B3, 0.16μM loop primers 5′-UTR-LF, VP1-LF and 5′-UTR-LB, VP1-LB, 120μM HNB, 2μL sample, prepare 10μL CRISPR system: 2×rCutSmart buffer, 100nM AsCas12a, 200nM crRNA, 1000nM DNA reporter molecule, and place the LAMP system at the bottom of the ep tube and the CRISPR system at the top of the ep tube; (2) LAMP reaction: Place the prepared LAMP system in a constant temperature incubator at 65°C for 40 min; (3) Interpretation of LAMP reaction results: Observe the LAMP reaction product under natural light. If it is sky blue, enterovirus is present; if it is violet, enterovirus is not detected. (4) CRISPR reaction: Invert the ep tube to mix the LAMP reaction product with the CRISPR system, and place it in a constant temperature incubator at 37°C for 10 min; (5) Interpretation of EV-A71 results in the CRISPR reaction: observe the CRISPR reaction product under blue light. If it is green fluorescence, EV-A71 is detected; if it is red fluorescence, EV-A71 is not detected.

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