Method for distinguishing PVC2 virus and PEDV virus and primer of same
A differential diagnosis, PEDV-F technology, applied in the direction of biochemical equipment and methods, recombinant DNA technology, microbial measurement/inspection, etc., can solve the problems of lack of simultaneous diagnosis, reduce sample contamination, reduce detection cost, and improve detection efficiency effect
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Embodiment 1
[0034] Embodiment 1 Construction of the method for differential diagnosis of PCV2 and PEDV virus simultaneously
[0035] 1PCV2, PEDV primer design and synthesis
[0036]According to the nucleotide sequences of porcine epidemic diarrhea virus (PEDV) and porcine circovirus type 2 (PCV2) genes published in GenBank, a pair of specific primers PEDV-F, PEDV-R and PCV2-F, PCV2- R (see Table 1), the estimated size of the amplified products are 530bp and 278bp, synthesized by Sangon Biopower (Shanghai) Co., Ltd.
[0037] Table 1 Primer sequences used in multiplex PCR
[0038]
[0039] 2 Tissue processing and extraction of DNA and RNA
[0040] 2.1 Organization of processing:
[0041] Wipe the mortar clean with an alcohol cotton ball and sterilize it by flame, then fully pre-cool it for later use; select an appropriate amount of diseased tissue, and cut it into pieces as much as possible; put the tissue in the mortar, add an appropriate amount of liquid nitrogen, and grind the tiss...
Embodiment 2
[0069] Embodiment 2 clinical sample detection:
[0070] Total RNA and DNA were extracted from 67 diarrheal disease materials collected in Leshan, Yibin, Guangyuan and Suining, Sichuan, and tested according to the multiplex PCR amplification system and amplification conditions established in this experiment, and negative and positive controls were set up. Take 6μL of the PCR product and electrophoresis in 1.5% agarose gel to determine the results: the positive rate of PEDV single infection is 34.33%, the positive rate of PCV2 single infection is 73.13%; the "PEDV+PCV2" combined infection rate is 16.42%. The coincidence rate was 100% by comparing with the results of PCR for specific detection of PCV2 and RT-PCR for detection of PEDV (as shown in Table 3 and Table 4).
[0071] Table 1 Detection results of PEDV and PCV2
[0072]
[0073] Table 2 Comparison of detection results of clinical disease materials by single PCR and multiplex PCR
[0074]
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