基于单链置换的环介导间接PCR鉴别PEDV、TGEV 方法的建立及应用?

doi:10.3864/j.issn.0578-1752.2017.24.012

摘要/Abstract

摘要： 【目的】猪病毒性腹泻主要是由猪流行性腹泻病毒（PEDV）和猪传染性胃肠炎病毒（TGEV）所引起的一种急性肠道传染病，临床上以呕吐、腹泻、脱水、高发病率和死亡率为主要特征。PEDV与 TGEV同属α冠状病毒，可以感染各年龄段猪只，尤其对仔猪的危害最为严重，可导致大量仔猪死亡，给养殖业造成严重经济损失。由于PEDV和TGEV感染在临床症状、病理变化和流行病学上极为相似，临床上很难区分，因此，建立一种高效、特异的临床鉴别诊断方法对于预防病毒性腹泻的爆发流行具有重要意义。本试验旨在建立猪流行性腹泻病毒（PEDV）和猪传染性胃肠炎病毒(TGEV）环介导间接PCR检测方法，用于猪病毒性腹泻的病原学鉴别诊断。【方法】根据GenBank数据库中PEDV和TGEV基因组序列信息，在对两种病毒基因序列同源性比较的基础上，分别选取PEDV和TGEV核蛋白（N）基因序列的一段高度保守区，设计两条序列相邻的特异性探针，标记于不同大小的TOC1基因片段两端，作为特异性标记的报告基因。此报告基因与待测靶标基因经杂交、环化后，采用1对引物反向PCR扩增报告基因，建立PEDV/TGEV鉴别诊断方法，扩增片段大小分别为404、252bp。【结果】该方法特异性好，可单独或同时检测PEDV和TGEV，与猪圆环病毒2型（PCV2）、猪A型轮状病毒（RAV）、猪繁殖与呼吸综合征病毒（PRRSV）、猪瘟病毒（CSFV）、猪伪狂犬病毒（PRV）和猪细小病毒（PPV）等常见猪源性病毒无交叉扩增；检测灵敏度高，对PEDV和TGEV的最低极限浓度分别为1.6和8 pg·μL^-1的核酸样品，两种病原的同时检测不影响检测灵敏度；采用环介导间接PCR、常规RT-PCR和SybrGreen实时PCR对157份临床样本进行比较检测，3种方法的PEDV检出率分别为33.76%、21.66%和36.31%，TGEV检出率分别为26.75%、13.38%和28.03%；kappa检验结果，环介导间接PCR、SybrGreen实时PCR两种方法对PEDV和TGEV检测结果的符合度均为96.18%（κ≥0.90）【结论】环介导间接PCR可以用于鉴别诊断PEDV/TGEV，是一种快速、准确、灵敏、特异的病原学诊断工具。

关键词: 猪流行性腹泻病毒（PEDV）, 猪传染性胃肠炎病毒(TGEV）, 环介导间接PCR, 探针, 报告基因

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

郑鸣，李华玮，刘莹莹，王永芬，边传周，郭宏伟. 基于单链置换的环介导间接PCR鉴别PEDV、TGEV 方法的建立及应用?[J]. 中国农业科学, 2017, 50(24): 4790-4798.

ZHENG Ming, LI HuaWei, LIU YingYing, WANG YongFen, BIAN ChuanZhou, GUO HongWei. Establishment and Application of Loop-Mediated Indirect PCR Assay Based on Single-Strand Substitution for Detection and Differentiation of PEDV and TGEV[J]. Scientia Agricultura Sinica, 2017, 50(24): 4790-4798.

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