CN111996291A

CN111996291A - Reagent, kit, detection method and application for detecting West Nile virus

Info

Publication number: CN111996291A
Application number: CN202010852040.4A
Authority: CN
Inventors: 吴江; 林彦星; 黄超华; 史卫军; 花群义; 阮周曦; 贾鹏; 曹琛福; 曾少灵; 林永涛
Original assignee: Shenzhen Customs Animal and Plant Inspection and Quarantine Technology Center
Current assignee: Shenzhen Customs Animal and Plant Inspection and Quarantine Technology Center
Priority date: 2020-08-21
Filing date: 2020-08-21
Publication date: 2020-11-27

Abstract

The application discloses a reagent, a kit, a detection method and application for detecting West Nile virus. The reagent comprises a primer pair and a probe, wherein the upstream primer and the downstream primer of the primer pair are respectively a sequence shown in Seq ID No.1 and a sequence shown in Seq ID No.2, and the probe is a sequence shown in Seq ID No.3 or a reverse complementary sequence thereof; in the probe, the 30 th base modifies a fluorescent group 6-FAM-Dt, the 31 st base is replaced by a base analogue dSpacer, the 34 th base modifies a fluorescent quenching group BHQ1-dT, and the 3' end modifies C3 Spacer. The reagent can perform sensitive, specific and efficient detection on WNV through RT-RPA, has short time consumption, high sensitivity and low requirement on hardware equipment, does not need complex sample treatment, and is particularly suitable for field rapid detection; the method has great significance for WNV rapid prevention and control, epidemic situation propagation prevention, economic loss reduction and animal husbandry breeding production safety guarantee.

Description

Reagent, kit, detection method and application for detecting West Nile virus

Technical Field

The application relates to the field of West Nile virus detection, in particular to a reagent, a kit, a detection method and application for West Nile virus detection.

Background

West Nile Virus (WNV) is an arbovirus, belonging to the Flaviviridae family of Flaviviridae genus, a single-stranded RNA virus, affecting birds, humans and horses. The greatest harm of the disease is to cause fatal encephalitis to people and horses, death of small animals such as chickens and birds, and the like, which cause serious public health problems. Therefore, the rapid and accurate diagnosis of the West Nile virus has great significance for the prevention and control of epidemic diseases.

Wild birds are the natural hosts of west nile virus, culex is the main transmission medium, and the virus lives in nature through the biological transmission chain of bird-mosquito-bird, human and other animals. In 1937, the virus was discovered in Wuganda in West Nile, and was named accordingly. In 2000, 439 cases of neuroinvasive diseases were found in the middle and north of israel, of which 29 died. In 2017, more than 2000 cases of west nile virus infection occurred in the united states, with patients approaching 2/3 showing severe neuroinvasive disease. The disease causes great harm to the health of human beings and the development of animal husbandry, and is listed as one of the current global major epidemics by the world health organization.

With the economic development, frequent international trips, migratory birds and the trade of imported animals and animal products, the risk of WNV is present in all countries. In order to prevent the virus strain of the West Nile virus from being introduced into China from abroad, a simple, convenient, rapid and accurate diagnosis method is established, which is beneficial to cutting off the pathogen transmission as early as possible and reducing the economic loss.

At present, the pathogen detection method of the disease comprises virus separation test, RT-PCR, fluorescent quantitative PCR, nested PCR, LAMP, second-generation sequencing technology and the like, and the antibody detection method comprises ELISA and Plaque Reduction Neutralization Test (PRNT). However, these detection techniques are complicated to operate, have high technical requirements on operators, have long detection time, and are difficult to apply to field detection. Therefore, it is highly desirable to establish a WNV detection method that is better suited for rapid field detection to prevent the introduction or diffusion of WNV and avoid serious public health problems.

Disclosure of Invention

The application aims to provide a novel reagent, a kit, a detection method and application for detecting West Nile virus.

The following technical scheme is adopted in the application:

one aspect of the application discloses a reagent for detecting West Nile virus, which comprises a primer pair and a probe, wherein an upstream primer of the primer pair is a sequence shown by Seq ID No.1, a downstream primer of the primer pair is a sequence shown by Seq ID No.2, and the probe is a sequence shown by Seq ID No.3 or a reverse complementary sequence of the sequence shown by Seq ID No. 3;

Seq ID No.1：5’-CCTGATTGAATTGGAACCACCCTTTGGAGACTCAT-3’

Seq ID No.2：5’-AGTCCCAAGCTGTATCTCCTAGGGCGGCTAATCTC-3’

Seq ID No.3：

5’-CATAGTGGTGGGCAGAGGAGAACAACAGATTAATCACCATTGGCACAAGTC-3’

in the probe with the sequence shown in Seq ID No.3, the 30 th base modifies a fluorescent group 6-FAM-Dt, the 31 st base is replaced by a base analog dSpacer, the 34 th base modifies a fluorescence quenching group BHQ1-dT, and the 3' end modifies C3 Spacer.

It should be noted that, in the reagent of the present application, the primer pair and the probe are designed for the conserved segment of the west nile gene, and are particularly used for the primers and the probes for one-step reverse transcription recombinase polymerase amplification. One-step reverse transcription recombinase polymerase amplification, abbreviated as RT-RPA, is to add reverse transcriptase on the basis of a recombinase polymerase amplification reaction system, so that the reverse transcriptase can directly carry out one-step rapid amplification on an RNA template. Recombinase polymerase amplification, abbreviated as RPA, is a novel isothermal amplification technique of nucleic acids developed by twist dx corporation, uk; one of the keys to this technique is the design of appropriate amplification primer pairs and probes. However, conventional PCR primer pairs and probes are not suitable for RPA; the primers of the conventional PCR are relatively too short and have low recombination efficiency; conventional probe systems are also incompatible with RPA. Therefore, primer pairs and probes suitable for RPA cannot be directly output by conventional primer or probe design software. At present, RPA primer pairs and probes are designed, and a plurality of specific primers and probes are manually designed for experimental screening according to screening guidelines provided on websites of TwistDx companies, so that the primers and probes with high amplification efficiency, high sensitivity and strong specificity are obtained. In one implementation mode of the application, 2 RPA probes are respectively designed, and 6 upstream primers and 6 downstream primers are respectively designed for each probe, and are used for experimental screening, and finally, a primer group of a sequence shown by Seq ID No.1 and a sequence shown by Seq ID No.2 and a probe of a sequence shown by SEQ ID No.3 are screened out and used as the West Nile Virus detection reagent of the application.

It should be further explained that the principle of RPA is to use three enzymes to make a pair of primers exponentially amplify the target at a constant temperature; in the reagent of the present application, it is considered that the RPA amplification product is detected by means of fluorescence detection, and therefore, a corresponding specific probe is provided on the basis of a pair of specific primers. It is understood that the RPA amplification product may also be detected by other means, such as lateral flow test strips, biochips, gel electrophoresis, etc.; therefore, if fluorescence detection is not employed, the probe having the sequence shown in SEQ ID No.3 may not be used. That is, in the reagent of the present application, primers having sequences shown in Seq ID No.1 and Seq ID No.2 may be used alone or together with a probe, and the specific mode of use may be selected depending on the detection conditions and the environment. In one implementation of the present application, the RPA amplification product is detected by fluorescence method, preferably using a primer and a probe together, due to the use of a portable room temperature isothermal amplification fluorescence detector.

In one implementation of the present application, the 5 'end of the probe is labeled with FAM fluorophore, and the 3' end of the probe is labeled with BHQ1 quencher.

It is understood that FAM and BHQ1 are only fluorophores and quenchers specifically used in one implementation of the present application, and do not exclude that other fluorophores or quenchers may also be used, for example, TET, JOE, HEX, CY3, CY5, etc., and that the fluorescence quencher is selected according to the fluorophore, and that the absorption spectrum of the fluorescence quencher may be selected as long as it covers the emission spectrum of the fluorophore, for example, BHQ2, BHQ3, Dabcy1, Tamra, etc.

The application also discloses application of the reagent for detecting the West Nile virus in preparation of a West Nile virus detection kit, a detection test strip or a detection chip.

It can be understood that the reagent of the present application, in fact, can be prepared into various kits, test strips or detection chips for detecting west nile virus, with respect to the specific probe and primer pair designed for west nile virus.

The application also discloses a kit for detecting the West Nile virus, and the kit contains the reagent for detecting the West Nile virus.

In one implementation manner of the application, the kit further contains lyophilized enzyme powder, a rehydration buffer, a magnesium acetate solution, and deionized water. Wherein the freeze-dried enzyme powder is a mixed freeze-dried powder of various enzymes for RT-RPA; the rehydration buffer was the concentrated reaction for RT-RPA. When the detection kit is used, firstly, freeze-dried enzyme powder is prepared into an enzyme solution according to the use requirement, and meanwhile, deionized water is added into a rehydration buffer solution to prepare a reaction solution, then, a magnesium acetate solution, the primers and the probes are added, and finally, a sample to be detected is added, so that the detection reaction can be carried out.

The kit of the present invention is designed for recombinase polymerase amplification detection of west nile virus, and therefore, for convenience of use, the kit may further include a reaction solution, an enzyme, and a reaction additive for one-step reverse transcription recombinase polymerase amplification. Wherein, the reaction solution is a hydration Buffer in an implementation manner of the application, the enzyme can be RPA freeze-dried enzyme powder mixed with a plurality of enzymes, and the reaction additive is MgAc in an implementation manner of the application; in addition, considering the implementation of one-step Reverse transcription, one implementation mode of the application also comprises Reverse Transcriptase MMLV Reverse Transcriptase and RNase Inhibitor recombination RNase Inhibitor.

The application also discloses a method for detecting the West Nile virus for non-diagnosis treatment purposes, which comprises the steps of carrying out one-step reverse transcription recombinase polymerase amplification on a sample to be detected by adopting the reagent for detecting the West Nile virus or the kit for detecting the West Nile virus, and collecting fluorescence by adopting a fluorescence detector.

The detection method is used for rapidly detecting the West Nile virus through one-step reverse transcription recombinase polymerase amplification, and on one hand, the one-step reverse transcription recombinase polymerase amplification detection speed is high, and the detection can be completed only in 15-20 minutes; on the other hand, the whole detection can be completed only at relatively low constant temperature, for example, a portable normal-temperature isothermal amplification fluorescence detector is adopted. Therefore, the detection method is particularly suitable for the field rapid detection of the West Nile virus, and provides powerful scientific basis for the rapid detection, prevention and control of epidemic diseases.

It is understood that the detection reagent, kit and method for west nile virus of the present application, although may provide reference for the diagnosis or treatment of west nile fever; however, the present application is not intended to diagnose West Nile fever by the reagent or method, and the reagent, kit or method is intended to detect related animal products or animal-derived products so as to prevent the West Nile virus from being carried and causing the spread or spread of the West Nile virus. In addition, the reagent, the kit and the method can also be applied to basic research of the West Nile virus.

In one implementation of the present application, the one-step reverse transcription recombinase polymerase amplification reaction conditions are isothermal reaction at 39 ℃ for 15 minutes.

It should be noted that the activity of the enzyme used in the RPA is most suitable for the temperature range of 37-42 deg.C, and the RPA can be detected within twenty minutes; in the preferred scheme of the application, the amplification efficiency of the primer probe is higher, and the isothermal reaction at 39 ℃ is preferred for 15 minutes.

The beneficial effect of this application lies in:

the reagent for detecting the West Nile virus can carry out sensitive, specific and efficient detection on the West Nile virus through one-step reverse transcription recombinase polymerase amplification. The reagent is used for detecting the West Nile virus, has short time consumption, high sensitivity and low requirement on hardware equipment, does not need complex sample treatment, is particularly suitable for on-site rapid detection, and can rapidly obtain a detection result; the method has great significance for the rapid prevention and control of the West Nile virus, the prevention of epidemic spread, the reduction or avoidance of economic loss and the guarantee of human health and the production safety of animal husbandry breeding.

Drawings

FIG. 1 shows the specific detection result of West Nile virus RT-RPA in the present application;

FIG. 2 shows the result of RT-RPA sensitivity detection of West Nile virus in the example of the present application;

FIG. 3 is a graph showing the results of real-time fluorescent quantitative PCR amplification as a comparison in examples of the present application.

Detailed Description

In recent years, the appearance of various constant-temperature nucleic acid amplification technologies solves the limitations of high cost, long time consumption, dependence on a precise temperature cycling instrument and the like of the traditional PCR technology. Among them, Recombinase Polymerase Amplification (RPA) is a novel isothermal nucleic acid amplification technique, and is considered as an isothermal amplification technique that is "most likely to replace conventional PCR".

RPA technology relies primarily on three enzymes: single-stranded DNA binding protein (SSB), recombinase, strand-displacing DNA polymerase. The technical principle is that at a constant temperature, a recombinase is combined with a primer to form a complex, the primer is positioned on a homologous target sequence of a DNA double-stranded template through an enzyme, the template DNA is melted under the assistance of a single-stranded DNA binding protein, a new DNA complementary strand is formed under the action of DNA polymerase, and the cycle is carried out, so that the exponential growth of the DNA is realized. The addition of reverse transcriptase in the reaction system can perform one-step rapid amplification on the RNA template, namely the one-step reverse transcription recombinase polymerase amplification adopted by the application. The optimum reaction temperature range for the RPA is 37 ℃ to 42 ℃ and the reaction time is less than twenty minutes. The real-time RPA detection technology used by combining the fluorescent probe can realize the direct reading of the detection result in the portable constant-temperature amplification fluorescent detector, greatly simplifies the reaction procedure, has better detection time and convenience than the traditional PCR method, and is very suitable for the basic layer of equipment simplification or the field laboratory; therefore, the present application has developed reagents for rapid detection of west nile virus. The reagent comprises a West Nile virus specific primer pair and a probe for one-step reverse transcription recombinase polymerase amplification, wherein the probe is in an amplification target region of the primer pair, an upstream primer of the primer pair is a sequence shown by Seq ID No.1, a downstream primer of the primer pair is a sequence shown by Seq ID No.2, and the probe is a sequence shown by Seq ID No.3 or a reverse complementary sequence of the sequence shown by Seq ID No. 3; wherein, in the probe sequence shown in SEQ ID No.3, the 30 th base modifies a fluorescent group 6-FAM-Dt, the 31 st base is replaced by a base analog dSpacer, the 34 th base modifies a fluorescence quenching group BHQ1-dT, and the 3' end modifies C3 Spacer.

The west nile virus detection reagent of the present application, one of the present applicationIn an implementation mode, the sensitivity can reach 5 multiplied by 10²The copes/mu L can be used for on-site rapid detection of West Nile virus and prevention of epidemic situation introduction, thereby more effectively guaranteeing human health and animal breeding production safety.

The present application will be described in further detail with reference to specific examples. The following examples are intended to be illustrative of the present application only and should not be construed as limiting the present application.

Examples

Materials and methods

1. Plasmid and nucleic acid sample for test

In this example, according to the envelope protein (E) gene of West Nile virus published in NCBI GenBank, the genetic engineering Biotechnology (Shanghai) GmbH synthesized the gene fragment and cloned into PUC15 vector, named WNV-Seg5, as positive plasmid control. The inactivated antigenic nucleic acids used in this example include West Nile virus (WNV-1), Bovine Viral Diarrhea Virus (BVDV), porcine Japanese Encephalitis Virus (JEV), Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), Classical Swine Fever Virus (CSFV), and bluetongue virus (BTV).

2. Primary reagents and instruments

Twist Amp exo kit (twist Dx), MMLV Reverse Transcriptase Transcriptase (Takara), recombined RNase Inhibitor (Takara), constant temperature amplification instrument (Axin), 5415R type high speed centrifuge (Eppendorf), 7500Fast fluorescence PCR instrument (ABI), quantitative micronucleic acid Qubit 4 fluorometer (Invitrogen), and the like.

Design and screening of RT-RPA primers and probes

In this example, multiple specific primers and probes were designed based on the envelope protein (E) gene sequence of the sequence in the West Nile Virus genome, and after the primers and probes were designed, their specificity was determined by BLAST alignment, and they were then used in the subsequent screening experiments, and the specific sequences of the primers and probes are shown in Table 1. All primers and probes of this example were synthesized by Biotechnology (Shanghai) Inc.

TABLE 1 recombinase polymerase amplification primers and probes

Primer or Probe name	Sequence (5 '→ 3')	SEQ ID No.
			WNVF1	CTTTGGCTGGAGCCATTCCTGTGGAATTTTCAAGC		4
WNVF2	CAAGCTTTGGCTGGAGCCATTCCTGTGGAATTTTC
			5
WNVF3	CTCTGCATCAAGCTTTGGCTGGAGCCATTCCTGTG
			6
WNVF4	CCTGATTGAATTGGAACCACCCTTTGGAGACTCAT
			1
WNVF5	GTCCTGATTGAATTGGAACCACCCTTTGGAGACTC			7
		WNVF6	CAAGGTCCTGATTGAATTGGAACCACCCTTTGGAG		8
WNVR1	ACTTGAAAGCCTTTGAACAGACGCCGTAGGTTGTT			9
		WNVR2	CAAGAAACTTGAAAGCCTTTGAACAGACGCCGTAG
	10
		WNVR3	GAGTCCCAAGAAACTTGAAAGCCTTTGAACAGACG	11
WNVR4	CCAAGCTGTATCTCCTAGGGCGGCTAATCTCTGCG
			12
WNVR5	AGTCCCAAGCTGTATCTCCTAGGGCGGCTAATCTC				2
		WNVR6	CCTCCAACTGATCCAAAGTCCCAAGCTGTATCTCC			13
WNVRPA1	CACTGTCAAGTTGACGTCGGGTCATTTGAAGAGAGTGAAGATGGA				14
		WNVRPA2	CATAGTGGTGGGCAGAGGAGAACAACAGATTAATCACCATTGGCACAAGTC				3

In Table 1, WNVF1, WNVF2, WNVF3, WNVF4, WNVF5 and WNVF6 are upstream primers, WNVR1, WNVR2, WNVR3, WNVR4, WNVR5 and WNVR6 are downstream primers, and WNVRPA1 and WNVRPA2 are two designed specific probes; wherein, in the WNVRPAP1 probe with the sequence shown in SEQ ID No.14, the 32 th base modifies a fluorescent group 6-FAM-dT, the 33 th base is replaced by a base analog dSpacer, the 34 th base modifies a fluorescence quenching group BHQ1-dT, and the 3' end modifies C3 Spacer; in the WNVRPAP2 probe with the sequence shown in SEQ ID No.3, the 30 th base modifies a fluorescent group 6-FAM-Dt, the 31 st base is replaced by a base analog dSpacer, the 34 th base modifies a fluorescence quenching group BHQ1-dT, and the 3' end modifies C3 Spacer.

It should be noted that the design of RT-RPA primer and probe is practically the same as that of RPA primer and probe, but reverse transcriptase and RNase inhibitor are added in the reaction system, thereby realizing one-step reverse transcription recombinase polymerase amplification. In the case of detecting the actual sample, the RNA of the sample is extracted for detection, and the rest of the steps and conditions are the same as those of the RPA.

4. Reaction system and reaction conditions

RT-RPA amplification was performed using the twist Amp exo Kit in this example.

The reaction system was 50. mu.L. The method comprises the steps of uniformly mixing 29.5 mu L of RehydrationBuffer, 2.1 mu L of each of two primers with the concentration of 10 mu M, 0.6 mu L of probe with the concentration of 10 mu M, 1 mu L of MMLV Reverse Transcriptase, 9.2 mu L of 40U/mu L of recombined RNase Inhibitor 1 mu L, DEPC water and 2 mu L of nucleic acid template, adding the mixture into a reaction tube of RPA freeze-dried enzyme powder, uniformly mixing, finally adding 2.5 mu L of MgAc solution with the concentration of 280mM, and uniformly mixing. And (3) placing the reaction tube in an Axxin constant-temperature amplification instrument, reacting for 15 minutes at 39 ℃, and reading a fluorescence signal in real time in the reaction process.

Wherein, the two primers with the concentration of 10 μ M refer to an upstream primer and a downstream primer. The reaction system of this example is different from the conventional RPA reaction system in that Reverse Transcriptase MMLV Reverse Transcriptase and RNase Inhibitor recombination RNase Inhibitor are added, and the rest components and reaction conditions are the same as those of RPA. The reaction system of this example can detect DNA or RNA directly. If the DNA is to be tested, the reverse transcriptase and RNase inhibitor will not function. In the case of RNA detection, the reverse transcriptase simultaneously performs reverse transcription during the reaction, reverse transcribes RNA into cDNA, and then performs recombinase polymerase amplification, which is performed simultaneously in one reaction system.

5. Primer and probe screening

In the screening process, one of the upstream primers is adopted to screen the downstream primer, and then the upstream primer is screened according to the screened downstream primer so as to obtain the optimal primer probe combination for detecting the West Nile virus. The primer and probe screening adopts '4. reaction system and reaction conditions'.

6. Specificity test

And (3) detecting a plurality of nucleic acid templates such as WNV-Seg5, WNV-1, Bovine Viral Diarrhea Virus (BVDV), porcine Japanese Encephalitis Virus (JEV), Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), Classical Swine Fever Virus (CSFV), bluetongue virus (BTV) and the like by adopting a screened primer and probe combination according to a reaction system and reaction conditions of '4'. And setting a negative control and a blank water control, wherein the negative control refers to an RNA sample extracted from negative pig blood.

7. Sensitivity test

In this example WNV-Seg5 was diluted to 10-fold gradient^-6The diluted nucleic acid template with each gradient concentration is adopted to carry out the RPA test according to the 4. reaction system and reaction conditions, and a water blank control is arranged in the test to test the sensitivity of the primer probe.

Meanwhile, the method is compared with a real-time fluorescent quantitative PCR method established by the plum sea and the like, namely, the same diluted nucleic acid template is adopted, and the diluted nucleic acid templates are detected according to the real-time fluorescent quantitative PCR method established by the plum sea and the like. Wherein, the real-time fluorescent quantitative PCR method established by the plum sea and the like is described in the reference documents: establishment and application research of real-time fluorescence quantitative PCR detection method of Lilin sea Chenlidan, Liao Yang West Nile virus [ J ]. J.Utility medicine J.2012, 28(19): 3288-.

8. Repeatability test

By 10^-1～10^-3Diluted WNV-1 nucleic acids with three different concentrations are used as templates to carry out RPA test according to the '4 reaction system and reaction conditions',the stability of the established method was analysed 6 times in duplicate.

9. Detection of samples

159 parts of bovine blood and pig blood samples stored in a laboratory are detected according to a reaction system and reaction conditions of '4', WNV-1 positive control, negative control and water blank control are set, and meanwhile, real-time fluorescence RT-PCR method recommended by OIE is used for detection. All samples are provided and preserved by Shenzhen customs movement center. And (3) extracting RNA from each blood sample by using an RNA extraction kit and then directly detecting.

Second, results and analysis

1. Primer and probe screening

After screening, in this example, the upstream primer WNV-F4 with the sequence shown in Seq ID No.1, the downstream primer WNV-R5 with the sequence shown in Seq ID No.2 and the probe WNV-RPAP2 with the sequence shown in Seq ID No.3 are finally screened out from the primer probes in Table 1, and the combination of the primers and the probes can be used for specific detection of West Nile Virus.

As can be seen from the screening results, even the same probe, different combinations of the upstream and downstream primers have an effect on the amplification efficiency, specificity and sensitivity of the polymerase amplification by the recombinase.

2. Results of specificity test

The specific detection results are shown in FIG. 1, wherein curve 1 is the detection result of WNV-Seg5, and curves 2-8 are the detection results of Bovine Viral Diarrhea Virus (BVDV), porcine Japanese Encephalitis Virus (JEV), Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), Classical Swine Fever Virus (CSFV), bluetongue virus (BTV), negative control and water blank control, respectively. As can be seen, only the recombinant plasmid WNV-Seg5 has a positive fluorescence curve; and other pathogenic nucleic acids, negative control and water blank control have no fluorescence curve and are negative. Therefore, the primers and the probes can carry out specificity detection on WNV, have no cross reaction with inactivated antigen nucleic acids of BVDV, JEV, PRRSV, CSFV and BTV, and have good specificity.

3. Results of sensitivity test

The results of the sensitivity detection of WNV by the primers and probes of this example are shown in FIG. 2, in which WNV-Seg5 was diluted by 10 in the order of curve 1 to curve 4 in FIG. 2^-1、10^-2、10^-3、10^-4The amplification curves of (1), from curve 5 to curve 7 being in the order of 10^-5、10^-6And amplification curves for water blanks. As a result, WNV-Seg510 can be detected with the lowest RPA in this example^-4And (4) dilution degree. The original concentration of the plasmid WNV-Seg5 was determined and then converted into the copy number of 5.25X 10⁶copies/μL，10^-4The dilution corresponds to a plasmid concentration of 5.25X 10²copies/μL。

Sensitivity detection was performed on the same diluted nucleic acid template by using a real-time fluorescent quantitative PCR method established with Lilin sea or the like, and the detection results are shown in FIG. 3, in which curves 1 to 6 are WNV-Seg5 dilution 10 in sequence in FIG. 3⁰、10^-1、10^-2、10^-3、10^-4、10^-5The amplification curve of (1). As a result, WNV-Seg510 was detected at the lowest level by the real-time fluorescent PCR method^-5Dilution at 5X 10 plasmid concentration¹copies/μL。

Therefore, compared with a real-time fluorescent quantitative PCR method established by the plum sea and the like, the WNV RT-RPA detection method of the embodiment can achieve similar sensitivity; however, the detection method of the embodiment has short detection time, does not need large-scale instruments and equipment, greatly shortens the detection time and improves the detection efficiency.

4. Results of the repeatability test

Will 10^-1、10^-2、10^-3The RT-RPA method is respectively used for repeatedly detecting three kinds of diluted nucleic acid with different concentrations for 6 times, corresponding fluorescence curves can be observed, and the detection results of the nucleic acid templates with the same concentration are consistent, which shows that the method has good repeatability.

5. Detection of samples

The detection result of the sample shows that when the primers and the probes of the embodiment are adopted to perform RT-RPA detection on 159 bovine blood and pig blood samples, a fluorescence amplification curve appears in a positive control, no amplification curve appears in a negative control, a water blank control and the sample, and the detection result of the sample is negative and is the same as the detection result of the real-time fluorescence RT-PCR method recommended by OIE.

The West Nile virus detection method and the reagent have the advantages of short detection time, simple reaction conditions, no need of large-scale instruments and equipment, and greatly improved detection efficiency.

The foregoing is a more detailed description of the present application in connection with specific embodiments thereof, and it is not intended that the present application be limited to the specific embodiments thereof. It will be apparent to those skilled in the art from this disclosure that many more simple derivations or substitutions can be made without departing from the spirit of the disclosure.

SEQUENCE LISTING

<110> Shenzhen customs animal and plant inspection and quarantine technical center

<120> reagent, kit, detection method and application for West Nile virus detection

<130> 20I29588

<160> 14

<170> PatentIn version 3.3

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Claims

1. A reagent for detecting west nile virus, comprising: the reagent comprises a primer pair and a probe, wherein an upstream primer of the primer pair is a sequence shown by Seq ID No.1, a downstream primer of the primer pair is a sequence shown by Seq ID No.2, and the probe is a sequence shown by Seq ID No.3 or a reverse complementary sequence of the sequence shown by Seq ID No. 3;

Seq ID No.1：5’-CCTGATTGAATTGGAACCACCCTTTGGAGACTCAT-3’

Seq ID No.2：5’-AGTCCCAAGCTGTATCTCCTAGGGCGGCTAATCTC-3’

Seq ID No.3：5’-CATAGTGGTGGGCAGAGGAGAACAACAGATTAATCACCATTGGCACAAGTC-3’

2. The reagent for detecting west nile virus of claim 1, which is characterized in that: the 5 'end of the probe is marked with FAM fluorescent group, and the 3' end of the probe is marked with BHQ1 quenching group.

3. The use of the reagent for west nile virus detection according to claim 1 or 2 in the preparation of a west nile virus detection kit, a detection test strip or a detection chip.

4. A kit for detecting West Nile virus is characterized in that: the kit contains the reagent for detecting the West Nile virus as claimed in claim 1 or 2.

5. The kit for west nile virus detection of claim 4, wherein: the kit also contains freeze-dried enzyme powder, rehydration buffer solution, magnesium acetate solution and deionized water.

6. A method for detecting west nile virus for non-diagnostic therapeutic purposes, comprising: the method comprises the steps of carrying out one-step reverse transcription recombinase polymerase amplification on a sample to be detected by using the reagent for detecting the West Nile virus as claimed in claim 1 or 2 or the kit for detecting the West Nile virus as claimed in claim 4 or 5, and collecting fluorescence by using a fluorescence detector.

7. The method for detecting west nile virus of claim 6, wherein: the one-step reverse transcription recombinase polymerase amplification reaction condition is that the isothermal reaction is carried out for 15 minutes at 39 ℃.