猪流行性腹泻病毒S基因全序列扩增方法的建立及应用  

Establishment and application of RT-PCR for S gene full-length amplification in porcine epidemic diarrhea virus

作　　者：包银莉 许洵锴 郑灿阳 王燚 林鸿颖 戴爱玲[1,2] 曾旭健 黄翠琴 BAO Yin-li;XU Xun-kai;ZHENG Can-yang;WANG Yi;LIN Hong-ying;DAI Ai-ing;ZENG Xu-jian;HUANG Cui-qin(College of Life Science,Longyan University,Longyan 364012,China;Engineering Research Center for the Prevention and Control of Animal Original Zoonosis,Fujian Province University,Longyan 364012,China;College of Animal Sciences(College of Bee Science),Fujian Agriculture and Forestry University,Fuzhou 350002,China;Minxi Vocational&Technical College,Longyan 364021,China)

机构地区：[1]龙岩学院生命科学学院,福建龙岩364012 [2]动物源性人兽共患病防控福建省高校工程研究中心,福建龙岩364012 [3]福建农林大学动物科学学院(蜂学学院),福建福州350002 [4]闽西职业技术学院,福建龙岩364021

出　　处：《中国兽医科学》2023年第12期1563-1569,共7页Chinese Veterinary Science

基　　金：福建省自然科学基金资助项目(2020N0035);中央引导地方科技发展专项基金资助项目(2021L3028);上杭县奇迈科技创新基金资助项目(2020SHQM13);动物源性人兽共患病防控福建省高校工程研究中心开放基金资助项目(2022K011)。

摘　　要：为解决现有猪流行性腹泻病毒(PEDV)S基因全序列获得方法存在的成功率低、操作繁琐的问题,针对NCBI中已知PEDV S基因上下游序列,筛选保守区域用于RT-PCR扩增引物的设计,并从特异性、灵敏性以及临床应用效果等方面对该方法进行了验证。结果显示,成功建立了一种可通过1对引物实现PEDV S基因全序列及其上下游部分片段体外扩增的RT-PCR方法,最适退火温度为52℃。该方法具有高度的特异性,仅可与PEDV的cDNA发生反应,不与猪其他常见病原的cDNA或DNA发生交叉反应;具有良好的灵敏性,检测限为4.44×10^(4)copies/μL;具有良好的普适性,10份PEDV临床阳性样品均可扩增出预期大小的目的片段,并成功实现了S全基因测序和遗传进化分析。该方法的建立简化了S基因全序列体外扩增的方法,提高了体外扩增的成功率。Since the low success rate and complicity of the existing method for getting the full-length sequence of S gene in porcine epidemic diarrhea virus(PEDV),the upstream and downstream se-quences of S gene from known PEDV strains in NCBI were downloaded and screened,and the conserved re-gions were used to design a pair of primers for the reverse transcription polymerase chain reaction(RT-PCR)amplification.Then validation of the method was performed from specificity,sensitivity and clinical application.The results showed that an RT-PCR method was successfully developed to amplify the complete S gene with partial upstream and downstream sequences in vitro,with the optimal annealing temperature of 52℃.This method was highly specific,reacting only with the cDNA of PEDV,and did not cross-react with the cDNAs or DNAs of other common porcine pathogens.It was sensitive,with a detection limit of 4.44 ×10^(4) copies/μL.In addition,it is generally applicable.Target fragments of expected sizes were obtained by amplifying 1o PEDV clinical positive samples.Besides,the sequencing of the S gene and the analysis of genetic evolution were realized.The development of this method simplifies the in vitro amplification of complete S gene in PEDV and improves the success rate.

关 键 词：猪流行性腹泻病毒 S基因 RT-PCR 全长序列 进化分析 

分 类 号：S852.659.6[农业科学—基础兽医学]