CrRNA group and primer group for simultaneously detecting EV71 and CVA16 and application of crRNA group and primer group

Abstract

The invention discloses a crRNA group and a primer group for simultaneously detecting EV71 and CVA16 and application of the crRNA group and the primer group, and belongs to the technical field of gene detection. The crRNA group and the primer group are designed aiming at conserved regions of EV71 and CVA16 genes, and the nucleotide sequence of the EV71-crRNA is shown as SEQ ID NO.11; the nucleotide sequence of the CVA16-crRNA is as shown in SEQ ID NO. 12; the nucleotide sequences of the EV71 primer pair are as shown in SEQ ID NO. 1 and SEQ ID NO. 6; and the nucleotide sequences of the CVA16 primer pair are as shown in SEQ ID NO. 9 and SEQ ID NO. 8. The kit prepared by the invention comprises the crRNA group and the primer group, and further comprises a probe group, a Cas12a protein, a Cas13a protein and RNA extraction kit, an in-vitro constant-temperature RPA reverse transcription kit and a colloidal gold immune test strip. According to the invention, the kit is combined with an RPA / Crispr Cas12a / 13a system to realize simultaneous detection of EV71 and CVA16 by a one-tube method, and the sensitivity and the specificity are high; the detection result is presented by a fluorescence method, and is directly subjected to color development through test paper by utilizing a lateral flow chromatography technology, so that the operation is quicker and more convenient.

Description

Technical Field

[0001] The present application belongs to the field of gene detection technology, and specifically relates to a crRNA group, a primer group and their application for simultaneously detecting EV71 and CVA16. Background Art

[0002] Hand, foot and mouth disease (HFMD) is a common childhood disease prevalent worldwide, characterized by fever and rashes or herpes on the hands, feet, mouth and other parts. A small number of children may develop serious complications such as acute flaccid paralysis, neurogenic pulmonary edema, aseptic meningoencephalitis, and secondary skin infection. Some severe children develop rapidly and even die. Hand, foot and mouth disease is mainly caused by enterovirus, among which Coxsackievirus A16 (CVA16) and enterovirus 71 (EV71) are the most common. CVA16 can infect the human body through the respiratory and digestive tracts, mainly through the mouth and respiratory tract, and can also be transmitted from the mother to the fetus through the placenta. The incubation period is generally 4 to 7 days, and the highest amount of toxins are excreted within 2 weeks of onset. The main symptoms of viral infection are fever and maculopapular rashes and herpes on the hands, feet, mouth and other parts. EV71 is highly contagious, and it causes a particularly high rate of neurological complications, with a high rate of severe illness and mortality, and the proportion of severe cases is more common in infants under 3 years old. The early clinical manifestations are atypical, but the disease progresses rapidly. Once pulmonary edema and pulmonary hemorrhage occur, it can cause death in a short period of time. Therefore, combined with clinical manifestations, timely identification of children infected with hand, foot and mouth disease virus subtypes at an early stage and targeted anti-related virus treatment can effectively control the disease as soon as possible.

[0003] At present, the detection methods of EV71 and CVA16 are: 1) tissue culture method; 2) virus antibody detection method; 3) polymerase chain reaction (PCR) technology; 4) nucleic acid hybridization method. Among them: tissue culture method is to inoculate the patient's body fluid or biopsy organ tissue specimens into monkey kidney, human embryonic kidney, human amniotic membrane and other cells for tissue culture to observe cell pathological changes, and then use different types of immune serum for neutralization test for positive samples for type identification. This detection method is suitable for high virus concentrations. The disadvantage is that tissue culture is quite time-consuming and difficult to popularize; virus antibody detection method can adopt neutralization test, hemagglutination inhibition test, complement fixation test and enzyme-linked immunosorbent assay (ELISA). At present, ELISA method is widely used in China. This method involves multi-step antibody binding and signal detection, requires multiple washings, is time-consuming and labor-intensive, and the specimens are generally tested in batches, and the results are slow; PCR technology and nucleic acid hybridization method are molecular detection methods, which have the advantages of rapidity, sensitivity and strong specificity, but these methods still need to rely on equipment such as PCR instrument and fluorescent quantitative PCR instrument. The complex experimental operation and expensive equipment make it difficult to promote and apply them in different places.

[0004] With the development of more sensitive and rapid disease detection technologies, recombinase polymerase amplification (RPA) technology combined with clustered regularly interspaced short palindromic repeats (CRISPR) and its related protein detection technology (Cas) has been widely praised for its ability to quickly and accurately identify and cut specific nucleic acid sequences, providing a powerful platform for molecular diagnosis; in addition, the above-mentioned test results can be visualized by combining with lateral flow chromatography (LFD) technology, and the results can be directly read by color development of the test paper, which reduces the dependence on instruments in molecular diagnosis, saves detection costs, and is suitable for instant detection at the grassroots and on-site.

[0005] However, there have been no reports on RPA / Crispr Cas detection technology for EV71 and CVA16, common viruses in hand, foot and mouth disease, and the problem of how to efficiently and quickly detect EV71 and CVA16 simultaneously in clinical practice remains unresolved. Summary of the invention

[0006] 1. Purpose of the Invention

[0007] For EV71 and CVA16, two common viruses in hand, foot and mouth disease, an RPA / Crispr Cas system and a specific detection method with rapid detection, high sensitivity and high accuracy are provided.

[0008] 2. Technical solution

[0009] In order to achieve the above-mentioned invention object, the technical solution adopted in this application is as follows:

[0010] An EV71-crRNA for detecting EV71, whose nucleotide sequence is shown in SEQ ID NO.11.

[0011] The present application also provides the use of the above-mentioned EV71-crRNA in the preparation of reagents for detecting EV71.

[0012] The present application also provides a primer pair for detecting EV71, the primer pair comprising an upstream primer and a downstream primer, wherein:

[0013] The upstream primer is selected from any of the following:

[0014] EV71-Primer-F1: the nucleotide sequence is shown in SEQ ID NO.1;

[0015] EV71-Primer-F2: the nucleotide sequence is shown in SEQ ID NO.3;

[0016] EV71-Primer-F3: the nucleotide sequence is shown in SEQ ID NO.5;

[0017] The downstream primer is selected from any of the following:

[0018] EV71-Primer-R1: the nucleotide sequence is shown in SEQ ID NO.2;

[0019] EV71-Primer-R2: the nucleotide sequence is shown in SEQ ID NO.4;

[0020] EV71-Primer-R3: The nucleotide sequence is shown in SEQ ID NO.6.

[0021] Furthermore, the primer pair for detecting EV71 includes EV71-Primer-F1 and EV71-Primer-R3, and the nucleotide sequences thereof are shown in SEQ ID NO.1 and SEQ ID NO.6.

[0022] The present application also provides the use of the above primer pair in preparing a reagent for detecting EV71.

[0023] The present application also provides a reagent for detecting EV71, which comprises the above-mentioned EV71-crRNA and the above-mentioned primer pair.

[0024] Furthermore, the above-mentioned reagent for detecting EV71 also includes a probe.

[0025] Furthermore, the above-mentioned probe includes a first ssDNA probe or a second ssDNA probe, wherein

[0026] The nucleotide sequence of the first ssDNA probe is shown in SEQ ID NO.13;

[0027] The nucleotide sequence of the second ssDNA probe is shown in SEQ ID NO.14;

[0028] The first ssDNA probe is used for detection by fluorescence method; the second ssDNA probe is used for visual detection on a test strip using lateral flow chromatography technology.

[0029] Furthermore, the above reagents also include: Buffer A, ddH2O, Buffer B, Cas12a protein, HEPES buffer, RNase Inhibitor, MgCl2, DEPC water, and dried reaction mixture (provided by in vitro constant temperature RPA reverse transcription kit, purchased from Le Shang Bio).

[0030] The present application also provides a kit for detecting EV71, which includes the above reagents.

[0031] The present application also provides the use of the above reagent or kit in detecting EV71, which comprises the following steps:

[0032] S1: Obtain the RNA of the sample to be tested;

[0033] S2: using the above RNA as a template and the above primer pair to perform RPA isothermal amplification to obtain an RPA amplification product;

[0034] S3: reacting the above RPA amplification product, probe, and EV71-crRNA in the Crispr Cas12a system;

[0035] S4: Detect the reaction results of the Crispr Cas12a system mentioned above; when the first ssDNA probe is used in S3, the fluorescence method is used for detection; when the second ssDNA probe is used in S3, the lateral flow chromatography technology is used for visual detection on the test paper.

[0036] Furthermore, the primer concentration in the above S2 is 100nM to 400nM.

[0037] Furthermore, the primer concentration in the above S2 is 300nM.

[0038] Furthermore, the total volume of the above RPA isothermal amplification system is 50 μL to 10 μL.

[0039] Furthermore, the total volume of the above RPA isothermal amplification system is 20 μL.

[0040] Furthermore, the RPA isothermal amplification time is 10 min to 30 min.

[0041] Furthermore, the RPA isothermal amplification time is 20 minutes.

[0042] Furthermore, the above-mentioned RPA isothermal amplification system includes: 10 μL buffer A, 0.6 μL EV71-Primer-F1 (10 μM), 0.6 μL EV71-Primer-R3 (10 μM), 2 μL RNA template and 1 μL Buffer B, 5.8 μL ddH2O, with a total volume of 20 μL.

[0043] Furthermore, in the above Crispr Cas12a system, Mg 2+ The concentration is 0mM~20mM.

[0044] Furthermore, in the above Crispr Cas12a system, Mg 2+ The concentration is 4mM~20mM.

[0045] Furthermore, in the Crispr Cas12a system, Mg 2+ The concentration is 8mM.

[0046] Furthermore, the reaction time of the above-mentioned Crispr Cas12a system is 10 minutes to 30 minutes.

[0047] Furthermore, the reaction time of the above-mentioned Crispr Cas12a system is 20 minutes.

[0048] Furthermore, the above-mentioned Crispr Cas12a system includes: ssDNA probe (10μM) 0.25μL, Cas12a protein (10μM) 0.25μL, EV71-crRNA (10μM) 0.25μL, HEPES buffer (1M, pH7.2-7.4) 0.5μL, RNaseInhibitor (40IU / μL) 1μL, MgCl2 (1M) 0.2μL, RPA amplification product 2.5μL, DEPC water 20.05μL, and the total volume is 25μL.

[0049] The present application also provides a method for detecting EV71, the method comprising the following steps:

[0050] S1: Obtain the RNA of the sample to be tested;

[0051] S2: using the above RNA as a template and the above primer pair to perform RPA isothermal amplification to obtain an RPA amplification product;

[0052] S3: reacting the above RPA amplification product, probe, and EV71-crRNA in the Crispr Cas12a system;

[0053] S4: Detect the reaction results of the Crispr Cas12a system mentioned above; when the first ssDNA probe is used in S3, the fluorescence method is used for detection; when the second ssDNA probe is used in S3, the lateral flow chromatography technology is used for visual detection on the test paper.

[0054] The present application also provides the application of the above detection method in detecting EV71.

[0055] The present application also provides a CVA16-crRNA for detecting CVA16, whose nucleotide sequence is shown in SEQ ID NO.12.

[0056] The present application also provides the use of the above-mentioned CVA16-crRNA in the preparation of a reagent for detecting CVA16.

[0057] The present application also provides a primer pair for detecting CVA16, the primer pair comprising an upstream primer and a downstream primer, wherein:

[0058] The upstream primer is selected from any of the following:

[0059] CVA16-Primer-F1: the nucleotide sequence is shown in SEQ ID NO.7;

[0060] CVA16-Primer-F2: the nucleotide sequence is shown in SEQ ID NO.9;

[0061] The downstream primer is selected from any of the following:

[0062] CVA16-Primer-R1: the nucleotide sequence is shown in SEQ ID NO.8;

[0063] CVA16-Primer-R2: The nucleotide sequence is shown in SEQ ID NO.10.

[0064] Furthermore, the primer pair for detecting CVA16 includes CVA16-Primer-F2 and CVA16-Primer-R1, and the nucleotide sequences thereof are shown in SEQ ID NO.9 and SEQ ID NO.8.

[0065] The present application also provides the use of the above primer pair in preparing a reagent for detecting CVA16.

[0066] The present application also provides a reagent for detecting CVA16, which comprises the above-mentioned CVA16-crRNA and the above-mentioned primer pair.

[0067] Furthermore, the above-mentioned reagent for detecting CVA16 also includes a probe.

[0068] Furthermore, the above-mentioned probe includes a first ssRNA probe or a second ssRNA probe, wherein

[0069] The nucleotide sequence of the first ssRNA probe is shown in SEQ ID NO.15;

[0070] The nucleotide sequence of the second ssRNA probe is shown in SEQ ID NO.16;

[0071] The first ssRNA probe is used for detection by fluorescence method; the second ssRNA probe is used for visual detection on a test strip using lateral flow chromatography technology.

[0072] Furthermore, the above reagents also include: Buffer A, ddH2O, Buffer B, Cas13a protein, HEPES buffer, rNTP, RNase Inhibitor, T7 polymerase, MgCl2, DEPC water, and dried reaction mixture (provided by in vitro constant temperature RPA reverse transcription kit, purchased from Le Shang Bio).

[0073] The present application also provides a kit for detecting CVA16, which comprises the above-mentioned reagents.

[0074] The use of the above reagent or kit in detecting CVA16 comprises the following steps:

[0075] S1: Obtain the RNA of the sample to be tested;

[0076] S2: using the above RNA as a template and the above primer pair to perform RPA isothermal amplification to obtain an RPA amplification product;

[0077] S3: react the above RPA amplification product, probe, and CVA16-crRNA in the Crispr Cas13a system;

[0078] S4: Detect the reaction results of the Crispr Cas13a system mentioned above; when the first ssRNA probe is used in S3, the fluorescence method is used for detection; when the second ssRNA probe is used in S3, the lateral flow chromatography technology is used for visual detection on the test paper.

[0079] Furthermore, the primer concentration in the above S2 is 100nM to 400nM.

[0080] Furthermore, the primer concentration in the above S2 is 100 nM.

[0081] Furthermore, the total volume of the above RPA isothermal amplification system is 50 μL to 10 μL.

[0082] Furthermore, the total volume of the above RPA isothermal amplification system is 20 μL.

[0083] Furthermore, the RPA isothermal amplification time is 10 min to 30 min.

[0084] Furthermore, the RPA isothermal amplification time is 20 minutes.

[0085] Furthermore, the above-mentioned RPA isothermal amplification system includes: 10 μL buffer A, 0.2 μL CVA16-Primer-F2 (10 μM), 0.2 μL CVA16-Primer-R1 (10 μM), 2 μL RNA template and 1 μL Buffer B, 6.6 μL ddH2O, with a total volume of 20 μL.

[0086] Furthermore, in the Crispr Cas13a system, Mg 2+ The concentration is 0mM~20mM.

[0087] Furthermore, in the Crispr Cas13a system, Mg 2+ The concentration is 4mM~20mM.

[0088] Furthermore, in the Crispr Cas13a system, Mg 2+ The concentration is 8mM.

[0089] Furthermore, the reaction time of the above-mentioned Crispr Cas13a system is 10 minutes to 30 minutes.

[0090] Furthermore, the reaction time of the above-mentioned Crispr Cas13a system is 20 minutes.

[0091] Furthermore, the above-mentioned Crispr Cas13a system includes: ssRNA probe (10μM) 0.5μL, Cas13a protein (10μM) 0.25μL, CVA16-crRNA (10μM) 0.25μL, HEPES buffer (1M, pH7.2-7.4) 0.5μL, rNTP 25mM / each2μL, RNase Inhibitor (40IU / μL) 1μL, T7 polymerase (50IU / μL) 0.5μL, MgCl2 (1M) 0.2μL, RPA amplification product 2.5μL, DEPC water 17.3μL, and the total volume is 25μL.

[0092] The present application also provides a method for detecting CVA16, the method comprising the following steps:

[0093] S1: Obtain the RNA of the sample to be tested;

[0094] S2: using the above RNA as a template and the above primer pair to perform RPA isothermal amplification to obtain an RPA amplification product;

[0095] S3: react the above RPA amplification product, probe, and CVA16-crRNA in the Crispr Cas13a system;

[0096] S4: Detect the reaction results of the Crispr Cas13a system mentioned above; when the first ssRNA probe is used in S3, the fluorescence method is used for detection; when the second ssRNA probe is used in S3, the lateral flow chromatography technology is used for visual detection on the test paper.

[0097] The present application also provides the application of the above detection method in detecting CVA16.

[0098] The present application also provides a crRNA group for detecting EV71 and CVA16, including EV71-crRNA and CVA16-crRNA, wherein the nucleotide sequence of EV71-crRNA is shown in SEQ ID NO.11; the nucleotide sequence of CVA16-crRNA is shown in SEQ ID NO.12.

[0099] The present application also provides the use of the above-mentioned crRNA group in the preparation of reagents for detecting EV71 and CVA16.

[0100] The present application also provides a primer set for detecting EV71 and CVA16, the primer set comprising an EV71 primer pair and a CVA16 primer pair, wherein:

[0101] The EV71 primer pair includes an EV71 upstream primer and an EV71 downstream primer.

[0102] The EV71 upstream primer is selected from any of the following:

[0103] EV71-Primer-F1: the nucleotide sequence is shown in SEQ ID NO.1;

[0104] EV71-Primer-F2: the nucleotide sequence is shown in SEQ ID NO.3;

[0105] EV71-Primer-F3: the nucleotide sequence is shown in SEQ ID NO.5;

[0106] The EV71 downstream primer is selected from any one of the following:

[0107] EV71-Primer-R1: the nucleotide sequence is shown in SEQ ID NO.2;

[0108] EV71-Primer-R2: the nucleotide sequence is shown in SEQ ID NO.4;

[0109] EV71-Primer-R3: the nucleotide sequence is shown in SEQ ID NO.6;

[0110] The CVA16 primer pair includes a CVA16 upstream primer and a CVA16 downstream primer.

[0111] The CVA16 upstream primer is selected from any of the following:

[0112] CVA16-Primer-F1: the nucleotide sequence is shown in SEQ ID NO.7;

[0113] CVA16-Primer-F2: the nucleotide sequence is shown in SEQ ID NO.9;

[0114] The CVA16 downstream primer is selected from any of the following:

[0115] CVA16-Primer-R1: the nucleotide sequence is shown in SEQ ID NO.8;

[0116] CVA16-Primer-R2: The nucleotide sequence is shown in SEQ ID NO.10.

[0117] Furthermore, the primer pair for detecting EV71 includes EV71-Primer-F1 and EV71-Primer-R3, whose nucleotide sequences are shown in SEQ ID NO.1 and SEQ ID NO.6, respectively; the primer pair for detecting CVA16 includes CVA16-Primer-F2 and CVA16-Primer-R1, whose nucleotide sequences are shown in SEQ ID NO.9 and SEQ ID NO.8, respectively.

[0118] The present application also provides the use of the above primer set in preparing reagents for detecting EV71 and CVA16.

[0119] The present application also provides a reagent for detecting EV71 and CVA16, which comprises the above-mentioned crRNA group and the above-mentioned primer group.

[0120] Furthermore, the above reagents also include a probe set.

[0121] Furthermore, the probe group includes a first probe group or a second probe group, wherein

[0122] The first probe group includes a first ssDNA probe and a first ssRNA probe, the nucleotide sequence of the first ssDNA probe is shown in SEQ ID NO.13, and the nucleotide sequence of the first ssRNA probe is shown in SEQ ID NO.15;

[0123] The second probe set includes a second ssDNA probe and a second ssRNA probe, the nucleotide sequence of the second ssDNA probe is shown in SEQ ID NO.14, and the nucleotide sequence of the second ssRNA probe is shown in SEQ ID NO.16;

[0124] The first probe group is used for detection by fluorescence method; the second probe group is used for visual detection on a test strip using lateral flow chromatography technology.

[0125] Furthermore, the above reagents also include: Buffer A, ddH2O, Buffer B, Cas12a protein, Cas13a protein, HEPES buffer, rNTP, RNase Inhibitor, T7 polymerase, MgCl2, DEPC water, and dried reaction mixture (provided by in vitro constant temperature RPA reverse transcription kit, purchased from Le Shang Bio).

[0126] The present application also provides a kit for detecting EV71 and CVA16, which comprises the above reagents.

[0127] The present application also provides the use of the above reagent or kit in detecting EV71 and CVA16, which comprises the following steps:

[0128] S1: Obtain the RNA of the sample to be tested;

[0129] S2: using the above RNA as a template and the above primer set to perform RPA isothermal amplification to obtain an RPA amplification product;

[0130] S3: reacting the above RPA amplification products, probe set, and crRNA set in the Crispr Cas12a / Cas13a system;

[0131] S4: Detect the reaction results of the Crispr Cas12a / 13a system; when the first probe group is used in S3, the fluorescence method is used for detection; when the second probe group is used in S3, the lateral flow chromatography technology is used for visual detection on the test paper.

[0132] Furthermore, in the above-mentioned RPA isothermal amplification reaction system, the concentration and ratio of the primer set are EV71 400nM:CVA16100nM to EV71 100nM:CVA16 400nM.

[0133] Furthermore, in the above RPA isothermal amplification reaction system, the primer set concentration and ratio are EV71 300nM: CVA16 100nM.

[0134] Furthermore, in the above RPA isothermal amplification reaction system, the total volume of the system is 50 μL to 10 μL.

[0135] Furthermore, in the above RPA isothermal amplification reaction system, the total volume of the system is 20 μL.

[0136] Furthermore, in the above RPA isothermal amplification reaction system, the amplification time is 10 min to 30 min.

[0137] Furthermore, in the above RPA isothermal amplification reaction system, the amplification time is 20 minutes.

[0138] Furthermore, the above-mentioned RPA isothermal amplification system includes: 10 μL buffer A, 0.6 μL EV71-Primer-F1 (10 μM), 0.6 μL EV71-Primer-R3 (10 μM), 0.2 μL CVA16-Primer-F2 (10 μM), 0.2 μL CVA16-Primer-R1 (10 μM), 5.4 μL ddH2O, 2 μL RNA template and 1 μL Buffer B, with a total volume of 20 μL.

[0139] Furthermore, in the reaction system of the Crispr Cas12a / 13a system, Mg 2+ The concentration is 0mM~20mM.

[0140] Furthermore, in the reaction system of the Crispr Cas12a / 13a system, Mg 2+ The concentration is 4mM~20mM.

[0141] Furthermore, in the reaction system of the Crispr Cas12a / 13a system, Mg 2+ The concentration is 8mM.

[0142] Furthermore, in the reaction system of the above-mentioned Crispr Cas12a / 13a system, the reaction time is 10 min to 30 min.

[0143] Furthermore, in the reaction system of the above-mentioned Crispr Cas12a / 13a system, the reaction time is 20 minutes.

[0144] Furthermore, the above-mentioned Crispr Cas12a / 13a system includes: ssDNA probe (10μM) 0.25μL, ssRNA probe (10μM) 0.5μL, Cas12a protein (10μM) and Cas13a protein (10μM) 0.25μL each, EV71-crRNA (10μM) and CVA16-crRNA (10μM) 0.25μL each, HEPES buffer (1M, pH7.2-7.4) 0.5μL, rNTP 25mM / each2μL, RNase Inhibitor (40IU / μL) 1μL, T7 polymerase (50IU / μL) 0.5μL, MgCl2 (1M) 0.2μL, DEPC water 16.55μL, RPA amplification product 2.5μL, and a total volume of 25μL.

[0145] The present application also provides a method for detecting EV71 and CVA16, the method comprising the following steps:

[0146] S1: Obtain the RNA of the sample to be tested;

[0147] S2: using the above RNA as a template and the above primer set to perform RPA isothermal amplification to obtain an RPA amplification product;

[0148] S3: reacting the above RPA amplification products, probe set, and crRNA set in the Crispr Cas12a / 13a system;

[0149] S4: Detect the reaction results of the Crispr Cas12a / 13a system;

[0150] In S3, the first probe group is used to perform detection by fluorescence method; and the second probe group is used to perform visual detection on a test paper using lateral flow chromatography technology.

[0151] The present application also provides the application of the above detection method in detecting EV71 and CVA16.

[0152] 3. Beneficial effects

[0153] Compared with the prior art, the present application has the following beneficial effects:

[0154] (1) The present application provides a crRNA group, a primer group and their application for the simultaneous detection of EV71 and CVA16. The crRNA group and primer group are designed for the conserved regions of the EV71 and CVA16 genes. For the first time, the RPA / Crispr Cas12a / 13a system is combined to realize the one-tube method for the simultaneous detection of EV71 and CVA16 viruses in hand, foot and mouth disease. By optimizing the conditions such as the concentration ratio of the primer group, a kit for the simultaneous detection of EV71 and CVA16 is prepared for the first time. The kit has high accuracy and strong specificity.

[0155] (2) The present application provides a crRNA group, a primer group and their applications for the simultaneous detection of EV71 and CVA16. Two sets of probe groups are designed for the fluorescence detection method and the test strip detection method of EV71 and CVA16 genes, respectively, so as to achieve the one-tube method for the simultaneous detection of EV71 and CVA16 without cross-reactivity, and the sensitivity can be as low as 1×10^0 copies / μL.

[0156] (3) The present application provides a crRNA group, a primer group and their applications for simultaneously detecting EV71 and CVA16. On the one hand, the detection results of the RPA / Crispr Cas12a / 13a system are presented by using a fluorescent signal (FD) through a fluorescence method. On the other hand, the reaction results of the RPA / Crispr Cas12a / 13a system are directly displayed through a test strip in combination with lateral flow chromatography technology (LFD), thereby realizing visualization of the results. BRIEF DESCRIPTION OF THE DRAWINGS

[0157] Figure 1 It is a screening diagram of the conservative regions of EV71 and CVA16 genes of the present invention; wherein, Figure A is a screening diagram of the conservative regions of EV71 genes, and Figure B is a screening diagram of the conservative regions of CVA16 genes.

[0158] Figure 2 It is a graph of gel electrophoresis results of amplification of EV71 and CVA16 with different primer pairs of the present invention; wherein, Figure A is obtained by amplification with the EV71 primer pair, and Figure B is obtained by amplification with the CVA16 primer pair.

[0159] Figure 3 It is a graph showing the amplification efficiency results of different primers for EV71 and CVA16 of the present invention; wherein, Figure A is the amplification result of the EV71 primer pair, and Figure B is the amplification result of the CVA16 primer pair.

[0160] Figure 4 This is a graph showing the specific detection results of the EV71 and CVA16 probe groups of the present invention.

[0161] Figure 5 This is a graph showing the results of optimizing the concentration ratio of the EV71 and CVA16 primer sets of the present invention.

[0162] Figure 6 Mg in Crispr Cas12a / 13a of the present invention 2+ Concentration optimization results.

[0163] Figure 7 It is a volume optimization result diagram of the RPA reaction system of the present invention.

[0164] Figure 8 It is a diagram of the reaction time optimization results of the RPA and Crispr Cas12a / Cas13a systems of the present invention; wherein Figure A is the RPA reaction time optimization result, and Figure B is the Crispr Cas12a / Cas13a system reaction time optimization result.

[0165] Fig. 9 This is a flow chart of the RPA / Crispr Cas12a / 13a system for detecting EV71 and CVA16.

[0166] Fig.10 This is a graph showing the detection results of EV71 and CVA16 in clinical samples using the RPA / Crispr Cas12a / Cas13a system of the present invention combined with fluorescence method.

[0167] Fig.11 This is a graph showing the accuracy of the RPA / Crispr Cas12a / 13a system combined with fluorescence detection of EV71 and CVA16.

[0168] Fig.12 This is a graph showing the sensitivity results of the RPA / Crispr Cas12a / 13a system of the present invention combined with fluorescence detection of EV71 and CVA16.

[0169] Fig.13 This is a graph showing the specific results of detecting EV71 and CVA16 using the RPA / Crispr Cas12a / 13a system of the present invention combined with fluorescence.

[0170] Fig.14 This is a graph showing the detection results of EV71 and CVA16 in clinical samples using the RPA / Crispr Cas12a / Cas13a system of the present invention combined with lateral flow chromatography technology.

[0171] Fig.15 This is a graph showing the sensitivity results of the RPA / Crispr Cas12a / 13a system of the present invention combined with lateral flow chromatography technology in detecting EV71 and CVA16.

[0172] Fig.16 This is a graph showing the specific results of detecting EV71 and CVA16 using the RPA / Crispr Cas12a / 13a system of the present invention combined with lateral flow chromatography technology. DETAILED DESCRIPTION

[0173] The present application is further described below in conjunction with specific embodiments.

[0174] It should be noted that the terms such as "upper", "lower", "left", "right", "middle", etc. cited in this specification are only for the convenience of description and are not used to limit the scope of implementation. Changes or adjustments to their relative relationships should be regarded as the scope of implementation of this application without substantially changing the technical content.

[0175] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this application belongs; the term "and / or" used herein includes any and all combinations of one or more of the associated listed items.

[0176] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. If the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0177] As used herein, the term "about" is used to provide flexibility and imprecision associated with a given term, measurement or value. The degree of flexibility for a particular variable can be easily determined by one skilled in the art.

[0178] As used herein, the term "at least one of" is intended to be synonymous with "one or more of." For example, "at least one of A, B, and C" expressly includes only A, only B, only C, and combinations of each thereof.

[0179] Concentration, amount and other numerical data can be presented in range format herein.It should be understood that such range format is only used for convenience and simplicity, and should be flexibly interpreted as not only including the numerical value clearly described as range limit, but also including all single numerical values ​​or sub-ranges contained in the range, just as each numerical value and sub-range are clearly described.For example, the numerical range of about 1 to about 4.5 should be interpreted as not only including the limit value of 1 to about 4.5 clearly described, but also including single numerals (such as 2,3,4) and sub-ranges (such as 1 to 3,2 to 4, etc.).The same principle is applicable to the scope of only narrating a numerical value, such as "less than about 4.5", which should be interpreted as including all the above-mentioned values ​​and ranges.In addition, no matter how the breadth of the described range or feature is, this explanation should be applicable.

[0180] Example 1

[0181] This embodiment provides the design of crRNA groups, primer groups and probe groups of EV71 and CVA16, including the following steps:

[0182] (1) Screening of conserved regions of EV71 and CVA16 genes

[0183] The VP1 gene, as the coding gene of EV71 and CVA16, contains a major immunogenic epitope and can be used as a diagnostic marker. The VP1 gene was searched from the GenBank database of the National Center for Biotechnology Information (NCBI), and 4,500 EV71 VP1 sequences and 3,200 CVA16 VP1 sequences from China were screened. MEGA7.0 and Jalview software were used for comparison and analysis to identify the conserved regions. The results are as follows: Figure 1 As shown, the conserved region nucleotide sequences of EV71 and CVA16 were finally selected as shown in SEQ ID NO.17 and SEQ ID NO.18. The specific sequences can be found in Table 1 below.

[0184] (2) Design of crRNA set, primer set and probe set

[0185] The above-mentioned EV71 and CVA16 conserved region sequences were selected according to the PAM sequences that crRNAs should have in the nucleic acid sequences, and the final designed EV71-crRNA and CVA16-crRNA nucleotide sequences were shown in SEQ ID NO.11 and SEQ ID NO.12;

[0186] Based on the above-mentioned conserved region sequences of EV71 and CVA16, three pairs of primers were designed for EV71 and two pairs of primers were designed for CVA16. For the primer pair of CVA16, a T7 RNA polymerase promoter sequence was added to the 5' end of its upstream primer. The nucleotide sequences of the above-mentioned EV71 and CVA16 primer pairs are shown in SEQ ID NO.1-10. The above-mentioned primers were synthesized by Shanghai Bioengineering.

[0187] Design the probe set as follows:

[0188] The first probe group: the nucleotide sequence of the ssDNA probe is shown in SEQ ID NO.13, and the nucleotide sequence of the ssRNA probe is shown in SEQ ID NO.15; the first probe group is detected by fluorescence method;

[0189] The second probe group: the nucleotide sequence of the ssDNA probe is shown in SEQ ID NO.14, and the nucleotide sequence of the ssRNA probe is shown in SEQ ID NO.16; the second probe group uses lateral flow chromatography technology to perform visual detection on a test paper.

[0190] The specific nucleotide sequences of the above crRNA group, primer group and probe group are shown in Table 1.

[0191] Table 1 List of EV71 and CVA16 crRNA groups, primer groups, probe groups and their conserved region nucleotide sequences

[0192]

[0193] Example 2

[0194] This example provides a study of the amplification results of different primer sets for EV71 and CVA16, including the following steps:

[0195] (1) Acquisition of EV71 and CVA16 template RNA

[0196] The conserved region sequences (about 200 base pairs) of EV71 and CVA16 were respectively integrated with the gene fragment of the T7 RNA polymerase recognition site into the pUC19 plasmid vector, and the recombinant vector was transformed into competent cells for amplification to obtain positive clones. Then, the required EV71 and CVA16 target fragments were specifically amplified by polymerase chain reaction (PCR), and transcribed in vitro using an RNA synthesis kit (New England Biolabs, MA, United States) to obtain EV71 and CVA16 template RNA fragments, respectively (the above work was completed by GenScript Biotech Co., Ltd.).

[0197] (2) RPA constant temperature amplification screening primer set

[0198] The primers of EV71 and CVA16 designed in Example 1 were combined in pairs to perform RPA constant temperature amplification on the template RNA. The specific RPA operation steps were as follows: 13.5 μL of nuclease-free water, 25 μL of buffer A and 2 μL of each upstream and downstream primer (10 μM) were mixed with the dry reaction mixture in the detection unit tube (provided by the in vitro constant temperature RPA reverse transcription kit, purchased from Le Shang Bio), and then 5 μL of RNA template was added, and then 2.5 μL of Buffer B (280nM magnesium acetate) was added to the tube cap, with a total volume of 50 μL; then gently vortexed 5 to 6 times, centrifuged briefly for 10 seconds, and finally incubated at 42°C in a metal bath for 30 minutes to produce amplification products. The above amplification products were purified using a universal DNA purification kit (DP214 provided by Tiangen Biochemical Technology Beijing Co., Ltd.), separated by 2.0% agarose gel electrophoresis, and the primer combination with the highest amplification efficiency was screened.

[0199] The results are as follows Figure 2 and Figure 3As shown, among them, EV71-Primer-F1 and EV71-Primer-R3 performed best, and their nucleotide sequences were shown in SEQ ID NO.1 and SEQ ID NO.6, respectively; CVA16-Primer-F2 and CVA16-Primer-R1 performed best, and their nucleotide sequences were shown in SEQ ID NO.9 and SEQ ID NO.8, respectively.

[0200] Example 3

[0201] This example performs probe set specific detection of EV71 and CVA16, including the following steps:

[0202] (1) The EV71 and CVA16 template RNAs were obtained in the same manner as in Example 2.

[0203] (2) Preparation of RPA solution A and Crispr Cas12a / 13a solution B

[0204] Solution A: 13.5 μL of nuclease-free water, 25 μL of buffer A, 2 μL of upstream primer (10 μM), and 2 μL of downstream primer (10 μM) were mixed with the dry reaction mixture in the detection unit tube (provided by the in vitro constant temperature RPA reverse transcription kit, purchased from Le Shang Biotechnology), and the dry reaction mixture was completely resuspended by vortexing, wherein the nucleotide sequences of the upstream primer and the downstream primer of EV71 are shown in SEQ ID NO.1 and SEQ ID NO.6, respectively; the nucleotide sequences of the upstream primer and the downstream primer of CVA16 are shown in SEQ ID NO.9 and SEQ ID NO.8, respectively;

[0205] Solution B: 0.25 μL ssDNA probe (10 μM), 0.5 μL ssRNA probe (10 μM), 0.25 μL Cas12a protein (10 μM), 0.25 μL Cas13a protein (10 μM), 0.25 μL EV71-crRNA (10 μM), 0.25 μL CVA16-crRNA (10 μM), 0.5 μL HEPES buffer (1 M, pH 7.2-7.4), 2 μL rNTP 25 mM / each, 1 μL RNase Inhibitor (40 IU / μL), 0.5 μL T7 polymerase (50 IU / μL), 0.2 μL MgCl2 (1 M) and 16.55 μL DEPC water, wherein the ssDNA probe nucleotide sequence is shown in SEQ ID NO.13; the ssRNA probe nucleotide sequence is shown in SEQ ID NO.15.

[0206] (3) RPA isothermal amplification

[0207] Add 5 μL RNA template to solution A, and then add 2.5 μL Buffer B (280 nM magnesium acetate) on the tube cap; then gently vortex mix 5 to 6 times, briefly centrifuge for 10 seconds, and finally incubate at 42°C in a thermostat or metal bath for 30 minutes to obtain the amplified product.

[0208] (4) Crispr Cas12a / 13a system detection

[0209] 2.5 μL of the above RPA amplification product was pipetted into solution B, and the centrifuge tube was inverted upside down to mix the two thoroughly. The Crispr reaction was performed at 37°C using the Q5 real-time PCR system (Thermo Fisher Scientific, USA) and the fluorescence changes were monitored.

[0210] The results are as follows Figure 4 As shown, when the EV71 target gene was present, only the FAM fluorescence signal was detected; when the CVA16 target gene was present, only the VIC fluorescence signal was detected, and with the increase in the number of amplification cycles, the intensities of the FAM and VIC fluorescence signals increased proportionally, indicating that the above-mentioned ssDNA probes and ssRNA probes were effectively cut and detected in the Crispr Cas12a / 13a system, and there was no cross-reactivity between the two cutting reactions.

[0211] Example 4

[0212] This example optimizes the concentration ratio of the EV71 and CVA16 primer sets, including the following steps:

[0213] (1) The EV71 and CVA16 template RNAs were obtained in the same manner as in Example 2.

[0214] (2) Optimization of primer set concentration ratio

[0215] To ensure the best amplification results of EV71 and CVA16, the concentration ratio of their primer sets was optimized, and the primer concentration ratio of EV71 and CVA16 was set from EV71 400nM:CVA16 100nM to EV71 100nM:CVA16 400nM; the nucleotide sequences of the upstream primer and downstream primer of EV71 are shown in SEQ ID NO.1 and SEQ ID NO.6, respectively; the nucleotide sequences of the upstream primer and downstream primer of CVA16 are shown in SEQ ID NO.9 and SEQ ID NO.8, respectively.

[0216] (3) RPA isothermal amplification

[0217] 25 μL of buffer A and 10 μL of nuclease-free water were mixed with the dried reaction mixture in the detection unit tube (provided by the in vitro constant temperature RPA reverse transcription kit, purchased from Le Shang Bio), and the dried reaction mixture was completely resuspended by vortexing. Subsequently, 5 μL of template RNA and different concentrations and ratios of EV71 and CVA16 primers were added in a stepwise manner, and nuclease-free water was added to a volume of 47.5 μL. After thorough mixing, 2.5 μL of Buffer B (280 mM magnesium acetate) was added to the reaction mixture, briefly centrifuged, and then immediately incubated at 42 ° C in a metal heat block for 30 min.

[0218] (4) Detection of Crispr Cas12a system and Crispr Cas13a system

[0219] For the detection of EV71 amplification results, a Crispr Cas12a-mediated cleavage experiment was used. Take 2 μL of the RPA amplification product in step 3, add 2 μL of 10×Cas12a reaction buffer, 0.2 μL of Cas12a enzyme (10 μM), 0.2 μL of crRNA (10 μM), 0.2 μL of fluorescence quenching single-stranded DNA (ssDNA, 10 μM) probe and 0.5 μL of RNase inhibitor in sequence, and add sterile water to a total volume of 20 μL.

[0220] The nucleotide sequence of the ssDNA probe is shown in SEQ ID NO.13.

[0221] For the detection of CVA16 amplification results, a Crispr Cas13a-mediated cleavage experiment was used. 2 μL of the RPA amplification product in step 3 was taken, and 0.5 μL of HEPES (1 M), 0.2 μL of MgCl2 (1 M), 0.2 μL of Cas13a enzyme (10 μM), 0.2 μL of crRNA (10 μM), 0.2 μL of fluorescence quenched single-stranded RNA (ssRNA, 10 μM) probe, 2 μL of rNTPs mixture (New England Biolabs, USA; N0466 L), 1 μL of mouse RNase inhibitor (New England Biolabs, USA; M0314 L) and 0.5 μL of T7 RNA polymerase (New England Biolabs, USA; M0251 L) were added in sequence, and sterile water was added to a total volume of 20 μL.

[0222] The nucleotide sequence of the ssRNA probe is shown in SEQ ID NO.15.

[0223] The above reaction was carried out at 37°C for 30 min using the Q5 Real-Time PCR System (Thermo Fisher Scientific, USA), and fluorescence readings were recorded every 30 sec. The fluorescence intensity of the probe at different primer concentration ratios was evaluated.

[0224] The results are as follows Figure 5 As shown, when the concentration of EV71 primers was 300 nM and the concentration of CVA16 primers was 100 nM, FAM and VIC showed higher fluorescence intensities.

[0225] Example 5

[0226] This example is used to perform Mg in the Crispr Cas12a / Cas13a system 2+ The optimization of concentration includes the following steps:

[0227] (1) The EV71 and CVA16 template RNAs were obtained in the same manner as in Example 2.

[0228] (2)Mg 2+ Concentration optimization

[0229] Given that Mg 2+ The concentration plays an important role in the cutting efficiency. To improve the performance of the CRISPR-Cas12a / Cas13a detection system, we set up 6 groups of Mg 2+ The concentrations are 0mM, 4mM, 8mM, 12mM, 16mM and 20mM respectively.

[0230] (3) Preparation of RPA solution A and Crispr Cas12a / 13a solution B

[0231] Solution A: 13.5 μL nuclease-free water, 25 μL buffer A, 1.5 μL EV71 upstream and downstream primers (10 μM) and 0.5 μL CVA16 upstream and downstream primers (10 μM) were mixed with the dry reaction mixture in the detection unit tube (provided by the in vitro constant temperature RPA reverse transcription kit, purchased from Le Shang Biotechnology), and the dry reaction mixture was completely resuspended by vortexing, wherein the nucleotide sequences of the EV71 upstream primer and downstream primer are shown in SEQ ID NO.1 and SEQ ID NO.6, respectively; the nucleotide sequences of the CVA16 upstream primer and downstream primer are shown in SEQ ID NO.9 and SEQ ID NO.8, respectively;

[0232] Solution B is different from Example 3 using the above 6 groups of different concentrations of Mg 2+ , the rest is the same as Example 3.

[0233] (4) RPA isothermal amplification was the same as in Example 3.

[0234] (5) Crispr Cas12a / 13a system detection

[0235] 2.5 μL of the above RPA amplification product was pipetted into solution B, and the centrifuge tube was inverted upside down to mix the two thoroughly. The Crispr reaction was performed at 37°C using the Q5 real-time PCR system (Thermo Fisher Scientific, USA) for 30 min, and the fluorescence readings were recorded every 30 seconds.

[0236] The results are as follows Figure 6 As shown, the CRISPR Cas12a / Cas13a system was activated in 8 mM Mg 2+ At the concentration, the fluorescence intensity of FAM and VIC was the most significant.

[0237] Example 6

[0238] In this embodiment, the volume optimization of the RPA reaction system is performed, including the following steps:

[0239] (1) The EV71 and CVA16 template RNAs were obtained in the same manner as in Example 2.

[0240] (2) Volume optimization of RPA reaction system

[0241] In order to optimize the RPA reaction system, we established a RPAMIX reaction system gradient from 50 μL to 10 μL and set up 5 groups of reaction systems, namely 50 μL, 40 μL, 30 μL, 20 μL and 10 μL. The composition of each system can be found in Table 2 RPA MIX reaction system gradient table.

[0242] (3) Preparation of RPA solution A and Crispr Cas12a / 13a solution B

[0243] Solution A: Prepare the RPA reaction system with reference to the RPA MIX reaction system gradient table in Table 2 (RNA template and Buffer B are not added here), mix the above different system solutions with the dry reaction mixture in the detection unit tube (provided by the in vitro constant temperature RPA reverse transcription kit, purchased from Le Shang Bio), and ensure that the dry reaction mixture is completely resuspended by vortexing;

[0244] The preparation of solution B is the same as in Example 3.

[0245] Table 2 RPA MIX reaction system gradient table

[0246]

[0247] The nucleotide sequence of EV71-Primer-F1 is shown in SEQ ID NO.1, and the nucleotide sequence of EV71-Primer-R3 is shown in SEQ ID NO.6; the nucleotide sequence of CVA16-Primer-F2 is shown in SEQ ID NO.9, and the nucleotide sequence of CVA16-Primer-R1 is shown in SEQ ID NO.8.

[0248] (4) RPA isothermal amplification

[0249] Add RNA template and Buffer B (280 nM magnesium acetate) to Solution A, quickly mix and centrifuge. To avoid system contamination caused by subsequent centrifuge tube opening, add all of the above Solution B to the cap of the centrifuge tube containing the above Solution A, and quickly place it in a thermostat or metal bath at 42°C for incubation for 30 minutes to obtain the amplified product.

[0250] (5) Crispr Cas12a / 13a system detection

[0251] After RPA amplification, the tube was centrifuged briefly to mix the RPA amplification product in the tube and the Crispr Cas12a / 13a system on the tube cap. The Crispr reaction was performed at 37°C for 30 minutes using the Q5 real-time PCR system (Thermo Fisher Scientific, USA), and the fluorescence readings were recorded every 30 seconds.

[0252] The results are as follows Figure 7 As shown, FAM and VIC fluorescence signals can be effectively detected under the above five RPA systems.

[0253] Example 7

[0254] This embodiment optimizes the reaction time of the RPA / Crispr Cas12a / Cas13a system.

[0255] (1) The EV71 and CVA16 template RNAs were obtained in the same manner as in Example 2.

[0256] (2) Optimization of reaction time

[0257] In order to optimize the reaction time of RPA and CRISPR Cas12a / Cas13a systems, we set five groups of different reaction times, namely 10 min, 15 min, 20 min, 25 min and 30 min.

[0258] (3) Preparation of RPA solution A and Crispr Cas12a / 13a solution B

[0259] Solution A selected 20 μL RPA system in Table 2 of Example 6 above (RNA template and Buffer B were not added here): 10 μL buffer A, 0.6 μL EV71-Primer-F1 (10 μM), 0.6 μL EV71-Primer-R3 (10 μM), 0.2 μL CVA16-Primer-F2 (10 μM), 0.2 μL CVA16-Primer-R1 (10 μM) and 5.4 μL ddH2O, mixed with the dry reaction mixture in the detection unit tube (provided by the in vitro constant temperature RPA reverse transcription kit, purchased from Le Shang Bio), and vortexed to ensure that the dry reaction mixture was completely resuspended, wherein the nucleotide sequences of the EV71 upstream primer and the downstream primer are shown in SEQ ID NO.1 and SEQ ID NO.6, respectively; the nucleotide sequences of the CVA16 upstream primer and the downstream primer are shown in SEQ ID NO.9 and SEQ ID NO.8, respectively;

[0260] The preparation of solution B is the same as in Example 3.

[0261] (4) Optimization of RPA constant temperature amplification time

[0262] Add 2 μL RNA template and 1 μL Buffer B (280 nM magnesium acetate) to solution A, mix quickly and centrifuge. To avoid system contamination caused by subsequent centrifuge tube opening, add all of the above solution B to the cap of the centrifuge tube containing the above solution A, and quickly place it in a thermostat or metal bath and incubate at 42°C for the above 5 different times to obtain the amplified product; after the RPA reaction is completed, centrifuge briefly immediately, and use the Crispr Cas12a / 13a system to detect the RPA amplification effect at different times (the Crispr detection time is set to the conventional 1h). The test results are as follows Figure 8 As shown in A, the RPA amplification effect remained unchanged after 20 min.

[0263] (5) Optimization of Crispr Cas12a / 13a system detection time

[0264] RPA isothermal amplification was the same as step (4) in this embodiment, except that the amplification time was optimized to 20 min. After the RPA amplification was completed, the tube was centrifuged briefly to mix the RPA amplification product in the tube with the Crispr Cas12a / 13a system on the tube cap. The Crispr reaction was performed at 37°C using the Q5 real-time PCR system (Thermo Fisher Scientific, USA) according to the above 5 groups of different time periods, and the fluorescence intensity was monitored every 30 seconds. The results are shown in Figure 8As shown in B, the fluorescence intensity of the Crispr Cas12a / 13a reaction also showed relative stability after 20 minutes, so the total reaction time of the RPA / Crispr Cas12a / Cas13a system was set to 40 minutes.

[0265] Example 8

[0266] This embodiment provides a method and application of RPA / Crispr Cas12a / Cas13a system to detect EV71 and CVA16. Fig. 9 As shown, the following steps are included:

[0267] (1) Extraction of total RNA from samples to be tested;

[0268] (2) Preparation of RPA solution A and Crispr Cas12a / 13a solution B

[0269] Solution A was prepared as shown in Table 3 RPA reaction system (RNA template and Buffer B were not added here), and the above solution was mixed with the dry reaction mixture in the detection unit tube (provided by the in vitro constant temperature RPA reverse transcription kit, purchased from Le Shang Bio) and the dry reaction mixture was completely resuspended by vortexing;

[0270] Solution B was prepared as shown in Table 4 Crispr Cas12a / 13a reaction system;

[0271] Table 3 RPA reaction system

[0272]

[0273] The nucleotide sequence of EV71-Primer-F1 is shown in SEQ ID NO.1, and the nucleotide sequence of EV71-Primer-R3 is shown in SEQ ID NO.6; the nucleotide sequence of CVA16-Primer-F2 is shown in SEQ ID NO.9, and the nucleotide sequence of CVA16-Primer-R1 is shown in SEQ ID NO.8.

[0274] Table 4 Crispr Cas12a / 13a reaction system

[0275]

[0276] The above-mentioned probe group adopts the first probe group in Example 1, wherein the nucleotide sequence of the ssDNA probe is shown in SEQ ID NO.13; the nucleotide sequence of the ssRNA probe is shown in SEQ ID NO.15; the EV71-crRNA nucleotide sequence is shown in SEQ ID NO.11, and the CVA16-crRNA nucleotide sequence is shown in SEQ ID NO.12.

[0277] (3) RPA isothermal amplification

[0278] Add 2 μL RNA template and 1 μL Buffer B (280 nM magnesium acetate) to solution A, quickly mix and centrifuge. To avoid system contamination caused by subsequent centrifuge tube opening, add all of the above solution B to the cap of the centrifuge tube containing the above solution A, and quickly place it in a thermostat or metal bath at 42°C for incubation for 20 minutes to obtain the amplified product.

[0279] (4) Crispr Cas12a / 13a system detection

[0280] After RPA amplification, the tube was centrifuged briefly to mix the RPA amplification product in the tube with the Crispr Cas12a / 13a system on the tube cap. The Crispr reaction was performed at 37°C using the Q5 real-time PCR system (Thermo Fisher Scientific, USA) for 20 minutes, and the fluorescence reading was recorded every 30 seconds. When the target gene is present, the trans-cleavage activity of Cas12a / Cas13a is triggered, resulting in the cleavage of ssDNA or ssRNA probes, thereby emitting a fluorescent signal; conversely, when the target gene is not present, no fluorescent signal is observed.

[0281] (5) Clinical application of the above detection methods

[0282] a. Accuracy

[0283] Total RNA was extracted from 29 clinical blood samples (RNA extraction kit purchased from Shanghai Bioengineering) and tested using the above method. The results are as follows: Fig.10 As shown, among the 29 clinical samples, 18 EV71-positive samples, 10 CVA16-positive samples, 6 samples that were both EV71 and CVA16-positive, and 7 samples that were both EV71 and CVA16-negative were detected.

[0284] The above 29 clinical blood samples were tested by qRT-PCR method. The results are as follows: Fig.11 As shown, among the 29 clinical samples, 18 EV71 positive samples, 10 CVA16 positive samples, 6 samples positive for both EV71 and CVA16, and 7 samples negative for both EV71 and CVA16 were detected. The results are completely consistent with the above test results, indicating that the method of the present invention has a high accuracy in the detection of EV71 and CVA16.

[0285] b. Sensitivity: The RNA template was diluted 10-fold in series, with the concentration ranging from 10^5 to 10^0 copies / μl, and the above method was used for detection. The results are as follows Fig.12As shown, the detection sensitivity of this method can reach 1×10^0 copies / μl.

[0286] c. Specificity: The above method was used to detect samples involving a variety of viral specimens, including EV71, CVA16, human cytomegalovirus, Epstein-Barr virus (EB), influenza A virus (FluA), influenza B virus (FluB), Mycoplasma pneumoniae (MP) and adenovirus (ADV). The results are as follows Fig.13 As shown, fluorescence signals were observed only in the target gene presence group, demonstrating that the method of the present invention is specific for the detection of EV71 and CVA16.

[0287] Example 9

[0288] This embodiment provides another method and application of RPA / Crispr Cas12a / Cas13a system to detect EV71 and CVA16, such as Fig. 9 As shown, the following steps are included:

[0289] (1) Extraction of total RNA from samples to be tested;

[0290] (2) Preparation of RPA solution A and Crispr Cas12a / 13a solution B

[0291] Solution A was prepared as in Example 8;

[0292] The preparation of solution B is different from that of Example 8, which uses the second probe set in Example 1, wherein the nucleotide sequence of the ssDNA probe is shown as SEQ ID NO.14, the nucleotide sequence of the ssRNA probe is shown as SEQ ID NO.16, and the rest is the same as in Example 8.

[0293] (3) RPA isothermal amplification was the same as in Example 8.

[0294] (4) Crispr Cas12a / 13a system detection

[0295] After RPA amplification, centrifuge briefly to mix the RPA amplification product in the tube with the Crispr Cas12a / 13a system on the tube cap. Incubate the Crispr reaction in a metal bath at 37°C for 20 min. When the reaction is complete, add 20 μL of RNase-free water to the reaction tube and place the test strip ( -JY0308) is placed in a reaction tube and reacted for 3min-10min. The result can be directly displayed and read. When the C line of the test strip is colored and the T1 line appears red, it means that the sample to be tested is CVA16 positive, and the T2 line appears blue, which means that the sample to be tested is EV71 positive. When only the C line of the test strip is colored, it means that the samples to be tested are both CVA16 and EV71 negative.

[0296] (5) Clinical application of the above detection methods

[0297] a. Accuracy

[0298] The same 29 clinical blood samples in Example 8 were tested using the above method, and the color development of the test line on the test strip was statistically analyzed. The results are as follows: Fig.14 As shown, among the 29 clinical samples, 18 EV71-positive samples, 10 CVA16-positive samples, 6 samples positive for both EV71 and CVA16, and 7 samples negative for both EV71 and CVA16 were detected. This result is highly consistent with the method used in Example 8 and the qRT-PCR test results, proving that the method of the present invention has a high accuracy in the detection of EV71 and CVA16.

[0299] b. Sensitivity: The RNA template was diluted 10-fold in series, with concentrations ranging from 10^5 to 10^0 copies / μl, and the above method was used for detection. The results are shown in Figure 15. The detection sensitivity of this method can reach 10^1 copies / μl.

[0300] c. Specificity: The above method was used to detect samples involving a variety of viral specimens, including EV71, CVA16, human cytomegalovirus, Epstein-Barr virus (EB), influenza A virus (FluA), influenza B virus (FluB), Mycoplasma pneumoniae (MP) and adenovirus (ADV). The results are as follows Fig.16 As shown, when the target gene is missing, only the control line (C) appears on the test strip, and the test lines T1 and T2 have no color, indicating that the above detection method is specific.

Claims

1. A crRNA group for detecting EV71 and CVA16, characterized in that: It includes EV71-crRNA and CVA16-crRNA, the nucleotide sequence of the EV71-crRNA is shown in SEQ ID NO.11; the nucleotide sequence of the CVA16-crRNA is shown in SEQ ID NO.

12.

2. A primer set for detecting EV71 and CVA16, characterized in that: It includes EV71 primer pair and CVA16 primer pair, wherein: The EV71 primer pair includes an EV71 upstream primer and an EV71 downstream primer. The EV71 upstream primer is selected from any one of the following: EV71-Primer-F1: the nucleotide sequence is shown in SEQ ID NO.1; EV71-Primer-F2: the nucleotide sequence is shown in SEQ ID NO.3; EV71-Primer-F3: the nucleotide sequence is shown in SEQ ID NO.5; The EV71 downstream primer is selected from any one of the following: EV71-Primer-R1: the nucleotide sequence is shown in SEQ ID NO.2; EV71-Primer-R2: the nucleotide sequence is shown in SEQ ID NO.4; EV71-Primer-R3: the nucleotide sequence is shown in SEQ ID NO.6; The CVA16 primer pair includes a CVA16 upstream primer and a CVA16 downstream primer. The CVA16 upstream primer is selected from any one of the following: CVA16-Primer-F1: the nucleotide sequence is shown in SEQ ID NO.7; CVA16-Primer-F2: the nucleotide sequence is shown in SEQ ID NO.9; The CVA16 downstream primer is selected from any one of the following: CVA16-Primer-R1: the nucleotide sequence is shown in SEQ ID NO.8; CVA16-Primer-R2: The nucleotide sequence is shown in SEQ ID NO.

10.

3. A primer set for detecting EV71 and CVA16 according to claim 2, characterized in that: The primer pair for detecting EV71 includes EV71-Primer-F1 and EV71-Primer-R3, and the nucleotide sequences thereof are shown in SEQ ID NO.1 and SEQ ID NO.6, respectively; The primer pair for detecting CVA16 includes CVA16-Primer-F2 and CVA16-Primer-R1, and the nucleotide sequences thereof are shown in SEQ ID NO.9 and SEQ ID NO.8, respectively.

4. Use of the crRNA group described in claim 1 and / or the primer group described in claim 2 or 3 in the preparation of reagents for detecting EV71 and CVA16.

5. A reagent for detecting EV71 and CVA16, characterized in that: Comprising the crRNA group and the primer group described in claim 4.

6. A reagent for detecting EV71 and CVA16 as claimed in claim 5, characterized in that: The reagents also include a probe set; The probe group includes a first probe group or a second probe group; The first probe group includes a first ssDNA probe and a first ssRNA probe, the nucleotide sequence of the first ssDNA probe is shown in SEQ ID NO.13, and the nucleotide sequence of the first ssRNA probe is shown in SEQ ID NO.15; The second probe group includes a second ssDNA probe and a second ssRNA probe, the nucleotide sequence of the second ssDNA probe is shown in SEQ ID NO.14, and the nucleotide sequence of the second ssRNA probe is shown in SEQ ID NO.

16.

7. A kit for detecting EV71 and CVA16, characterized in that: The kit comprises the reagent according to claim 5 or claim 6.

8. Use of the reagent according to claim 5 or 6, and / or the kit according to claim 7 in detecting EV71 and CVA16, characterized in that: The application comprises the following steps: S1: Obtain the RNA of the sample to be tested; S2: using the RNA as a template and the primer set to perform RPA isothermal amplification to obtain an RPA amplification product; S3: reacting the RPA amplification product, probe set, and crRNA set in a Crispr Cas12a / Cas13a system; S4: Detecting the reaction results of the Crispr Cas12a / 13a system; In S3, the first probe group is used to perform detection by fluorescence method; in S3, the second probe group is used to perform visual detection on a test paper using lateral flow chromatography technology.

9. A method for detecting EV71 and CVA16, the method comprising the following steps: S1: Obtain the RNA of the sample to be tested; S2: using the RNA as a template and the primer set to perform RPA isothermal amplification to obtain an RPA amplification product; S3: reacting the RPA amplification product, probe set, and crRNA set in a Crispr Cas12a / 13a system; S4: Detecting the reaction results of the Crispr Cas12a / 13a system; In S3, the first probe group is used to perform detection by fluorescence method; in S3, the second probe group is used to perform visual detection on a test paper using lateral flow chromatography technology.

10. The use according to claim 8 or the detection method according to claim 9, characterized in that: In the RPA isothermal amplification reaction system, The primer set concentration ratio is EV71 400nM:CVA16 100nM to EV71 100nM:CVA16 400nM; and / or The total volume of the system is 50 μL to 10 μL; and / or The amplification time is 10 min to 30 min; and / or In the reaction system of the Crispr Cas12a / 13a system, Mg 2+ The concentration is 0mM to 20mM; and / or The reaction time is 10min to 30min.

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