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| Establishment and Application of Multiplex Ligation-dependent Probe Amplification Assay for Identification of Pathogens Causing Porcine Viral Diarrhea Diseases |
| HE Hai-Jian1, *, LIU Zheng-Kui2, *, WU Yuan1, Wang Zhi-Peng2, CHEN Lin2, WANG Lei2, ZHOU Ying-Shan2, JIANG Chun-Yan1, SONG Hou-Hui2, **, WANG Xiao-Du2, ** |
1 College of Agricultural and Biological Engineer, Jinhua Polytechnic, Jinhua 321007, China;
2 College of Animal Science and Technology·College of Veterinary Medicine, Zhejiang A&F University, Hangzhou 311300, China; |
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Abstract Diarrhea of piglets in large-scale farms is often caused by different viruses, which have similar clinical symptoms in winter and spring. In order to distinguish the pathogens in clinical samples of viral diarrhea, it is urgent to establish a rapid and simultaneous detection technology for viral nucleic acid of six viral diarrhea diseases. In this study, the probes and pre-amplification primers for multiple restriction probe amplification (MLPA) were designed countering for the conserved regions of nucleic acids of Porcine epidemic diarrhea virus (PEDV) S gene, Transmissible gastroenteritis virus (TGEV) N gene, Porcine delta coronavirus (PDCoV) N gene, Porcine Bocavirus (pBCaV) NS1 gene, Porcine norovirus (pNov) RdRp gene, Porcine rotavirus (PRV) NSP1 gene. The method of detecting nucleic acid of 6 diseases was established using MLPA and capillary electrophoresis. The results showed that the detection of the 6 mixed templates by a single probe showed a high specificity. When the total concentration of the mixed probes were 1.33 nmo/L, and the specific amplified bands of various diseases were the same as expected, with the products of pBCaV (102 bp), pNov (110 bp), PDCoV (117 bp), PEDV (124 bp), pRV(131 bp) and TGEV (138 bp). This method had no cross reaction with other clinical diseases about Porcine reproductive and respiratory syndrome virus (PRRSV), Classic swine fever virus (CSFV), Pseudorabies virus (PRV) and Porcine circovirus type 2 (PCV2). The minimum limit values for detecting nucleic acid of pBCaV, pNoV, PDCoV, PEDV, pRV, TGEV were 7.58×101, 7.56×100, 7.54×100, 7.53×100, 7.50×101 and 7.49×100 copies/µL, respectively. The repeatability between and within groups was well. The results of 67 clinical and simulated samples by this method showed that PEDV were 100% in accordance with the virus isolation method, pBCaV, pNoV, PRV, TGEV and PDCoV were 100% in accordance with simulated virus. This study provides a new technology for detecting 6 clinical diarrhea pathogens simultaneously, and for the rapid response of clinical disease prevention and control.
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Received: 25 November 2019
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Corresponding Authors:
**, xdwang@zafu.edu.cn; songhh@zafu.edu.cn
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