Dual RT-PCR method and special reagent case for checking cattle rotavirus and cattle virus diarrhea virus simultaneously

A technology for bovine viral diarrhea and bovine rotavirus, which is applied in the directions of biochemical equipment and methods, microbial determination/inspection, etc., can solve the problem of less research, poor sensitivity and specificity, and difficulty in the rapid diagnosis of bovine diarrhea. Distinguish and other problems to achieve the effect of reducing detection cost and detection time, good specificity and sensitivity, and accurate diagnosis

Inactive Publication Date: 2007-10-31
SHANDONG OX LIVESTOCK BREEDING CO LTD
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Problems solved by technology

In addition, the symptoms of diarrhea caused by these two pathogens are similar, and it is difficult to distinguish them clinically.
[0003] At present, there are many methods for detecting BRV and BVDV, such as virus isolation, neutralization test, agar expansion test, electron microscope observation, ELISA, nucleic acid probe, RT-PCR, etc. There are few studies on diagnostic methods. Traditional methods such as virus isolation, electron microscope observation, and neutralization test take a long time, and it takes 7-10 days to get a diagnostic result. However, methods such as ELISA, agar amplification test, and nucleic acid probes are less sensitive and specific. Difference
In addition, there is no research on the diagnostic method for the simultaneous detection of bovine rotavirus and bovine viral diarrhea virus in domestic literature.

Method used

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  • Dual RT-PCR method and special reagent case for checking cattle rotavirus and cattle virus diarrhea virus simultaneously
  • Dual RT-PCR method and special reagent case for checking cattle rotavirus and cattle virus diarrhea virus simultaneously
  • Dual RT-PCR method and special reagent case for checking cattle rotavirus and cattle virus diarrhea virus simultaneously

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Experimental program
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Embodiment 1

[0018] 1. Sample collection and processing

[0019] Take about 10g of diarrhea cow feces sample, prepare 1:5 fecal suspension with sterile normal saline, centrifuge at 4000rpm / min for 10min, take the supernatant for later use; or take mesenteric lymph nodes, intestinal mucosa or spleen during autopsy Store it at low temperature and send it to the laboratory. Take about 5g of the above tissue sample, cut it into pieces, add sterile saline and grind, then centrifuge at 4000rpm / min for 10min, and take the supernatant for use.

[0020] 2. Extraction of sample RNA template

[0021] (1) Take 500μl of the supernatant from step 1 into a 1.5ml centrifuge tube, then add 500μl of TRIZOL LS, mix well, and place at room temperature for 10 minutes.

[0022] (2) Add 200 μl of chloroform, shake vigorously to make the solution appear milky white without phase separation, and leave it at room temperature for 10 minutes.

[0023] (3) Centrifuge at 4°C at 13000 rpm / min for 15 minutes, and transfer th...

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Abstract

The invention discloses a double RT-PCR method to check bull rotavirus and bull virus diarrhea virus and private agent box, which is characterized by the following steps: collecting sample; extracting virus RNA; designing special primer; proceeding RT-PCR augment; proceeding agarose jel electrophoresis; judging result; comprising virus RNA extracting agent, reverse transcription agent box, PCR agent box, positive matching product and negative matching product. This invention can check out two dull loose bowel movement viruses at the same time in one RT-PCR reaction, which decreases checking cost and time.

Description

Technical field [0001] The invention relates to a method for simultaneously detecting bovine rotavirus and bovine viral diarrhea, in particular to a dual RT-PCR method and a special kit for simultaneously detecting bovine rotavirus and bovine viral diarrhea virus. Background technique [0002] Bovine rotavirus (BRV) and bovine viral diarrheavirus (BVDV) are two important pathogens that cause bovine viral diarrhea. BRV can cause severe diarrhea in newborn calves. A survey in the United Kingdom shows that its incidence is 60-80%, causing great economic losses to the cattle industry. BVDV is also a pathogen that widely exists in cattle herds around the world. Domestic Wang Xinping conducted epidemiological investigations in Inner Mongolia, Jilin, Ningxia and other regions and found that its infection rate was 16.5-89%. At present, with the increase of the scale and intensification of my country's dairy farming industry, bovine diarrhea, especially newborn calf diarrhea, has become o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68
Inventor 杨少华王长法高运东杨宏军
Owner SHANDONG OX LIVESTOCK BREEDING CO LTD
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