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Fluorescent quantitative rt-pcr detection kit for detecting bovine viral diarrhea virus and its application

A bovine viral diarrhea, detection kit technology, applied in the directions of microorganism-based methods, microorganism determination/inspection, microorganisms, etc., can solve the problems of unfavorable large-scale application detection, time-consuming serological methods, and low virus isolation rate, etc. Achieve the effect of improving detection time and efficiency, good specificity, and high detection sensitivity

Active Publication Date: 2011-12-07
WUHAN CHOPPER BIOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Most serological methods have the disadvantages of time-consuming, labor-intensive, and low accuracy
my country's national standard technology for diagnosing BVDV adopts the method of culturing cells to isolate viruses, but this method is time-consuming and laborious, coupled with the low rate of virus isolation, and some non-cytopathic BVDVs do not appear cytopathic in cell culture, so it is not conducive to large-scale application detection
Although the conventional methods of molecular biology can make up for it in some aspects, the shortcomings of false positives, environmental pollution, and low repeatability limit the popularization and application.

Method used

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  • Fluorescent quantitative rt-pcr detection kit for detecting bovine viral diarrhea virus and its application
  • Fluorescent quantitative rt-pcr detection kit for detecting bovine viral diarrhea virus and its application
  • Fluorescent quantitative rt-pcr detection kit for detecting bovine viral diarrhea virus and its application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Composition and reagent preparation of bovine viral diarrhea virus fluorescent quantitative RT-PCR detection kit

[0046] 1. RT-PCR reaction solution: each reaction includes 10×RT-PCR Buffer 2.5 μL, 25mM dNTP Mix 1 μL, 10 μM TaqMan probe PB 0.25 μL, 10 μM upstream primer PF 0.5 μL, 10 μM downstream primer PR 0.5 μL, DEPC water 16.25 μL, a total of 21 μL. 48 reactions totaling 1008 μL were dispensed into 1 tube.

[0047] 2. RNA extraction solution: 30mL / bottle, aliquot into brown bottles, and store at 4°C.

[0048] 3. DEPC water: Add diethyl pyrocarbonate at a final concentration of 0.1% to deionized water, overnight at room temperature, pressurize at 15 lbs for 20 min, and dispense into 1.5 mL centrifuge tubes, 1 mL / tube.

[0049] 4. DNA polymerase: Taq DNA polymerase (5U / μL) was aliquoted in 25 μL / tube and stored at -20°C.

[0050] 5. Reverse transcriptase: Aliquot AMV reverse transcriptase (5U / μL) in 25 μL / tube and store at -20°C.

[0051] 6. Positive control subs...

Embodiment 2

[0054] Application method of fluorescent quantitative RT-PCR rapid detection kit for bovine viral diarrhea virus

[0055] 1 Nucleic acid extraction from samples

[0056] 1.1 Take several 1.5mL sterilized (depending on the number of samples to be tested) centrifuge tubes without RNase contamination and mark them. Add 600 μL of RNA extraction solution, and then add 200 μL each of the sample and the negative and positive controls in the kit, and mix by pipetting; then add 200 μL of chloroform, and shake to mix. Let stand for 5 minutes, then centrifuge at 12,000rpm for 10 minutes.

[0057] 1.2 Pipette the upper liquid phase in each tube (do not inhale the lower liquid) and transfer it to a new 1.5ml RNase-free centrifuge tube, add 200μL-20℃ pre-cooled isopropanol, mark it, and mix it upside down. uniform. Let stand for 5 minutes, then centrifuge at 12,000rpm for 10 minutes.

[0058] 1.3 Gently pour off the supernatant, put it upside down on absorbent paper, and dry the liquid;...

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Abstract

The invention provides a fluorescence quantitative reverse transcription-polymerase chain reaction (RT-PCR) detection kit for detecting bovine viral diarrhea virus (BVDV) and application of the kit. The kit is used for the field of clinic and scientific research, including quick quantitative detection of BVDV infection and BVDV pollution monitoring of biological products such as bovine serum, bovine serum albumin and bovine testicle cell derived hog cholera lapinized virus vaccines and the like. The invention also provides a non-diagnostic fluorescence quantitative RT-PCR method for detectingBVDV infection.

Description

technical field [0001] The invention relates to a fluorescent quantitative RT-PCR detection kit for detecting bovine viral diarrhea virus and its application, which belongs to the field of viral nucleic acid detection, and is suitable for rapid quantitative detection of bovine viral diarrhea virus infection in clinical and scientific research. It also involves the detection of BVDV contamination in biological products such as bovine serum used for vaccine production and bovine testicular cell-derived attenuated hog fever vaccine. Background technique [0002] Bovine viral diarrhea is caused by bovine viral diarrhea virus of the genus Pestivirus in the Flaviviridae family, and is a widespread infectious disease of cattle and sheep worldwide. BVDV is one of the important pathogens that endanger the cattle, sheep and pig industries. It can cause a series of complex clinical symptoms in infected animals, including viral diarrhea, mucosal disease, persistent infection and immune ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
Inventor 薛霜刘汉平陈其兵孙庆歌张萍漆世华温文生谢红玲
Owner WUHAN CHOPPER BIOLOGY
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