摘要
为建立能同时快速鉴别新型鹅星状病毒和鹅星状病毒变异株的检测方法,根据新型鹅星状病毒的ORF2基因和鹅星状病毒变异株的ORF1a基因的保守区域序列,设计并合成2对特异性引物。通过优化反应体系及反应条件,建立了能同时检测2种鹅星状病毒的一步法双重RT-PCR检测方法。该方法可同时扩增出新型鹅星状病毒658 bp和鹅星状病毒变异株381 bp的特异性条带,对坦布苏病毒、鹅细小病毒、鹅副黏病毒、H9N2亚型禽流感病毒、大肠杆菌和沙门菌的扩增结果均为阴性,具有良好的特异性。对新型鹅星状病毒和鹅星状病毒变异株重组质粒的最低检测量分别是1μl 1.71×103拷贝和1μl 1.70×103拷贝,敏感性良好,并且具有良好的重复性。应用该方法对临床采集的雏鹅泄殖腔拭子进行检测,结果显示,124份样品中新型鹅星状病毒阳性率为45.16%,鹅星状病毒变异株阳性率为28.23%,2种鹅星状病毒均为阳性的占11.29%,与单一RT-PCR方法检测结果符合率为100.00%。本研究建立的一步法双重RT-PCR检测方法快速简便,特异性强、灵敏度高,可用于临床样品的鉴别诊断,该方法的建立对2种鹅星状病毒病的流行病学调查和有效防控以及混合感染的致病性研究具有重要意义。
To establish a method for the simultaneous detection of the novel goose astrovirus(nGoAstV) and the variant of goose astrovirus(vGoAstV), two pairs of specific primers were designed and synthesized according to the conserved sequence of the nGoAstV ORF2 gene and the vGoAstV ORF1a gene. By optimizing the reaction system and conditions, a one-step duplex RT-PCR method was established for simultaneous detection of two kinds of goose astroviruses. Two specific fragments of nGoAstV(658 bp) and vGoAstV(381 bp) were amplified simultaneously. Moreover, the amplification results of Tembusu virus(TMUV), goose parvovirus(GPV), goose paramyxovirus(GPMV), H9N2 subtype avian influenza virus(AIV), Escherichia coli and Salmonella were negative, indicating that this method had good specificity. The minimum detection amount of recombinant plasmids of nGoAstV and vGoAstV was 1.71×10~3 copies per microliter and 1.70×10~3 copies per microliter respectively, indicating that the method had good sensitivity and repeatability. The 124 clinical cloacal swabs samples collected from gosling were detected by the established one-step duplex RT-PCR method. The positive rates of nGoAstV and vGoAstV were 45.16% and 28.23%, respectively, and the rate of two kinds of goose astrovirus co-infection was 11.29%. The coincidence rate between this method and the single RT-PCR method was 100.00%. The one-step duplex RT-PCR method established in this study is rapid, simple, sensitive and specific, which can be used for clinical differential diagnosis of nGoAstV and vGoAstV. It is of great significance for epidemiological investigation, effective prevention and control, and the pathogenicity study of co-infection of two kinds of goose astroviruses.
作者
杨颖
肖亦辰
马平
赵冬敏
章丽娇
李银
刘青涛
杨婧
刘宇卓
韩凯凯
黄欣梅
YANG Ying;XIAO Yi-chen;MA Ping;ZHAO Dong-min;ZHANG Li-jiao;LI Yin;LIU Qing-tao;YANG Jing;LIU Yu-zhuo;HAN Kai-kai;HUANG Xin-mei(Institute of Veterinary Medicine,Jiangsu Academy of Agricultural Sciences,Nanjing 210014,China;Key Laboratory of Veterinary Biological Engineering and Technology,Ministry of Agriculture and Rural Affairs,Nanjing 210014,China;National Research Center of Engineering and Technology for Veterinary Biologicals,Nanjing 210014,China;College of Veterinary Medicine,Nanjing Agricultural University,Nanjing 210095,China)
出处
《江苏农业学报》
CSCD
北大核心
2022年第6期1612-1619,共8页
Jiangsu Journal of Agricultural Sciences
