A one-tube rapid detection kit for Zika virus and Japanese encephalitis virus and its application

Abstract

The invention relates to the technical field of pathogen detection, and discloses a kit for rapid detection and simultaneous identification of Zika virus and Japanese encephalitis virus by a one-tube method and an application thereof. The kit comprises an enzyme dry powder group, an RPA dual amplification system and a dual-target nucleic acid test strip, wherein the RPA dual amplification system comprises a ZIKV-F / ZIKV-R primer pair, a JEV-F / JEV-R primer pair, a ZIKV-Pb probe and a JEV-Pb probe; the ZIKV-F / ZIKV-R primer pair comprises SEQ ID NO.1 and SEQ ID NO.2; the JEV-F / JEV-R primer pair comprises SEQ ID NO.4 and SEQ ID NO.5; the ZIKV-Pb probe comprises SEQ ID NO.3; and the JEV-Pb probe comprises SEQ ID NO.6. This scheme designs specific primer pairs and probes for the genomic RNA of the two viruses, and modifies the primers / probes of the two viruses with different positioning antigen molecules and color antigen molecules, so as to facilitate the rapid and simultaneous detection of whether the sample contains one or both of the two viruses, thereby achieving simultaneous detection of the two viruses.

Description

Technical Field

[0001] The present invention relates to the technical field of pathogen detection, and in particular to a kit for rapid detection of Zika virus and Japanese encephalitis virus using a one-tube method and an application thereof. Background Art

[0002] Zika virus (ZIKV) and Japanese encephalitis virus (JEV) are both members of the Flaviviridae family, a class of enveloped single-stranded positive-sense RNA viruses with a genome size of approximately 11kb. Zika virus was first discovered in African monkeys in 1947 and has since been detected in humans in Uganda and Tanzania. Zika virus is mainly transmitted to humans by mosquitoes and can cause a range of symptoms, including rash, fever, conjunctivitis, etc. The virus is insidious in its activity and has caused many cases of neurological diseases and neonatal malformations in Africa, Asia and other places, attracting global attention. Japanese encephalitis virus is also a mosquito-borne flavivirus and is the main pathogen of human Japanese encephalitis in Asia. It has a high mortality rate and can cause a variety of neurological sequelae. Given the similar transmission routes and clinical symptoms of Zika virus and Japanese encephalitis virus, rapid and accurate detection and identification of these two viruses is crucial for timely prevention and control measures.

[0003] Currently, the commonly used methods for diagnosing Zika virus and Japanese encephalitis virus are based on RT-PCR and RT-qPCR techniques. In addition, isothermal amplification techniques such as RT-LAMP (reverse transcription loop-mediated isothermal amplification) are also used to detect the RNA of these two viruses. RT-LAMP technology uses 6 pairs of primers to react at 65-72℃ isothermal conditions for 40-60 minutes to amplify viral RNA.

[0004] Although technologies such as RT-qPCR and RT-LAMP have played an important role in diagnosing Zika virus and Japanese encephalitis virus, the existing technologies still have the following technical problems: (1) High false positive rate: RT-LAMP technology reacts at a relatively high temperature and has a large number of primers, which can easily produce false positive results. (2) Low detection efficiency: Given the similar transmission routes and clinical symptoms of Zika virus and Japanese encephalitis virus, the existing technology cannot quickly and accurately identify these two viruses at the same time. (3) Long detection time and non-portability: RT-PCR and RT-qPCR require complex thermal cycling equipment and cannot achieve rapid on-site detection, resulting in the inability to quickly and accurately identify and prevent the disease in the early stages of transmission.

[0005] In view of this, the development of a one-tube rapid detection kit for Zika virus and Japanese encephalitis virus not only effectively makes up for the shortcomings of existing technologies, but also can quickly detect and identify both at one time, which is of great significance for the accurate identification and prevention and control of the two viruses in the early stages of transmission. Summary of the invention

[0006] The present invention aims to provide a kit and application for rapid detection and identification of Zika virus and Japanese encephalitis virus in one tube, so as to solve the technical problem that the prior art cannot rapidly detect Zika virus and Japanese encephalitis virus at the same time, resulting in the inability to rapidly identify the cause of the disease for prevention and treatment.

[0007] To achieve the above object, the present invention adopts the following technical scheme: a kit for rapid detection of Zika virus and Japanese encephalitis virus by one-tube method, comprising an enzyme dry powder group, an RPA dual amplification system and a dual-target nucleic acid test strip, the RPA amplification system comprising a ZIKV-F / ZIKV-R primer pair, a JEV-F / JEV-R primer pair, a ZIKV-Pb probe and a JEV-Pb probe;

[0008] The ZIKV-F / ZIKV-R primer pair includes SEQ ID NO.1 and SEQ ID NO.2; the JEV-F / JEV-R primer pair includes SEQ ID NO.4 and SEQ ID NO.5; the ZIKV-Pb probe includes SEQ ID NO.3; and the JEV-Pb probe includes SEQ ID NO.6.

[0009] Preferably, as an improvement, the 5' ends of the ZIKV-R primer, ZIKV-Pb probe, JEV-R primer and JEV-Pb probe are connected with antigen molecules, and the antigen molecules connected to the 5' ends of ZIKV-R, ZIKV-Pb, JEV-R and JEV-Pb are different.

[0010] Preferably, as an improvement, the antigen molecule is any one of biotin, tetramethylrhodamine, FAM, FITC or other antigen molecules that match the antibody or specific binding molecule on the dual-target nucleic acid test strip.

[0011] Preferably, as an improvement, the antigen molecules are divided into colorimetric antigen molecules and localization antigen molecules, and both the colorimetric antigen molecules and the localization antigen molecules are biotin, tetramethylrhodamine, digoxigenin, FAM, FITC or any other antigen molecules that match the antibodies or specific binding molecules on the dual-target nucleic acid test strip.

[0012] Preferably, as an improvement, the 5' end modification of the ZIKV-F / ZIKV-R primer, ZIKV-Pb probe, JEV-F / JEV-R primer pair, and JEV-Pb probe is (a) or (b) or (c) or (d):

[0013] (a) The 5' ends of the ZIKV-R primer and the JEV-R primer are connected to the localization antigen molecule, and the 5' ends of the ZIKV-Pb probe and the JEV-Pb probe are connected to the color development antigen molecule;

[0014] (b) The 5' ends of the ZIKV-R primer and the JEV-Pb probe are connected to a color developing antigen molecule, and the 5' ends of the ZIKV-Pb probe and the JEV-R primer are connected to a localizing antigen molecule;

[0015] (c) The 5' ends of the ZIKV-R primer and the JEV-Pb probe are connected to the localization antigen molecule, and the 5' ends of the JEV-R primer and the ZIKV-Pb probe are connected to the color development antigen molecule;

[0016] (d) The 5' ends of ZIKV-R primer and JEV-R primer are connected to color antigen molecules, and the 5' ends of ZIKV-Pb probe and JEV-Pb probe are connected to localization antigen molecules.

[0017] Preferably, as an improvement, the dual-target nucleic acid test strip comprises colorimetric microspheres modified with two different antibodies corresponding to the colorimetric antigen molecules, colorimetric microspheres modified with immunoglobulin Y antibodies, two different antibodies corresponding to the positioning antigen molecules, and immunoglobulin Y.

[0018] Specifically, the dual-target nucleic acid test strip includes a bottom plate, a nitrocellulose membrane fixed in the middle of the bottom plate, a sample pad and a water-absorbing pad fixed at both ends of the bottom plate; the edges of the sample pad and the water-absorbing pad are respectively overlapped on both ends of the nitrocellulose membrane, and a binding pad is overlapped between the sample pad and the nitrocellulose membrane. There are detection line T1 line 1, detection line T2 line 2 and quality control line C line 3 on the nitrocellulose membrane, and immunoglobulin Y is fixed on the quality control line C line. Two different antibodies corresponding to the positioning antigen molecules are provided on the detection line T1 line and the detection line T2 line (such as streptavidin for biotin and digoxin antibody for digoxin), which are used to recognize the positioning antigen molecules on the amplification product and realize the positioning of the amplification product on the dual-target nucleic acid test strip. The dual-target nucleic acid test strip also contains color-developing microspheres modified with two different antibodies corresponding to the color-developing antigen molecules (such as FAM antibody for FAM and tetramethylrhodamine antibody for tetramethylrhodamine) and color-developing microspheres modified with immunoglobulin Y antibodies.

[0019] Preferably, as an improvement, the ZIKV-Pb probe and the JEV-Pb probe are modified with a cleavage recognition group, and a blocking group is connected to the 3' end; the blocking group is used to block the amplification of the 3' end of the probe, and the cleavage recognition group is used to be recognized and cleaved by the cleavage enzyme; the cleavage recognition group is alkaline furan, called dSpacer; or THF, called tetrahydrofuran residue; the blocking group is C3 spacer, i.e., C3 spacer.

[0020] Preferably, as an improvement, the enzyme dry powder group includes recombinase, single-stranded DNA binding protein, DNA polymerase, reverse transcriptase and exonuclease IV, and the exonuclease IV is used to recognize and cut the cutting recognition group in the ZIKV-Pb probe and JEV-Pb probe.

[0021] Preferably, as an improvement, the RPA amplification system further comprises a rehydration solution and a magnesium acetate solution.

[0022] The principles and advantages of this solution are:

[0023] The RPA reaction system includes recombinase, single-strand DNA binding protein, DNA polymerase, reverse transcriptase and endonuclease IV (nfo enzyme). When the RNA template is added, the reverse transcriptase synthesizes complementary DNA (cDNA) from the RNA template, and the complex formed by the recombinase and the specific primer binds to find the appropriate target site in the double-stranded DNA; after the complex is positioned on the template, the single-strand binding protein immediately binds to the displaced DNA chain and dissociates the recombinase; after the recombinase dissociates, the 3' end of the primer is exposed and recognized by the DNA polymerase, and the DNA polymerase starts to amplify the DNA chain at the 3' end of the primer according to the template sequence to form an amplified product.

[0024] When the nfo probe (i.e., the probe recognized and cut by exonuclease IV) is complementary to one chain of the amplified product, the nfo enzyme will specifically recognize the modified group tetrahydrofuran (THF: a base-free nucleotide analog, which can be added when synthesizing the probe) or the alkaline furan (dSpacer) site on the probe, and cut the furan site, rendering the blocking group at the 3' end of the probe ineffective, so that the polymerase can continue to extend, and finally obtain an amplified product with both a probe marker and a primer marker. Microspheres modified with antibodies corresponding to the marker at one end are fixed on the conjugation pad of the nucleic acid test strip, and antibodies or specific binding molecules that capture the marker at the other end of the amplified product are fixed on the test line and the control line. By fixing the microspheres and antibodies or specific molecules corresponding to the markers at both ends of the two amplified products on the test strip and forming a test line band through the chromatography of the test strip, it is possible to determine which type of viral RNA is contained in the sample, thereby achieving rapid detection and identification of the virus.

[0025] 1. Simultaneous detection of two viruses: Compared with the existing rapid detection technology that cannot detect Zika virus and Japanese encephalitis virus at the same time, this solution designs specific primer pairs and probes for the genomic RNA of the two viruses, and modifies the primers / probes of the two viruses with different positioning antigen molecules and color antigen molecules, so as to facilitate the simultaneous rapid detection of whether the sample contains one or both of the two viruses, thereby realizing the simultaneous detection of the two viruses.

[0026] 2. Prevent the two viruses from interfering with each other: This solution modifies the primer pairs and probes of the two viruses with different positioning antigen molecules and color-developing antigen molecules, so that the two do not interfere with each other in displaying the test results. At the same time, it effectively avoids the false positive test results of the prior art, and can also effectively avoid the "contamination" phenomenon caused by the simultaneous amplification of two DNAs, which makes the test results inaccurate, and effectively improves the accuracy of the test results.

[0027] 3. High detection accuracy: This solution performs high-fold specific amplification of the RNA of the two viruses, and displays the obtained amplified products with different labels on nucleic acid test strips, and the test results can be directly read. The applicant has found through long-term experiments that even if the viral genome nucleic acid content in the sample is as low as pg level, the test results can still be clearly read on the nucleic acid test strips, which has the advantages of high sensitivity, high specificity and rapid visual detection.

[0028] Preferably, as an improvement, the present scheme provides an application of a one-tube method kit for rapid detection of Zika virus and Japanese encephalitis virus, comprising adding the sample RNA to be tested to the RPA amplification system to amplify the amplified product, and then dropping the amplified product on a dual-target nucleic acid test strip to display the test result.

[0029] Technical effect: This scheme adopts the above-mentioned setting to facilitate the rapid detection of ZIKV and JEV. This scheme is the first to use a one-tube method to amplify and detect ZIKV and JEV, which takes only 25 minutes and can quickly identify ZIKV and JEV. It has the advantages of high sensitivity, good specificity, short time consumption, and no reliance on large-scale experimental equipment. These advantages make the one-tube method developed by the present invention a promising method for large-scale pathogen screening and identification diagnosis at ports and on-site sites. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Schematic diagram of the structure and detection principle of the dual-target nucleic acid test strip in Example 1 of the present invention.

[0031] Figure 2 The results of different samples detected by the one-tube rapid detection kit for Zika virus and Japanese encephalitis virus in Example 1 of the present invention are compared.

[0032] Figure 3 This is the sensitivity test result of the kit for rapid detection of Zika virus and Japanese encephalitis virus in one tube in Example 1 of the present invention when detecting genomic RNA containing ZIKV alone.

[0033] Figure 4 This is the sensitivity test result of the kit for rapid detection of Zika virus and Japanese encephalitis virus in one tube in Example 1 of the present invention when detecting genomic RNA containing JEV alone.

[0034] Figure 5 This is the sensitivity test result of the kit for rapid detection of Zika virus and Japanese encephalitis virus in one tube in Example 1 of the present invention when simultaneously detecting genomic RNA containing ZIKV and JEV.

[0035] Figure 6 The test results of the kit for rapid detection of Zika virus and Japanese encephalitis virus in one tube in Example 1 of the present invention are compared for samples containing unknown viruses at different concentrations. DETAILED DESCRIPTION

[0036] The present invention is further described in detail below in conjunction with examples, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the technical means used in the following examples and experimental examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used can be obtained from commercial sources.

[0037] Reference numerals: detection line T1 line 1, detection line T2 line 2, detection line C line 3.

[0038] Example 1

[0039] This scheme provides a one-tube rapid detection kit for Zika virus and Japanese encephalitis virus, including an enzyme dry powder group, an RPA amplification system and a dual-target nucleic acid test strip.

[0040] The enzyme dry powder set (purchased as a reference from Anpu Future Biotechnology Co., Ltd., model number RNA Constant Temperature Rapid Amplification Kit (Colloidal Gold Test Strip Type)) includes recombinase, single-stranded DNA binding protein, DNA polymerase, reverse transcriptase and nuclease IV. Nuclease IV is used to recognize and cut the cleavage recognition group in the ZIKV-Pb probe and JEV-Pb probe.

[0041] The RPA amplification system includes ZIKV-F / ZIKV-R primer pair, JEV-F / JEV-R primer pair, ZIKV-Pb probe and JEV-Pb probe, rehydration solution and magnesium acetate.

[0042] As a reference, the 48 μL amplification buffer system includes: 29.5 μL rehydration solution, ZIKV-F concentration of 340 nM, ZIKV-R concentration of 400 nM, ZIKV-Pb concentration of 40 nM, JEV-F concentration of 200 nM, JEV-R concentration of 300 nM, JEV-Probe concentration of 20 nM, and magnesium acetate concentration of 14 mM.

[0043] As a reference, in this scheme, the ZIKV-F / ZIKV-R primer pair includes SEQ ID NO.1 and SEQ ID NO.2; the JEV-F / JEV-R primer pair includes SEQ ID NO.4 and SEQ ID NO.5; the ZIKV-Pb probe includes SEQ ID NO.3; and the JEV-Pb probe includes SEQ ID NO.6.

[0044] As a reference, the 5' ends of ZIKV-R primer, ZIKV-Pb probe, JEV-R primer, and JEV-Pb probe are connected with antigen molecules, and the antigen molecules connected to the 5' ends of ZIKV-R, ZIKV-Pb, JEV-R, and JEV-Pb are all different. The antigen molecule is any one of biotin, tetramethylrhodamine, digoxigenin, FAM, FITC, or other antigen molecules that match the antibody or specific binding molecule on the dual-target nucleic acid test strip.

[0045] As a reference, antigen molecules are divided into color antigen molecules and localization antigen molecules, and both color antigen molecules and localization antigen molecules are any of biotin, tetramethylrhodamine, digoxigenin, FAM, FITC or other antigen molecules that match the antibodies or specific binding molecules on the dual-target nucleic acid test strip. The 5' end modification of ZIKV-R primer, ZIKV-Pb probe, JEV-R primer pair, and JEV-Pb probe is (a) or (b) or (c) or (d):

[0046] (a) The 5' ends of the ZIKV-R primer and the JEV-R primer are connected to the localization antigen molecule, and the 5' ends of the ZIKV-Pb probe and the JEV-Pb probe are connected to the color development antigen molecule;

[0047] (b) The 5' ends of the ZIKV-R primer and the JEV-Pb probe are connected to a color developing antigen molecule, and the 5' ends of the ZIKV-Pb probe and the JEV-R primer are connected to a localizing antigen molecule;

[0048] (c) The 5' ends of the ZIKV-R primer and the JEV-Pb probe are connected to the localization antigen molecule, and the 5' ends of the JEV-R primer and the ZIKV-Pb probe are connected to the color development antigen molecule;

[0049] (d) The 5' ends of ZIKV-R primer and JEV-R primer are connected to color antigen molecules, and the 5' ends of ZIKV-Pb probe and JEV-Pb probe are connected to localization antigen molecules.

[0050] As a reference, the ZIKV-Pb probe and the JEV-Pb probe are modified with a cleavage recognition group, and a blocking group is connected to the 3' end; the blocking group is used to block the amplification of the 3' end of the probe, and the cleavage recognition group is used to be recognized and cleaved by the cleavage enzyme; the cleavage recognition group is alkaline furan, also called dSpacer; or THF, called tetrahydrofuran residue; the blocking group is C3 spacer, i.e. C3 spacer.

[0051] Specifically, as a reference, in the present embodiment, the 5' end of the ZIKV-R primer is connected with tetramethylrhodamine (TAMRA), and the 5' end of the ZIKV-Pb probe is connected with the antigen molecule digoxin (DIG); the 5' end of the primer JEV-R primer is connected with the antigen molecule biotin (Biotin), and the 5' end of the JEV-Pb probe is connected with carboxyfluorescein (FAM). The 3' ends of the ZIKV-Pb probe and the JEV-Pb probe are both connected with the blocking group C3 spacer, and the middle parts of the ZIKV-Pb probe and the JEV-Pb probe are both modified with the cutting recognition group dSpacer. The nucleotide sequences of the ZIKV-F / ZIKV-R primer pair, the ZIKV-Pb probe, the JEV-F / JEV-R primer pair and the JEV-Pb probe in the present embodiment are shown in Table 1.

[0052] Table 1 Primer pair sequences in this protocol

[0053]

[0054] In addition, this scheme also provides a dual-target nucleic acid test strip, the structure of which is as follows: it includes a bottom plate, a nitrocellulose membrane fixed in the middle of the bottom plate, a sample pad and a water absorbent pad fixed at both ends of the bottom plate; the edges of the sample pad and the water absorbent pad are respectively overlapped on both ends of the nitrocellulose membrane, and a binding pad is overlapped between the sample pad and the nitrocellulose membrane; there are detection line T1 line 1, detection line T2 line 2 and quality control line C line 3 on the nitrocellulose membrane, immunoglobulin Y is fixed on the quality control line C line, and different positioning antibody molecules are fixed on the detection line T1 line 1 and the detection line T2 line 2, and the antibody molecules correspond to the positioning antigen molecules on the primer / probe. The dual-target nucleic acid test strip also contains color-developing microspheres modified with two different antibodies corresponding to the color-developing antigen molecules, and color-developing microspheres modified with immunoglobulin Y antibodies.

[0055] As a reference, Figure 1As shown, the dual-target nucleic acid test strip binding pad contains color microspheres modified with FAM antibodies, color microspheres modified with tetramethylrhodamine antibodies, and color microspheres modified with immunoglobulin Y antibodies. When the positioning antigen molecules on the primers / probes of JEV and ZIKV are digoxin or biotin, respectively, streptavidin is coated on the detection line T1 line 1, digoxin antibody is coated on the detection line T2 line 2, and immunoglobulin Y is coated on the control line C line 3, so that digoxin antibody captures digoxin, streptavidin captures biotin, and immunoglobulin Y captures color microspheres modified with immunoglobulin Y antibodies, so that the detection line T1 line 1 and the detection line T2 line 2 detect JEV or ZIKV respectively, and the control line C line 3 is used for positive control.

[0056] This solution also provides the application of a one-tube method kit for rapid detection of Zika virus and Japanese encephalitis virus, including adding the sample RNA to be tested to the RPA amplification system to amplify the amplification product, and then dropping the amplification product on a dual-target nucleic acid test strip to display the test result.

[0057] Experimental Example 1: Specificity test of this kit

[0058] The samples in this implementation case are RNA extracted from cells containing JEV, ZIKV, influenza virus A (influenza Avirus), adenovirus 5 (adenovirus), dengue virus (dengue virus), positive saliva containing the new coronavirus (COVID-19) and the saliva of healthy people.

[0059] The detection steps are as follows:

[0060] (1) Extraction of nucleic acid from the sample to be tested

[0061] RNA from serum, cell or tissue samples was extracted using a viral RNA extraction kit (purchased as a reference from Anpu Future Biotechnology Co., Ltd., model: RNA Constant Temperature Rapid Amplification Kit (Colloidal Gold Test Strip Type)).

[0062] (2) RT-RPA amplification

[0063] Add amplification buffer to enzyme powder, and after it is fully dissolved, add extracted RNA and react at 39°C for 20 minutes.

[0064] (3) Nucleic acid test strip detection

[0065] Take 10 μL of amplification product and mix it with 190 μL of distilled water, then immerse the dual-target nucleic acid test strip in the mixture, observe the control line and the detection line, and take out the test strip 2 minutes after the liquid reaches the top of the test strip.

[0066] Figure 2The test results are shown (image captions: 1, JEV; 2, ZIKV; 3, influenza virus A; 4, adenovirus 5; 5, new coronavirus; 6, dengue virus; 7, saliva of a healthy person; N, sterile water).

[0067] The experimental results show that the kit can effectively and specifically detect samples containing genomic RNA of ZIKV and JEV, and the test results can be quickly read through the test strips.

[0068] Experimental Example 2: Sensitivity test of the kit of this solution

[0069] In the sensitivity test case, 10.0 ng of genomic RNA of ZIKV and JEV were prepared and diluted 10 times respectively. The obtained genomic RNA was amplified by the kit of this protocol and tested by test strips. The steps are as follows:

[0070] (1)RPA amplification

[0071] Add amplification buffer to enzyme powder, and after it is fully dissolved, add extracted RNA and react at 39°C for 20 minutes.

[0072] (2) Nucleic acid test strip detection

[0073] Take 10 μL of amplification product and mix it with 190 μL of distilled water, then immerse the dual-target nucleic acid test strip in the mixture, observe the control line and the test line, and take out the test strip 2 minutes after the liquid reaches the top of the test strip. Figure 3 to Figure 5 As shown: Figure 3 It was demonstrated that when only ZIKV RNA was added to the detection system, the detection sensitivity was 1.0 pg; Figure 4 It was demonstrated that when only JEV RNA was added to the detection system, the detection sensitivity was 0.1 pg; Figure 5 It was demonstrated that when the genomic RNA of ZIKV and JEV were simultaneously added into the detection system, the detection sensitivities were 1.0pg and 10pg, respectively.

[0074] The experimental results show that when the genomic RNA containing ZIKV is detected alone, even if the genomic RNA content is as low as 1pg, the test result can still be clearly read on the test strip; and when the genomic RNA containing JEV is detected alone, even if the genomic RNA content is as low as 0.1pg, the test result can still be clearly read on the test strip, and the detection sensitivity of both is very high. Because the kit of the present invention is mainly used for the identification of ZIKV and JEV, the actual situation of containing two viruses at the same time is rare, so the sensitivity meets the actual needs.

[0075] Experimental Example 3: This kit detects viruses in saliva

[0076] In order to verify whether this kit can detect viruses in saliva, 200L of saliva from healthy people was taken, and 10μL, 20μL, 40μL and 80μL of cell suspension containing viruses were added respectively to simulate actual samples. The detection steps are as follows:

[0077] (1) Extraction of nucleic acid from the sample to be tested

[0078] Extract RNA from saliva samples using a viral RNA extraction kit.

[0079] (2)RPA amplification

[0080] Add amplification buffer to enzyme powder, and after it is fully dissolved, add extracted RNA and react at 39°C for 20 minutes.

[0081] (3) Nucleic acid test strip detection

[0082] Take 10 μL of amplification product and mix it with 190 μL of distilled water, then immerse the dual-target nucleic acid test strip in the mixture, observe the control line and the test line, and take out the test strip 2 minutes after the liquid reaches the top of the test strip. Figure 5 As shown: This kit can detect viral RNA contained in 10μL cell suspension, indicating that it can meet actual needs.

[0083] The above is only an embodiment of the present invention, and the common knowledge such as the known specific technical solutions and / or characteristics in the solution is not described in detail here. It should be pointed out that for those skilled in the art, without departing from the technical solution of the present invention, several modifications and improvements can be made, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.

Claims

1. A kit for rapid detection of Zika virus and Japanese encephalitis virus by one-tube method, characterized in that: It comprises an enzyme dry powder group, an RPA dual amplification system and a dual-target nucleic acid test strip, wherein the RPA amplification system comprises: a primer pair consisting of ZIKV-F and ZIKV-R, a primer pair consisting of JEV-F and JEV-R, a ZIKV-Pb probe and a JEV-Pb probe; the primer sequences of the ZIKV-F and ZIKV-R are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively, the primer sequences of the JEV-F and JEV-R are shown in SEQ ID NO.4 and SEQ ID NO.5, respectively, the sequence of the ZIKV-Pb probe is shown in SEQ ID NO.3, and the sequence of the JEV-Pb probe is shown in SEQ ID NO.6; The 5' end of the ZIKV-R is connected to tetramethyl rhodamine, and the 5' end of the ZIKV-Pb is connected to the antigen molecule digoxin; the 5' end of the JEV-R is connected to the antigen molecule biotin, and the 5' end of the JEV-Pb is connected to FAM; The structure of the dual-target nucleic acid test strip is as follows: it includes a bottom plate, a nitrocellulose membrane fixed in the middle of the bottom plate, and a sample pad and a water absorbent pad fixed at both ends of the bottom plate; the edges of the sample pad and the water absorbent pad are respectively overlapped on both ends of the nitrocellulose membrane, and a binding pad is overlapped between the sample pad and the nitrocellulose membrane; there are a detection line T1 line 1, a detection line T2 line 2 and a quality control line C line 3 on the nitrocellulose membrane, immunoglobulin Y is fixed on the quality control line C line 3, and different positioning antibody molecules are fixed on the detection line T1 line 1 and the detection line T2 line 2, and the positioning antibody molecules correspond to the positioning antigen molecules digoxin and biotin on the primer and the probe; The binding pad of the dual-target nucleic acid test strip contains color-developing microspheres modified with FAM antibody, color-developing microspheres modified with tetramethylrhodamine antibody, and color-developing microspheres modified with immunoglobulin Y antibody; The enzyme dry powder group comprises a recombinase, a single-stranded DNA binding protein, a DNA polymerase, a reverse transcriptase and a nuclease exonuclease IV, wherein the nuclease exonuclease IV is used for recognizing and cutting the cutting recognition groups in the ZIKV-Pb probe and the JEV-Pb probe.

2. The kit for rapid detection of Zika virus and Japanese encephalitis virus by one-tube method according to claim 1, characterized in that: The RPA amplification system also includes a rehydration solution and a magnesium acetate solution.

3. A method for using a kit for rapid detection of Zika virus and Japanese encephalitis virus using a one-tube method according to any one of claims 1 to 2, characterized in that: The method includes adding the sample RNA to be tested to the RPA amplification system to amplify and obtain the amplification product, and then dropping the amplification product on a dual-target nucleic acid test strip to display the test result; the method is not for diagnostic purposes.

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