Taqman real-time fluorescent PCR kit for detecting pig umbilical cord blood transmissible gastroenteritis virus and application thereof
A real-time fluorescent and infectious technology, applied in the direction of microbial measurement/inspection, biochemical equipment and methods, etc., can solve the problems of limited guiding significance, complicated detection process, difficult virus isolation, etc., and achieve reliable scientific and technological support, The effects of extensive and specific, direct and effective diagnosis
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Embodiment 1
[0065] The flow chart of the present invention's diagnosis of porcine umbilical cord blood porcine transmissible gastroenteritis virus is as follows figure 1 shown. Specifically include the following steps: (1) sample collection; (2) sample processing; (3) RNA extraction; (4) qPCR: use the specific primers and probes designed in the present invention to carry out qPCR reaction; (5) determine the result.
[0066] 1. Sample Collection
[0067] (1) Cord blood sample collection
[0068] a. Take a clean penicillin bottle and cork, clean it, boil and sterilize it for 30 minutes, dry it and collect it for later use;
[0069] b. When the piglets are born, squeeze the "cord blood" of all piglets delivered by each sow into a clean penicillin bottle, 3-5 drops per piglet, and squeeze the "cord blood" into penicillin bottle, sealed;
[0070] Precautions: ①It is necessary to collect all the piglets from the same litter sow to avoid missed detection caused by individual differences in the...
Embodiment 2
[0134] Embodiment 2 Sensitivity research
[0135] Evaluate the sensitivity of the kit of the present invention with positive control cloning plasmid, carry out 10 times gradient dilutions to cloning plasmid, detection range is 10 8 -10 1 copies / μl. The results show that the detection range of the method is 10 8 -10 1 Copies / μl, reliable results can be obtained for porcine transmissible gastroenteritis virus content in this range, that is, the sensitivity of the method can detect samples with 10 copies of porcine transmissible gastroenteritis virus virus content. See the test results Figure 4 .
Embodiment 3
[0136] Embodiment 3 specificity research
[0137] In order to detect the specificity of the kit of the present invention, utilize the kit of the present invention to detect porcine parvovirus, porcine rotavirus, porcine reproductive and respiratory syndrome virus, porcine circovirus, porcine epidemic diarrhea virus, porcine encephalitis B Viruses, porcine pseudorabies virus and other 7 kinds of viruses.
[0138] The test results show that the kit of the invention can only amplify the TGEV S gene in the piglet umbilical cord blood, indicating that the kit of the invention can specifically amplify TGEV without cross-reaction with other nucleic acids. See the test results Figure 5 .
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