Scientia Agricultura Sinica ›› 2017, Vol. 50 ›› Issue (24): 4790-4798.doi: 10.3864/j.issn.0578-1752.2017.24.012


Establishment and Application of Loop-Mediated Indirect PCR Assay Based on Single-Strand Substitution for Detection and Differentiation of PEDV and TGEV

ZHENG Ming, LI HuaWei, LIU YingYing, WANG YongFen, BIAN ChuanZhou, GUO HongWei   

  1. Henan University of Animal Husbandry and Economy, Zhengzhou 450046
  • Received:2017-06-01 Online:2017-12-16 Published:2017-12-16

Abstract: 【Objective】 The virus diarrhea of pigs is an acute and highly contagious enteric disease, which is mainly caused by the porcine epidemic diarrhea virus (PEDV) and the porcine transmissible gastroenteritis virus (TGEV). The clinical signs of the disease mainly include vomiting, diarrhea, dehydration, high morbidity, and mortality. PEDV and TGEV belong to two distinct species of the Alphacoronavirus genus within Coronaviridae. Pigs of all ages are susceptible, especially the piglets, which can lead to death of large numbers of piglets and cause huge economic losses to the swine industry. Due to the clinical symptoms, pathologic changes and epidemiology are very similar, it is difficult to distinguish them by clinical diagnosis. Therefore, the rapid, specific preclinical identification of PEDV/TGEV is of great significance for preventing the outbreak and spread of this disease. The aim of this study is to establish the method of loop-mediated indirect PCR assay for the detection of PEDV/TGEV and the etiologic diagnosis for viral diarrheas in piglets. 【Method】According to the genome sequence information of PEDV and TGEV in GenBank database, a highly conserved region of PEDV and TGEV nucleoprotein (N) gene sequences were selected based on the homology comparison of the two viral genes. Two pairs of specific adjacent probes were designed, which were labeled on both ends of TOC1 gene fragments of different sizes as specific probe-labeled reporter genes for loop-mediated indirect PCR. After hybridizing with the target genes and cyclizing, the probe-labeled reporter genes were amplified by reverse PCR. The loop-mediated indirect PCR assay had been developed for the detection of PEDV/TGEV, and the specific PCR products were 404bp for PEDV and 252bp for TGEV, respectively.【Result】 The experimental results showed that the assay could be useful for the specific detection of PEDV and TGEV, from a contaminated sample by any one of them or by both. The specificity and sensitivity of the loop-mediated indirect PCR assay were tested. The results showed that the specificity was high, and no cross-amplification was observed with other common swine-borne viruses such as PCV2, RAV, PRRSV, CSFV, PRV and PPV. The lowest detection limits of the loop-mediated indirect PCR assay were 1.6 pg /μL for PEDV and 8 pg /μL for TGEV, respectively. And simultaneous detection of both pathogens did not affect the detection sensitivity. The comparison test was conducted on 157 clinical samples collected from adjacent pig farms by loop-mediated indirect PCR, conventional PCR and SYBR Green real-time RT-PCR. The results showed that the PEDV positive detection rates of loop-mediated indirect PCR, conventional PCR and SYBR Green real-time RT-PCR were 33.76%, 21.66% and 36.31% respectively, and the TGEV detection the positive rates were 26.75%、13.38% and 28.03% respectively. Results of Kappa analysis showed that, for both PEDV and TGEV detection, overall agreements between the loop-mediated indirect PCR and the SYBR Green real-time RT-PCR were both 96.18% with a kappa value of greater than or equal to 0.90.【Conclusion】The study suggested that the established loop-mediated indirect PCR was a rapid, accurate, sensitive and specific etiologic diagnosis tool, suitable for the differential diagnosis of PEDV and TGEV.

Key words:  porcine epidemic diarrhea virus, transmissible gastroenteritis virus, loop-mediated indirect PCR, probe, reporter gene

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