A kit for enterovirus and ev-a71 based on hnb-lamp coupled crisper / cas12a and a method of use

Abstract

The application discloses an enterovirus and EV-A71 typing kit based on HNB-LAMP coupling CRISPR / Cas12a technology and a use method. The application overcomes the problem that traditional detection technology cannot simultaneously detect enterovirus and EV-A71 typing, successfully develops HNB-LAMP coupling CRISPR / Cas12a technology under the premise that the sensitivity and accuracy of the detection method are not reduced, can simultaneously detect two targets of enterovirus and EV-A71 typing within 50 min, the sensitivity of enterovirus is 10 1 copies / μL, the detection sensitivity of EV-A71 is 10 0 copies / μL, and the comprehensive accuracy is 95.7%, and the kit has the characteristics of rapidness, high sensitivity and high accuracy.

Description

Technical Field

[0001] This invention relates to a detection technology, and more particularly to a method and kit for detecting enteroviruses and their EV-A71 based on HNB-LAMP coupled with CRISPR / Cas12a. It belongs to the field of microbial testing technology. Background Technology

[0002] Enteroviruses belong to the genus Enterovirus in the family Picornaviridae. Many enteroviruses can cause human diseases, including hand-foot-and-mouth disease (HMFD), acute flaccid paralysis, acute hemorrhagic conjunctivitis, aseptic meningitis, and myocarditis. Enteroviruses are transmitted through the respiratory and digestive tracts and pose a potential threat of large-scale disease outbreaks. Enterovirus infection can occur at any age, with infants under one year old having a 7 times higher infection rate than children and adults. Summer and autumn are the main epidemic seasons for enteroviruses, during which the infection rate in newborns can reach as high as 13%. They are also prone to causing outbreaks in neonatal wards.

[0003] The International Committee on Taxonomy of Viruses classifies enteroviruses into groups A, B, C, and D based on their biological and genetic characteristics. Among these, severe and fatal cases of enterovirus infection are mostly caused by Enterovirus 71 (EV-A71). EV-A71 is a highly neurotropic virus that can cause serious central nervous system complications. EV-A71 infection has become a significant global public health issue and is a key focus of surveillance for enterovirus-related infections.

[0004] For the detection and identification of enterovirus infections, it is necessary not only to determine whether it is an enterovirus infection, but also to focus on whether it is EV-A71. Rapid, sensitive, and feasible diagnostic methods that can simultaneously detect and differentiate between EVs and EV-A71 are ideal auxiliary tools for EV surveillance. This helps in predicting the potential severity of an outbreak and informing appropriate epidemic response, greatly improving the timeliness of early detection and treatment, thereby initiating appropriate interventions.

[0005] Cell culture-based virus isolation and diagnosis is a routine method for enterovirus detection, but it is time-consuming and cumbersome, 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 enteroviruses, but they require complex thermal cycling equipment, expensive probes, nucleic acid extraction, and highly skilled operators, making them unsuitable for rapid clinical diagnosis.

[0006] Loop-mediated isothermal amplification (LAMP) technology is considered a potential alternative to RT-qPCR. LAMP is more resistant to inhibitors, and the Bst DNA polymerase used can perform extraction-free nucleic acid amplification under isothermal conditions. Furthermore, LAMP binds well to colorimetric indicators; for example, in the presence of hydroxynaphthol blue (HNB), positive LAMP amplification turns sky blue, and negative amplification turns violet, facilitating point-of-care visual evaluation of test results. In recent years, especially with the development of CRISPR / Cas molecular diagnostics, its coupling with LAMP (LAMP-CRISPR / Cas12) has further enhanced the sensitivity and specificity of detection, and is expected to become an ideal solution for point-of-care diagnostics. Guided by CRISPR RNA (crRNA), CRISPR / Cas12 can recognize a large number of LAMP amplification products and trigger trans-cleavage of the surrounding modified reporter fluorescent group ssDNA, generating a visible fluorescent signal. Nevertheless, the limitation of LAMP-CRISPR / Cas12 is that it can only detect one target per tube and cannot simultaneously screen for enteroviruses and EV-A71 subtypes. Summary of the Invention

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

[0008] To achieve the above and other related objectives, the technical solution provided by this invention is: a kit for simultaneous detection of enteroviruses and their EV-A71 typing 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

[0013] 5′-ATTAGCCGCATTCAGGGGCCACAGGGTGTGAAGAGCTAT-3′

[0014] 5′-UTR-BIP

[0015] 5′-TGTCGTAACGCGCAAGTCCGGAAACACGGACACCCAAAGT-3′

[0016] 5′-UTR-LF 5′-GGATTCTTATGTAGCCTC-3′

[0017] 5′-UTR-LB 5′-TGGCGGAACCGACT-3′ 。

[0018] VP1-F3 5′-GCGGAGTTCACTTTTGTTGC-3

[0019] VP1-B3 5′-CGCAGGTGACATGAATGGTA-3′

[0020] VP1-FIP 5′-GGCTCCAGGTGGCACAAACATCACACC CACAGGGGAAGT-3′

[0021] VP1-BIP

[0022] 5′-AGCCAGATTCCAGGGAATCCCTAGGGT CTGACAGCTTGACAA-3′

[0023] VP1-LB 5′- GTATTGGAGCAATTGTGGGACA-3′

[0024] VP1-LF 5′- CATGGCAAACCGCCACCAA-3′

[0025] Design crRNA targeting the EV-A71 VP1 sequence:

[0026] VP1-crRNA:

[0027] 5′-AATTTCTACTCTTGTAGATTGCCACCTGGAGCCCCTAAGCCA-3′

[0028] ssDNA reporter molecule: FAM-TTATT-BHQ1

[0029] 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, and CRISPR reagent components: 2×rCutSmart buffer, 100nM AsCas12a, 200nM crRNA, and 1000nM DNA reporter molecule.

[0030] The detection method includes the following steps:

[0031] Step 1: Sample preparation and configuration of the detection system. After sample collection, place the sample in the release preservation solution.

[0032] Step 2: Using the enterovirus nucleic acid sample in the test sample as a template, incubate at 65℃ for 40 min, and amplify the target fragment using loop-mediated isothermal amplification (LAMP) technology and specific primers to obtain the amplification product;

[0033] Step 3: Under natural light, determine the test results for enteroviruses using a colorimetric method. If the color is sky blue, enteroviruses are present; if the color is violet, enteroviruses are not present.

[0034] Step 4: Mix the amplification product obtained in Step 1 with CRISPR / Cas12a reagent, incubate at 37℃ for 10 min, and perform a specific cleavage reaction mediated by crRNA.

[0035] Step 5: Excite the reaction system after step 3 with blue light and observe the fluorescence color development to determine whether EV-A71 is present. If it is red fluorescence, EV-A71 is not present; if it is green fluorescence, EV-A71 is present.

[0036] Beneficial effects:

[0037] The detection targets of this invention include common enteroviruses (EVs) and enterovirus type 71 (EV-A71). First, LAMP primers were designed based on the conserved 5′-UTR sequence of enteroviruses for enterovirus detection. Then, LAMP primers, CRISPR crRNA sequences, and CRISPR reporter molecules were designed based on the VP1 sequence of enterovirus type 71 (EV-A71) for EV-A71 genotyping detection. A CRISPR reporter molecule was also designed. Based on this design, a detection system for enteroviruses and their type 71 was constructed. The detection methods include configuring the detection system, LAMP reaction and result interpretation, and CRISPR reaction and result interpretation.

[0038] This invention utilizes HNB as an indicator to develop an RT-LAMP-CRISPR / Cas12a detection method for the simultaneous detection of EVs and EV-A71. The method involves first performing RT-LAMP amplification on the genomes of EV-A71 and EVs, interpreting the results, then adding CRISPR / Cas12a reagent for trans-cleavage assay, and finally performing colorimetric analysis using a blue light transilluminator.

[0039] (1) Dual identification of target viruses: This invention can use a single sample to simultaneously and efficiently identify two targets, enterovirus and EV-A71, in a single tube, breaking the current limitation of single detection and single target, and has great application value.

[0040] (2) High sensitivity: For EV detection, using serially diluted EVs 5′-UTR gene for HNB-RT-LAMP assay, the colorimetric LOD under natural light can reach 10. 1 copies / μL ( Figure 4 This sensitivity is consistent with that of conventional LAMP assays. For EV-A71 detection, HNB-CRISPR assays were performed on serially diluted EV-A71 VP1 genes. As RNA concentration decreased, the product transitioned from green to red under blue light, with a Lod value reaching 10. 0 The number of copies / μL is more than six times higher than that of the classic CRISPR / Cas diagnostic system SHERLOCK (6.75 copies / μL).

[0041] (3) High precision: The LAMP primers for EVs in this invention are designed based on the highly conserved 5'-UTR sequence of EVs. LAMP-specific primers for EV-A71 are designed using Primer Explorer V5, utilizing the VP1 gene of EV-A71 (GenBank accession number: GQ855293.1). crRNA is designed based on the VP1 gene fragment amplified by the six self-designed primers and the PAM site requirements of CRISPRCRISPR / Cas12a. While achieving LAMP technology for EV detection, the detection product can be directly used for EV-A71 detection. Simultaneously, this invention develops a "One-tube" strategy to achieve simultaneous detection of EVs and EV-A71, avoiding aerosol contamination caused by opening the lid midway, reducing the false positive rate of EV-A71 detection, and greatly improving the accuracy and specificity of the detection method.

[0042] (4) Rapid identification of target viruses: The dual identification of enteroviruses and their EV-A71 type can be completed within 50 minutes, which is far longer than the 1-2 hours of traditional molecular detection technology PCR, greatly satisfying the rapid screening before precise clinical medication.

[0043] (5) Simple and easy to use: The test results are fluorescent and visible, with low technical threshold and no need for complicated machines. Users can complete high-precision testing at home, which is convenient for screening and prevention during the pandemic. Attached Figure Description

[0044] Figure 1 This is a schematic diagram of the HNB-LAMP coupled CRISPR / Cas12a detection principle in this invention;

[0045] Figure 2 This is a diagram of the EV-A71 LAMP primers and crRNA design in this invention (a: schematic diagram; b: actual sequence diagram).

[0046] Figure 3 The images show the LAMP primer and crRNA test results from this invention (a: EVsLAMP primer test image; b: EVs+EV-A71 LAMP primer test image; c: crRNA test).

[0047] Figure 4 These are the system test results diagrams from this invention (a: HNB-RT-LAMP system test diagram; b: HNB-CRISPR system test diagram).

[0048] Figure 5 These are the sensitivity test results of the present invention (a: HNB-RT-LAMP sensitivity test graph; b: HNB-CRISPR sensitivity test graph).

[0049] Figure 6The diagram shows the specificity test results of this invention (a: EVs LAMP primer test results; b: EV-A71 LAMP primer test results; c: crRNA test results).

[0050] Figure 7 This is a diagram of the clinical sample test results of the present invention. Detailed Implementation

[0051] Example 1: Design and synthesis of primers, crRNA, and reporter molecules

[0052] Primers for EVs were designed based on the highly conserved 5′-UTR sequence of enteroviruses used in previous studies. Using the VP1 gene of EV-A71 (GenBank accession No: GQ855293.1), six LAMP-specific primers for amplifying EV-A71 were designed using the free software PrimerExplorer V5 (http: / / primerexplorer.jp / lampv5e / index.html). crRNA was designed based on the VP1 gene fragment amplified by the six self-designed primers and the PAM site requirements of CRISPRCRISPR / Cas12a (see [link to crRNA design]). Figure 1 This technology enables the detection of EVs using LAMP, while the amplified products can be directly used for the detection of EV-A71.

[0053] Table 1 lists the sequences of the primers, crRNA, and reporter molecules:

[0054]

[0055] Example 2: Construction of the reaction system

[0056] The HNB-CRISPR detection system consists of an equal volume of HNB-RT-LAMP detection system and a CRISPR / Cas12a reverse cleavage system. The LAMP reagent components are: 1×WarmStart LAMP mixture, 1.6 μM inner primers 5′-UTR-FIP, VP1-FIP, 5′-UTR-BIP, VP1-BIP, 0.2 μM outer primers 5′-UTR-F3, VP1-F3, 5′-UTR-B3, VP1-B3, 0.16 μM loop primers 5′-UTR-LF, VP1-LF, 5′-UTR-LB, VP1-LB, and 120 μM HNB.

[0057] CRISPR reagent components: 2×rCutSmart buffer, 100nM AsCas12a, 200nM crRNA, 1000nM DNA reporter molecule.

[0058] 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 Tulugang Biotechnology Co., Ltd. DEPC water and DNA markers were purchased from Nanjing Novizan Medical Technology Co., Ltd. All sequences, including RNA template, primers, crRNA, and ssDNA reporter molecules, were synthesized by Beijing Qingke Biotechnology Co., Ltd. Sample release and preservation solution was purchased from Zhenshi Biosciences.

[0059] Specific steps for using the reagent kit

[0060] (1) Sample preparation and configuration of detection system

[0061] (2) LAMP reaction

[0062] (3) Interpretation of EVs results

[0063] (3) CRISPR reaction

[0064] (4) EV-A71 result interpretation

[0065] (1) The sample preparation and configuration system is as follows:

[0066] Sample preparation: Sample collection involves inserting a swab into the nostril parallel to the palate, leaving the swab in place for a few seconds to absorb the secretion sample, and then placing the sample into a release preservation solution for lysis and release.

[0067] Prepare a 10 μL LAMP system: 1×WarmStart LAMP mixture, 1.6 μM inner primers 5′-UTR-FIP, VP1-FIP and 5′-UTR-BIP, VP1-BIP, 0.2 μM outer primers 5′-UTR-F3, VP1-F3 and 5′-UTR-B3, VP1-B3, 0.16 μM loop primers 5′-UTR-LF, VP1-LF and 5′-UTR-LB, VP1-LB, 120 μM HNB, 2 μL sample;

[0068] Prepare a 10 μL CRISPR system: 2×rCutSmart buffer, 100 nM AsCas12a, 200 nM crRNA, and 1000 nM DNA reporter molecule. Place the LAMP system at the bottom of the ep tube and the CRISPR system at the top of the ep tube.

[0069] (2) The LAMP reaction is as follows: the prepared LAMP system is placed in a constant temperature incubator and incubated at 65°C for 40 min.

[0070] (3) EVs results interpretation: Observe the LAMP reaction product under natural light. If it is sky blue, enterovirus is present. If it is violet, enterovirus is not detected.

[0071] (4) The CRISPR reaction is as follows: Invert the CRISPR reaction 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.

[0072] (5) EV-A71 results interpretation: 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.

[0073] Example 3 Feasibility Test

[0074] (1) Feasibility test of LAMP primers

[0075] To test the availability of LAMP primers, the EV-A71 primer set alone was used for HNB-RT-LAMP reactions. Tubes containing the EV-A71 genome appeared sky blue under natural light, distinguishing them from other EVs and the negative control (NTC). Agarose gel electrophoresis also observed LAMP amplification bands for EV-A71 (see...). Figure 3 This indicates that the EV-A71 primer set can successfully amplify the VP1 gene.

[0076] When the EV-A71 primer set and the universal primer set for EVs were mixed, a multiplex HNB-RT-LAMP reaction was performed. Positive signals were observed for EV-A71 along with other EVs (see [link to HNB-RT-LAMP reaction]). Figure 3 ).

[0077] The mixed primer set of these two primer pairs will be used in the subsequent HNB-CRISPR system.

[0078] (2) Feasibility test of CRISPR crRNA

[0079] Next, to test the feasibility of using crRNA for CRISPR / Cas12a assays of EV-A71, the genomes of EV-A71 and EVs were first amplified by RT-LAMP, and then CRISPR / Cas12a reagents were added for trans-cleavage assays.

[0080] The results showed that the assay using RT-LAMP products was very rapid, and the fluorescence signal of the EV-A71 group could be observed within 10 minutes, which could be clearly distinguished from EVs (see [link to RT-LAMP assay]). Figure 4 The above results confirm the feasibility of the primer set and crRNA.

[0081] (3) Feasibility test of HNB-CRISPR system

[0082] This invention develops a "One-tube" strategy to simultaneously detect EVs and EV-A71, avoiding aerosol contamination caused by opening the lid midway. After LAMP amplification, with Mg... 2+ Pyrophosphatase (Mg2P2O7) precipitate is formed. EVs are detected by colorimetric analysis under natural light.

[0083] After mixing HNB-RT-LAMP and CRISPR / Cas12a systems, the amplified products of EV-A71 will be detected by the CRISPR / Cas12a system, generating a fluorescence signal under blue light excitation to detect EV-A71.

[0084] To test the feasibility of using EV-A71 and other EVs for HNB-CRISPR, after RT-LAMP amplification, EV-A71 and other EVs exhibited a sky-blue color, distinguishing them from the violet color of NTC. Simultaneously, under blue light excitation, HNB could be excited to red, but the red fluorescence of positive amplification was weaker than that of NTC (see...). Figure 4 ).

[0085] After 10 minutes of CRISPR / Cas12a trans-cleavage assay, EV-A71 was excited to green fluorescence under blue light excitation, while other EVs remained red, thus distinguishing EV-A71 (see [link to assay]). Figure 4 This demonstrates the feasibility of HNB-CRISPR for in-tube determination of EVs and EV-A71.

[0086] Example 4 Sensitivity Test

[0087] To test the sensitivity of the HNB-CRISPR system, for EV detection, serially diluted EVs 5′-UTR genes were used, and HNB-RT-LAMP assays were performed using the kit in Example 2. The colorimetric LOD under natural light reached 10. 1 copies / μL (see copies / μL) Figure 5 (a) This is consistent with the sensitivity of conventional LAMP assays.

[0088] For the detection of EV-A71, the kit in Example 2 was used to perform HNB-CRISPR assays on serially diluted EV-A71 VP1 genes. As the RNA concentration decreased, the product transitioned from green to yellow under blue light, and the Lod reached 10. 0 copies / μL (see copies / μL) Figure 5 b).

[0089] Example 5 Specificity Test

[0090] Simultaneously, we used the kit from 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 B virus, human metapneumovirus (HMPV), and human rhinovirus (HRV), to test the LAMP primer specificity of EVs and VP genes. The detection results for all five viruses were violet-blue and undetectable (see Example 2). Figure 6 a) The results showed that the LAMP primer sets of EVs and EV-A71 had excellent specificity.

[0091] We also selected five different EV-A71 viral fragments (excluding the VP1 gene) to test the specificity of crRNA. Under blue light irradiation, all five fragments showed a red "not detected" result (see [link to test results]). Figure 6 (b) The results showed that the crRNA designed for the VP1 gene fragment had excellent specificity.

[0092] Example 6: Clinical Sample Testing

[0093] The Qinhuai District Center for Disease Control and Prevention provided nasopharyngeal swab samples from 94 suspected cases of hand-foot-mouth disease in children under 12 years old. EVs and EV-A71 were tested using HNB-CRISPR, and standard RT-qPCR testing was also performed.

[0094] Ninety-four clinical samples were used for performance testing of HNB-CRISPR. These samples were nasopharyngeal swabs from children under 12 years old suspected of having hand-foot-mouth disease, provided by the CDC. Overall, 79 samples that initially tested positive for EVs after HNB-RT-LAMP underwent further CRISPR / Cas12a testing, and ultimately, 3 samples tested positive for EV-A71 (see [link to relevant documentation]). Figure 7 HNB-CRISPR determined an EV positivity rate of 84.0% and an EV-A71 positivity rate of 3.8% in children suspected of having HZMD (see HNB-CRISPR). Figure 7 The HNB-CRISPR achieved genotyping accuracy of 95.7% for EVs and 100% for EV-A71, demonstrating its excellent detection performance.

[0095] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the invention. Any person skilled in the art can make many possible variations and modifications to the technical solutions of the present invention, or modify them into equivalent embodiments, without departing from the spirit and technical essence of the present invention. Therefore, any simple modifications, equivalent substitutions, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention, without departing from the content of the technical solutions of the present invention, shall still fall within the scope of protection of the present invention.

Claims

1. A kit for detecting enteroviruses and their EV-A71 typing based on HNB-LAMP coupled with CRISPR / Cas12a technology, characterized in that: The system includes an HNB-CRISPR system suitable for the detection of enteroviruses and their 71 genotypes. The HNB-CRISPR system includes LAMP primer set 1 designed for the conserved 5′-UTR sequence of enteroviruses, LAMP primer set 2 designed for the EV-A71 VP1 sequence, and crRNA and ssDNA reporter molecules designed for the EV-A71 VP1 sequence. The LAMP primer set 1 designed for the conserved 5′-UTR sequence of enteroviruses contains primers with nucleotide sequences as shown in SEQ ID NO. 1-6: The 5′-UTR-F3 nucleotide sequence is shown in SEQ ID NO.1; The 5′-UTR-B3 nucleotide sequence is shown in SEQ ID NO.2; The 5′-UTR-FIP nucleotide sequence is shown in SEQ ID NO.3; The 5′-UTR-BIP nucleotide sequence is shown in SEQ ID NO.4; The 5′-UTR-LF nucleotide sequence is shown in SEQ ID NO.5; The 5′-UTR-LB nucleotide sequence is shown in SEQ ID NO. 6; The LAMP primer set 2 designed for the EV-A71 VP1 sequence contains nucleotide sequences as shown in SEQ ID NO. 7-12 below: 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 VP1-BIP nucleotide sequence is shown in SEQ ID NO.10; The VP1-LB nucleotide sequence is shown in SEQ ID NO.11; The VP1-LF nucleotide sequence is shown in SEQ ID NO.12; The crRNA nucleotide sequence designed for the EV-A71 VP1 sequence is shown in SEQ ID NO.13; ssDNA reporter molecule: FAM-TTATT-BHQ1.

2. The enterovirus and its 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 LAMP mixture, 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-F3 and 5′-UTR-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, and 0.16 μM loop primers VP1-LF and VP1-LB as shown in SEQ ID NO.11 and SEQ ID NO.

12. 120μM HNB Hydroxynaphthol Blue.

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

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