CAJ | 학술논문

针对草鱼呼肠孤病毒(grass carp reovirus, GCRV)各类型毒株的保守区域分别设计3对引物,优化反应条件和体系,建立了草鱼呼肠孤病毒三重 RT-PCR 检测方法,通过 PCR 扩增分别获得大小约为530 bp、297 bp 和196 bp的 DNA 片段.进一步分析表明,该检测方法具有良好的敏感性、特异性和稳定性,可以检测Ⅰ、Ⅱ、Ⅲ型核酸最低量分别约为260、190与230拷贝的病毒量,且某一类型的引物只能检测到同一类型的 GCRV.用建立的多重RT-PCR 方法对2008—2011年采集自江西、广东、湖北、浙江、重庆、四川、福建等地的草鱼(Ctenopharyngodon idellus)主养区的86份草鱼出血病样品进行检测和初步流行病学分析.结果表明,Ⅰ型阳性率为9.3%,Ⅱ型阳性率为45.3%,Ⅲ型阳性率为2.3%,Ⅰ型和Ⅱ型混合感染阳性率为5.8%,Ⅱ和Ⅲ型混合感染阳性率为2.3%,其他类型的混合感染未检测到.表明目前主要流行的草鱼呼肠孤病毒为Ⅱ型.本研究建立的 GCRV 三重 PCR 检测方法可以特异性的检测各种类型的草鱼呼肠孤病毒,且具有较高的敏感性;利用该方法进行草鱼出血病初步的流行病学分析表明,目前主要流行的草鱼呼肠孤病毒为Ⅱ型,不同毒株类型混合感染的现象也普遍存在.本方法的建立旨在对草鱼出血病的快速诊断和分子流行病学调查提供实用的可操作手段.
침대초어호장고병독(grass carp reovirus, GCRV)각류형독주적보수구역분별설계3대인물,우화반응조건화체계,건립료초어호장고병독삼중 RT-PCR 검측방법,통과 PCR 확증분별획득대소약위530 bp、297 bp 화196 bp적 DNA 편단.진일보분석표명,해검측방법구유량호적민감성、특이성화은정성,가이검측Ⅰ、Ⅱ、Ⅲ형핵산최저량분별약위260、190여230고패적병독량,차모일류형적인물지능검측도동일류형적 GCRV.용건립적다중RT-PCR 방법대2008—2011년채집자강서、엄동、호북、절강、중경、사천、복건등지적초어(Ctenopharyngodon idellus)주양구적86빈초어출혈병양품진행검측화초보류행병학분석.결과표명,Ⅰ형양성솔위9.3%,Ⅱ형양성솔위45.3%,Ⅲ형양성솔위2.3%,Ⅰ형화Ⅱ형혼합감염양성솔위5.8%,Ⅱ화Ⅲ형혼합감염양성솔위2.3%,기타류형적혼합감염미검측도.표명목전주요류행적초어호장고병독위Ⅱ형.본연구건립적 GCRV 삼중 PCR 검측방법가이특이성적검측각충류형적초어호장고병독,차구유교고적민감성;이용해방법진행초어출혈병초보적류행병학분석표명,목전주요류행적초어호장고병독위Ⅱ형,불동독주류형혼합감염적현상야보편존재.본방법적건립지재대초어출혈병적쾌속진단화분자류행병학조사제공실용적가조작수단.
@@@@Three specific primer pairs were designed based on the conserved sequences of different genotypes of the grass carp reovirus (GCRV). 530 bp, 297 bp, and 196 bp DNA fragments were amplified and through optimizing the reaction conditions and system, a multiplex-PCR was established to detect the three genotypes simultaneously. Further studies indicate that the multiplex-PCR has good amplification efficiency, specificity, and sensitivity. The detection limit was 260 copies/μL, 190 copies/μL, and 230 copies/μL for types I, II, and III, respectively. The specificity experiment indicated that the primers were strictly genotype-specific. This method provides a significant improvement in the detection, genotyping, and molecular epidemiological study of GCRV. The detection results of 86 samples collected from 16 cities in the main grass carp breeding area in China using the established multiplex-PCR method revealed that the positive rates of genotypes I, II, and III were 9.3%, 45.3%, and 2.3%, respectively. The positive co-infection rate of types I and II was 5.8%, that of types II and III was 2.3%, while co-infection of types I and III was not detected. The multiplex PCR for the three genetypes of GCRV were highly specific for the corresponding virus genotypes, with high sensitivity. Preliminary epidemiological data analysis by multiplex PCR indicates that type II is the most common genotype, and the phenomenon of combined infection of different genotypes are in general population of grass carp.