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Bovine viral diarrhea virus (BVDV) fluorescent quantitative RT-PCR (reverse transcription-polymerase chain reaction) detection kit

A technology for bovine viral diarrhea and a detection kit, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, etc., and can solve problems such as laborious, time-consuming, and low sensitivity

Active Publication Date: 2013-11-20
CHINA INST OF VETERINARY DRUG CONTROL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, BVDV detection mainly includes fluorescent antibody detection method, green monkey kidney (vero) passage cell detection method, cytopathic and / or red blood cell adsorption foreign virus detection method (Ministry of Agriculture of the People's Republic of China. Veterinary Biology of the People's Republic of China Product regulations. Beijing: Chemical Industry Press, 2000, 180-192.), etc., these three methods are relatively time-consuming, laborious and less sensitive

Method used

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  • Bovine viral diarrhea virus (BVDV) fluorescent quantitative RT-PCR (reverse transcription-polymerase chain reaction) detection kit
  • Bovine viral diarrhea virus (BVDV) fluorescent quantitative RT-PCR (reverse transcription-polymerase chain reaction) detection kit
  • Bovine viral diarrhea virus (BVDV) fluorescent quantitative RT-PCR (reverse transcription-polymerase chain reaction) detection kit

Examples

Experimental program
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Effect test

Embodiment 1

[0109] ——Clinical testing

[0110] The BVDV-MGB-RT-PCR detection kit was used to detect 94 finished and semi-finished samples of swine fever vaccines from 8 manufacturers of live swine fever vaccines. At the same time, the RT-PCR method (see note) was used as the conformity to evaluate the method. Feasibility of clinical promotion.

[0111] (1) Extraction of RNA

[0112] RNA was extracted using commercial kits or conventional RNA extraction methods. The extracted RNA must be amplified within 2 hours.

[0113] (2) Amplification reagent preparation

[0114] Take out each component from the kit, melt at room temperature, and centrifuge at 2000r / min for 5s. The preparation of each sample test reaction system is shown in Table 3.

[0115] (3) Nucleic acid amplification and detection

[0116] Put the above-mentioned PCR tubes into the fluorescent PCR detector, and record the order in which the samples are placed.

[0117] Loop condition settings:

[0118] In the first stage,...

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Abstract

The invention relates to a bovine viral diarrhea virus (BVDV) fluorescent quantitative RT-PCR (reverse transcription-polymerase chain reaction) detection kit. According to 21 BVDV-I genome complete sequences, 6 BVDV-II genome complete sequences and 34 CSFV (classical swine fever virus) full-length sequences which are published by GeneBank, one pair of degenerate primers specific to BVDV and one probe are designed in a conserved region of BVDV by utilizing software such as DANMAN and Primer Express3.0. By optimizing reverse transcriptase concentration, DNA (deoxyribonucleic acid) polymerase concentration, upstream and downstream primer concentration and probe concentration, the BVDV fluorescent quantitative RT-PCR detection kit is invented. The BVDV fluorescent quantitative RT-PCR detection kit can specifically detect all the strains of BVDV and has no cross reaction with other pathogens; the BVDV fluorescent quantitative RT-PCR detection kit has good sensitivity which is one order of magnitude higher than that of the conventional RT-PCR; tests on repeatability and stability of 20 quality control samples show that the BVDV fluorescent quantitative RT-PCR detection kit has good repeatability and stability; detection results of 94 clinical samples show that coincidence rate of the method and a BVDV RT-PCR detection kit is 97.9%.

Description

technical field [0001] The bovine viral diarrhea virus fluorescent quantitative RT-PCR detection kit involved in the invention belongs to the field of veterinary biological products. Background technique [0002] Bovine viral diarrhea / mucosal disease is an infectious disease caused by bovine viral diarrhea virus (Bovine Viral Diarrhea Virus, BVDV). BVDV is widely distributed in the world, bringing huge economic losses to global dairy and beef cattle, goats (TOPLIFFCL, SMITHDR, CLOWSER SL, et al.Prevalence of bovine viral diarrhea virus infections in alpacas in the United States.J Am Vet Med Assoc,2009,234(4):519-529.), sheep, pigs (TERPSTRA C, WENSVOORT G. Acongenital persistent infection of bovine virus diarrhea virus in pigs: clinical, virological and immunological observations. Vet Q, 1997, 19( 3):97-101.), deer and wallabies and other animals are the hosts and sources of infection of BVDV (LINDBERG A, BROWNLIE J, GUNN GJ, et al. The control of bovine viral diarrhea viru...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68
Inventor 徐璐王琴范学政赵启祖朱元源邹兴启陈锴
Owner CHINA INST OF VETERINARY DRUG CONTROL
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