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One-step RT-PCR kit and method for identifying serotype of bluetongue virus

A technology of RT-PCR and bluetongue virus, which is applied in the field of one-step RT-PCR identification method and kit for BTV serotypes, can solve the problems of cumbersome process, high experimental cost, and long time consumption, and achieve low cost and high sensitivity The effect of high and good specificity and sensitivity

Pending Publication Date: 2021-03-09
YUNNAN ANIMAL SCI & VETERINARY INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The kit overcomes the shortcomings of the traditional serum neutralization test such as long time-consuming, cumbersome process, and high cost of the test, and has the advantages of simple operation, less time-consuming, low cost, and accurate test results. - Blank for PCR detection kit

Method used

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  • One-step RT-PCR kit and method for identifying serotype of bluetongue virus
  • One-step RT-PCR kit and method for identifying serotype of bluetongue virus
  • One-step RT-PCR kit and method for identifying serotype of bluetongue virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Embodiment 1 one-step RT-PCR kit and method

[0066] 1 material

[0067] The 12 popular serotypes BTV (BTV-1, -2, -3, -4, -5, -7, -9, -12, -15, -16, -21 and - 24) See Table 1 for information on representative strains; 24 BTV serotype reference viruses such as BTV-1 to BTV-24 are from the World Organization for Animal Health (OIE) BTV reference laboratory Onderstepoort Veterinary Institute: South Africa; BTV Seg obtained by in vitro transcription -10 Single strand RNA (Single strand RNA, ssRNA) nucleic acid concentration is 1.48×10 12 Copy / μL (Yang Zhenxing, et al. Establishment and application of dual fluorescence quantitative RT-PCR detection method for bluetongue virus and epidemic hemorrhagic disease virus [J]. Chinese Veterinary Science, 2019, 49(09): 1104-1111) .

[0068] Table 1 Virus isolation information and sequence similarity of representative strains of 12 BTV serotypes prevalent in China

[0069]

[0070]

[0071] 2 Main reagents and instruments

...

Embodiment 2

[0108] Example 2 BTV serotype-specific RT-PCR detection kit and method specificity experiment

[0109] Utilize 5 μ L of the nucleic acids of 24 kinds of serotype BTV (BTV-1~BTV-24) reference strains extracted in Example 1 as a template, respectively with the BTV serotype-specific RT-PCR primers in Table 2 of Example 1, according to The reaction conditions of Example 1 were used for PCR amplification, and 5 μL of the PCR amplification product was taken for electrophoresis detection at the end of the reaction. A negative control was set up for the reaction at the same time, and the specificity of RT-PCR primers for BTV serotypes among different serotype strains was analyzed.

[0110] The results showed that the RT-PCR primers for each BTV serotype only produced specific amplification bands with the viral nucleic acid templates of the corresponding serotype, and the nucleic acid amplification results for other serotype strains were all negative. It shows that the BTV serotype id...

Embodiment 3

[0111] Example 3 Sensitivity experiment of BTV serotype RT-PCR detection kit and method

[0112] Take 1 μ L of nucleic acid of 12 kinds of serotype BTV representative strains in Table 1 of Example 1 as a template, and carry out absolute quantitative qRT-PCR of BTV nucleic acid with BTV group-specific qRT-PCR detection method (Yang Zhenxing, etc. Bluetongue virus Establishment and application of a dual fluorescence quantitative RT-PCR detection method for epidemic hemorrhagic disease virus [J]. Chinese Veterinary Science, 2019, 49(09): 1104-1111), using Seg-10ssRNA as a template for qRT-PCR reaction to obtain The standard curve of the 12 serotype BTV strain nucleic acid was calculated for BTV serotype qRT-PCR sensitivity analysis

[0113] The BTV RNA of different serotypes with known nucleic acid copy numbers was diluted 10 times as a template, and the corresponding BTV serotype-specific RT-PCR primers were selected, and one-step RT-PCR amplification was performed according to ...

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Abstract

The invention discloses an RT-PCR kit and method for identifying the serotype of BTV (Bluetongue virus, BTV), and belongs to the technical field of veterinary infectious disease detection. The RT-PCRkit is mainly used for serotype identification of the BTV of 12 serotypes, and the 12 serotypes are BTV-1, BTV-2, BTV-3, BTV-4, BTV-5, BTV-7, BTV-9, BTV-12, BTV-15, BTV-16, BTV-21 and BTV-24 respectively. The RT-PCR kit comprises specific RT-PCR amplification primers of 12 serotypes of BTV, and the nucleotide sequences of the specific RT-PCR amplification primers are as shown in SEQ ID No. 1 to SEQ ID No. 24. The kit has the advantages of being good in specificity, rapid, simple, convenient and low in cost, accurate typing of the 12 BTV serotypes can be conducted, the genetic characteristics of a BTV gene segment 2 can be obtained through sequencing of the amplification products, and data is provided for analysis of evolutionary characteristics of viruses.

Description

technical field [0001] The invention belongs to the technical field of veterinary infectious disease detection, and in particular relates to a one-step RT-PCR identification method and kit for BTV serotypes. Background technique [0002] Bluetongue (Bluetongue, BT) is a severe hemorrhagic arboviral disease transmitted by the bite of susceptible animals by blood-sucking Culicoides spp., and its pathogen is Bluetongue virus (BTV). BT is distributed worldwide, with outbreaks in tropical, subtropical and temperate regions of Africa, North America, South America, Oceania and Asia. BTV can infect almost all ruminants. After being infected with the virus, cattle and goats often have no obvious clinical symptoms and become the storage host and source of transmission of the virus. Susceptible sheep infected with BTV may have obvious clinical symptoms of bluetongue, manifested as high fever, emaciation, lip congestion, swelling and erosion, nasal cavity and gastrointestinal mucosal b...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/686C12N15/11
CPCC12Q1/701C12Q1/686C12Q2521/107C12Q2527/127C12Q2565/125
Inventor 杨恒李占鸿廖德芳李卓然杨振兴李华春
Owner YUNNAN ANIMAL SCI & VETERINARY INST
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