三种猪腹泻相关病原微芯片检测方法研究及初步应用  

A Study on and Preliminary Application of Microchip Detection Method for Three Kinds of Swine Diarrhea Related Pathogens

作　　者：高志强 汪琳 蒲静 赵相鹏 尹羿 任彤 张伟 刘凤华[2] GAO Zhi-Qiang;WANG Lin;PU Jing;ZHAO Xiang-Peng;YIN Yi;REN Tong;ZHANG Wei;LIU Feng-Hua(Beijing Customs Testing Center,Beijing 101113;Beijing College of Agriculture,Beijing 102206)

机构地区：[1]北京海关技术中心,北京100026 [2]北京农学院,北京102206

出　　处：《中国口岸科学技术》2020年第10期4-10,共7页China Port Science and Technology

基　　金：国家重点研发计划2017YFF0305;北京市科技计划课题(Z171100001317011)。

摘　　要：为同时快速筛查猪传染性胃肠炎病毒(Transmissible gastroenteritis virus,TGEV)、猪流行性腹泻病毒(Porcine epidemic diarrhea virus,PEDV)以及猪圆环病毒2型(Porcine circovirus type 2,PCV2)3种可致猪腹泻的猪病病原,本研究基于微芯片技术,针对3种病毒特异性基因分别建立微量实时荧光(RT-)PCR检测体系,并将其组合在微芯片上。结果显示,该方法可特异性检出以上3种病毒核酸,但不能检出本实验室保存的其他5种猪病病毒核酸,而且相互之间无交叉反应;灵敏度试验显示TGEV和PEDV检测灵敏度均可达到1TCID_(50)/0.1 mL,对于含PCV2全基因组质粒,检测极限为41 fg/μL。对237份送检样品的检测结果显示,PEDV和PCV2检出率分别为0.8%和7.2%,但未检出TGEV,与常规管式荧光(RT-)PCR检测结果一致。该项技术可大大降低筛查的试剂成本,因为每个反应池只需要1.2μL的(RT-)PCR试剂混合物,而且比传统的实时(RT-)PCR系统的热平衡和热均匀性更快。本研究表明该技术可用于猪相关疫病的快速检测和筛查。In order to quickly and simultaneously detect 3 kinds of porcine pathogens including transmissible gastroenteritis virus(TGEV),porcine epidemic diarrhea virus(PEDV),and porcine circovirus type 2(PCV2)that can cause porcine diarrhea,based on realtime(RT-)PCR technology,a microchip real-time(RT-)PCR detection system targeting three virus-specific genes was established.The results showed that the method could specifically detect the 3 kinds of viral nucleic acids above,but could not detect the other 5 kinds of virus nucleic acids stored in our laboratory with no cross reaction among them.Sensitivity test showed that the detection limits were about 1 TCID50/0.1 mL for TGEV and PEDV,but 41 fg/μL for plasmid containing complete PCV2 genome.A total of 237 delivered samples were detected by the developed methods,and positive rates of PEDV and PCV2 were 0.8%and 7.2%respectively,while all the results for TGEV were negative,which was consistent with the results of traditional real-time(RT-)PCR.This technology can greatly reduce the cost of screening reagents since only 1.2μl of PCR reagent mixture is needed for each reaction pool,and compared with the traditional real-time PCR system,it allows faster thermal equilibrium and thermal uniformity.This study shows that this technology can be used for rapid detection and screening of swine-diarrhea-related diseases.

关 键 词：猪腹泻相关病毒 微芯片 荧光(RT-)PCR 

分 类 号：S858.28[农业科学—临床兽医学]