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RT-qPCR (Reverse Transcription-Quantitative Polymerase Chain Reaction) detection kit and method for identifying bluetongue virus serotype

A serotype, BTV-3 technology, applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., can solve problems such as low sensitivity, high cost, and low detection throughput

Pending Publication Date: 2021-02-19
YUNNAN ANIMAL SCI & VETERINARY INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In view of the shortcomings of the existing serum neutralization test and conventional RT-PCR in the detection of BTV serotypes, such as time-consuming, high cost, low detection throughput and low sensitivity, the purpose of the present invention is to provide a 12 A real-time fluorescent quantitative RT-PCR (real-time quantitative polymerase chain reaction, RT-qPCR) detection kit and corresponding method for BTV serotypes, so as to realize rapid and accurate identification of serotypes of BTV strains popular in China

Method used

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  • RT-qPCR (Reverse Transcription-Quantitative Polymerase Chain Reaction) detection kit and method for identifying bluetongue virus serotype
  • RT-qPCR (Reverse Transcription-Quantitative Polymerase Chain Reaction) detection kit and method for identifying bluetongue virus serotype
  • RT-qPCR (Reverse Transcription-Quantitative Polymerase Chain Reaction) detection kit and method for identifying bluetongue virus serotype

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067]Example 1 RT-qPCR kit for identification of bluetongue virus serotype

[0068]1. Material

[0069]The 12 popular serotypes of BTV in China (BTV-1, BTV-2, BTV-3, BTV-4, BTV-5, BTV-7, BTV-9, BTV-12, BTV-15, BTV-16, BTV -2 and BTV-24) representative strains were isolated and preserved by the applicant. See Table 4 for strain information; BTV-6, -8, -10, -11, -13, -14, -17, -18,- 19, -10, -22, -23 and other 12 international standard reference viruses for BTV serotypes originated from the OIE South Africa Reference Laboratory (Onderstepoort Veterinary Institute); the recombinant plasmid pLB-BTV-NS3-T7 was prepared and preserved by the applicant (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).

[0070]Table 4 Virus isolation information of 12 representative strains of BTV serotypes circulating in China

[0071] Sero...

Embodiment 2

[0113]Example 2 RT-qPCR detection method sensitivity test and standard curve drawing

[0114]Take the 12 serotypes BTV (BTV-1, BTV-2, BTV-3, BTV-4, BTV-5, BTV-7, BTV-9, BTV-12) prepared in Example 1 to determine the number of nucleic acid copies. , BTV-15, BTV-16, BTV-21 and BTV-24) representative strain cDNA as standard, 10-fold serial dilution to 10 copies0~107Copy / μL and other 8 gradients, use the corresponding BTV serotype primers and TaqMan probes to detect according to the method in Example 1, and set 3 replicates for each gradient. Use the logarithmic value of the viral nucleic acid copy number as the abscissa and the average value of the Ct value as the ordinate to establish a standard curve, and calculate the correlation coefficient (R2) And amplification efficiency (E value), analyze the minimum detection copy number of BTV nucleic acid of different serotypes by qPCR to confirm the sensitivity of the kit.

[0115]The test results show that the amplification curves of the nucleic...

Embodiment 3

[0119]Example 3 Serotyping of BTV isolates by RT-qPCR detection kit

[0120]1. Viruses

[0121]A total of 163 BTV strains of 12 serotypes isolated by the applicant from 1979 to 2020. The virus information is shown in Table 7. The serotype of the virus was confirmed by RT-PCR and sequencing.

[0122]2. Nucleic acid preparation

[0123]Take 50μL of BTV virus solution, and use the automatic nucleic acid extractor "King Fisher Flex" to extract nucleic acids according to the instructions of the RNA extraction kit "MagMAX-96Viral RNA Isolation Kit". The extracted nucleic acid was denatured at 94°C for 3min, immediately ice bathed for 5min, and stored at -80°C for later use; the normal BHK-21 cell culture medium was simultaneously extracted as a negative control.

[0124]3. Sample detection

[0125]Take the denatured viral nucleic acid as a template, perform RT-qPCR detection according to the method in Example 1, identify the virus serotype, and verify whether the BTV serotype specific RT-qPCR kit provided ...

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Abstract

The invention discloses an RTqPCR (Reverse Transcription Quantitative Polymerase Chain Reaction) kit for identifying a BTV (Bluetongue Virus) serotype, and belongs to the technical field of veterinaryinfectious disease detection. The RTqPCR kit is mainly used for identifying the serotypes of 12 serotypes of bluetongue viruses, and the 12 serotypes of bluetongue viruses 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-qPCR kit comprises a primer pair combination and a corresponding probe combination, wherein the primer pair combination is used for specifically amplifying Seg-2 gene sequences of BTV strains of 12 serotypes. The detection kit and method provided by the invention have the characteristics of high sensitivity, strong specificity, high detection flux and simple, convenient and rapid operation, and can be used for identifying the BTV serotype so as to realize accurate identification of the BTV serotype in a temporary sample.

Description

Technical field[0001]The present invention belongs to the field of veterinary infectious disease detection, and in particular, the present invention relates to an RT-QPCR detection trial cartridge and detection method for blue tongue virus serotype identification.Background technique[0002]Bluetongue, BT) caused by Bluetongue Virus (BTV) infection is a highly infringed bleeding insectovirus disease. The outbreak of BT can bring huge economic losses to the cattle and sheep farm industry, and is listed as a legal reported animal diseases by the World Animal Health Organization (OIEE ", my country is also listed as a class of animal diseases. BTV is mainly transmitted through the bite of Culicoides SPP., BTV can be infected with most domestic and wild ruminants. After the BTV is infected with BTV, cattle and goats often have a hidden infection and become an important storage host and infective source of virus. BTV infection sheep can cause obvious BT clinical symptoms, mainly in high fe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6851C12N15/11
CPCC12Q1/701C12Q1/6851C12Q2600/166C12Q2531/113C12Q2521/107C12Q2563/107C12Q2545/113
Inventor 廖德芳杨恒李占鸿宋子昂李卓然杨振兴李华春
Owner YUNNAN ANIMAL SCI & VETERINARY INST
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