China Animal Husbandry and Veterinary Medicine ›› 2021, Vol. 48 ›› Issue (10): 3855-3863.doi: 10.16431/j.cnki.1671-7236.2021.10.038

• Basic Veterinary Medicine • Previous Articles     Next Articles

Establishment and Preliminary Application of Duplex Real-time Quantitative PCR for Detection of PDCoV and TGEV

WANG Zhengfan1,2,3, ZHU Lisai2,3, WANG Juan2,3, LI Yiyun1,2,3, XIANG Rui1,2,3, YING Biyun1,2,3, WANG Guiping2,3,4, JIA Aiqing2,3,4, BAI Aiquan1   

  1. 1. Foshan University, Foshan 528231, China;
    2. Guangdong Haid Institute of Animal Husbandry & Veterinary, Guangzhou 511400, China;
    3. Guangdong Provincial Key Laboratory of Pig Breeding and Pig Disease Prevention and Control Technology Research Enterprise, Guangzhou 511400, China;
    4. Guangdong Haid Group Co., Ltd., Guangzhou 511400, China
  • Received:2021-04-11 Online:2021-10-20 Published:2021-09-30

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Abstract: This study was aimed to establish a fast, efficient and sensitive duplex Real-time quantitative PCR method for differential diagnosis of Porcine deltacoronavirus (PDCoV) and Transmissible gastroenteritis virus (TGEV). By drawing the standard curve of duplex Real-time quantitative PCR, the specificity, sensitivity and repeatability of the method were tested, and clinical samples were tested. The results showed that there was a good linear relationship between the cycle threshold of this method and the logarithm of the copy number of PDCoV and TGEV plasmids, and the corresponding correlation coefficients were R(P)2=0.9994 and R(T)2=0.996, respectively. It could specifically detect PDCoV and TGEV, but had nocross-react with PEDV, PRV, PRRSV, CSFV and RV, and had strong specificity. The minimum detection limits of the PDCoV and TGEV plasmid standard products reached 2 and 20 copies/μL, respectively, and were 1 000 and 100 times higher than conventional RT-PCR, respectively, with higher sensitivity. The average Ct values of intra-assay and inter-assay reproducibility of PDCoV and TGEV were basically the same, and the coefficient of variation (CV) was less than 2%, which had good reproducibility. The results of 114 piglet diarrhea samples tested by this method showed that the positive rates of PDCoV and TGEV were 5.6% (6/114) and 8.8% (10/114), respectively, and the detection rate of mixed infection was 4.6% (5/114), duplex Real-time quantitative PCR had a higher detection rate and sensitivity than conventional RT-PCR. The result indicated that the duplex Real-time quantitative PCR had the advantages of strong specificity, high sensitivity, good repeatability and stability, it was suitable for early virus diagnosis and batch clinical sample detection, and provided technical support and data reference for disease prevention and control, epidemiological investigation and correlation research.

Key words: Porcine delta coronavirus(PDCoV); Transmissible gastroenteritis virus(TGEV); TaqMan probe; duplex Real-time quantitative PCR

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