Scientia Agricultura Sinica ›› 2013, Vol. 46 ›› Issue (20): 4370-4377.doi: 10.3864/j.issn.0578-1752.2013.20.021

• ANIMAL SCIENCE·VETERINARY SCIENCERE·SOURCE INSECT • Previous Articles     Next Articles

Development of a RT-PCR Method for Differentiation of the Wild-Type PEDVs and the Attenuated PEDVs

 WU  Yu-Lu-1, CHENG  Qun-1, YU  Ling-Xue-1, HOU  Yi-Xuan-2, WANG  Kang-1, LIU  Guang-Qing-1, TONG  Guang-Zhi-1, ZHOU  Yan-Jun-1   

  1. 1.Division of Swine Infectious Diseases, Shanghai Veterinary Research Institute of Chinese Academy of Agricultural Sciences, Shanghai 200241
    2.School of Agricultural and Biology, Shanghai Jiao Tong University, Shanghai 200240
  • Received:2013-04-12 Online:2013-10-15 Published:2013-08-08

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Abstract: 【Objective】The objective of this study is to establish an effective tool for differentiating diagnosis of the PEDVs in epidemiological investigations by the method of RT-PCR. 【Method】According to the truncation in the ORF3 gene of PEDV reference strains in GenBank, the wild-type PEDV isolates and attenuated isolates, two pairs of primers were designed. The clinical samples from different farms occurred with diarrhea epidemic were tested, and their ORF3 gene was analyzed. Some representative samples were selected and amplified by the primers ORF3-JD1/2. The procedure of the RT-PCR was perfected. A large number of clinical samples were collected to carry out the specificity and sensitivity test.【Result】The sequences of ORF3 gene of 11 PEDV isolates were obtained. No deletion in ORF3 gene of 9 strains was found, and it indicated that they were wild-type PEDVs. And the results showed 95.8%-97.1% nucleotide sequence homology identity between wild isolates and attenuated ones. The RT-PCR was shown to specifically amplify a 300 bp fragment from the wild-type PEDVs or a 250 bp fragment from the attenuated PEDVs. This method showed no cross-amplification between PEDVs and other porcine virus. And the sensitivity of detection of viral was upto 100 TCID50. The clinical samples were tested by using this RT-PCR method , and the results showed a 65.4% PEDV positive rate.【Conclusion】 The RT-PCR could be used as an effective tool for differentiating diagnosis of the PEDVs in epidemiological investigations.

Key words: PEDV , ORF3 gene , RT-PCR , differential diagnosis

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