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Fluorescent quantitation RT-PCR detection kit used for detecting bovine viral diarrhea virus and application thereof

A bovine viral diarrhea and RT-PCR technology, applied in the field of fluorescence quantitative RT-PCR detection kits, can solve the problems of time-consuming, labor-intensive, low sensitivity, etc., and achieve the effects of accurate detection results, high sensitivity and good repeatability

Inactive Publication Date: 2015-01-28
成都史纪生物制药有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Conventional detection methods have the disadvantages of time-consuming, laborious and low sensitivity.

Method used

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  • Fluorescent quantitation RT-PCR detection kit used for detecting bovine viral diarrhea virus and application thereof
  • Fluorescent quantitation RT-PCR detection kit used for detecting bovine viral diarrhea virus and application thereof
  • Fluorescent quantitation RT-PCR detection kit used for detecting bovine viral diarrhea virus and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Embodiment 1: Fluorescent quantitative RT-PCR detection kit for detecting bovine viral diarrhea virus

[0025] 1. A pair of BVDV-specific primers: the upstream sequence (BVDV-P1) of the primer pair is 5'-GGG(A / G / T)AGTCGTCA(A / G)TGGTTCG-3', the downstream sequence (BVDV-P2) is 5'-CCTATCAGGC TGT(A / G)T(C / T)C(A / G)TA(A / G)C-3';

[0026] 2. One specific probe: the specific probe sequence (BVDV-MGB) is 5'-FAM-CGTCCAC(A / G)TGGCATCTCGAG-NFQ-MGB-3';

[0027] 3. RT-PCR reaction solution components (total volume is 25 μL):

[0028] 2×One step RT-PCR BufferⅢ: 12.5μL;

[0029] 5U / μL Takara Ex Taq HS: 0.5μL;

[0030] PrimeScript RT enzyme Mix II: 0.5 μL;

[0031] 10 μM upstream primer BVDV-P1: 1.5 μL;

[0032] 10μM downstream primer BVDV-P2: 1.5μL;

[0033] Specific probe: 1 μL;

[0034] Total RNA: 5 μL,

[0035] RNase Free dH 2 O: 2.5 μL.

[0036] 4. Positive control: BVDV C24V strain MDBK cell line product, its virus titer is 10 6.5 TCID 50 / ml, inactivated and extracted RN...

Embodiment 2

[0038] Example 2: Establishment of Fluorescent Quantitative RT-PCR Detection Method for Bovine Viral Diarrhea Virus

[0039] 1. Materials

[0040] According to the BVDV nucleotide sequence, DNAStar software was used for sequence analysis, and a pair of primers and probes were designed by using Oligo 6.0 primer design software to identify BVDV and CSFV viruses. Primers were synthesized by Shanghai Yingjun Company, and probes were synthesized by ABI Company. One Step PrimeScriptTM RT-PCR Kit (Perfect Real Time) was purchased from Bao Biology (Dalian) Co., Ltd.; virus RNA extraction kit was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.

[0041] 2. Methods and results

[0042] (1) Establishment of optimal reaction conditions

[0043] In order to determine the best reaction system of primers, the concentrations of primers and probes were optimized accordingly. The reaction program was: 42°C, 5min (one cycle); 95°C, 10sec; 95°C, 5sec; 60°C, 30sec, the reacti...

Embodiment 3

[0052] Embodiment 3: Fluorescent quantitative RT-PCR detects bovine viral diarrhea virus

[0053] 1. Materials

[0054] Semi-finished products and finished products of CSF heat-resistant cell seedlings: provided by Chengdu Tianbang Biological Products Co., Ltd.; CSF Spleen Virus and BVDV Seed Virus (C24V strain) were purchased from the China Supervision Institute; Tissue Virus RNA Extraction Kit was purchased from OMEGA Company, Cat. No. : R6934-01; One Step PrimeScript TM RT-PCR Kit (Perfect Real Time) was purchased from Treasure Bio (Dalian) Co., Ltd., product number: DRR064A; virus RNA extraction kit was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd., product number: DP315-R.

[0055] 2. Methods and Results

[0056] (1) Extraction of mixed sample RNA

[0057] Spleen virus of classical swine fever, semi-finished products and finished products of heat-resistant cell seedlings and BVDV seed virus were mixed according to different proportions, a total of ...

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PUM

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Abstract

The invention discloses a fluorescent quantitation RT-PCR detection kit used for detecting the bovine viral diarrhea virus and an application of the fluorescent quantitation RT-PCR detection kit. The fluorescent quantitation RT-PCR detection kit is applied to the fields of clinics and scientific research and can be widely used for rapid detection of the BVDV, especially rapid detection of BVDV infection and rapid monitoring of polluting BVDV in bovine source raw materials and hog cholera attenuated live vaccines. The invention further provides a fluorescent quantitation RT-PCR method used for detecting bovine viral diarrhea virus inflection, and the method is accurate in detection result and has the advantages of being high in specificity, high in flexibility and good in repeatability, finishing amplification and detection at a time, being easier to operate and being capable of finishing detection within two hours.

Description

technical field [0001] The invention belongs to the field of veterinary biological products, and in particular relates to a fluorescent quantitative RT-PCR detection kit for detecting bovine viral diarrhea virus and an application thereof. Background technique [0002] Bovine viral diarrhea virus (BVDV), also known as bovine viral diarrhea-mucosal disease virus (Bovine viral diarrhea-mucosal disease virus, BVD-MDV), belongs to Flaviviridae (Flavivixidae), Pestivirus ( Pestivirus). The virus belongs to the same genus as Classical swine fever virus (CSFV). Bovine-derived products such as bovine serum, frozen embryos, and bovine semen may contaminate BVDV and cause the spread of BVDV. In the production of veterinary vaccines, bovine-derived materials such as bovine serum, bovine testicular cells, and pancreatic enzymes are often used as raw materials. Therefore, if there is BVDV pathogen in these raw materials, it will lead to vaccine contamination, especially after pig vacc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
CPCC12Q1/701C12Q1/6851C12Q2600/112C12Q2545/114C12Q2561/101
Inventor 徐宏军岳丰雄黄杰张奕强柏菊王洁清胡来根王家福
Owner 成都史纪生物制药有限公司
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