摘要
以新发生的兔出血症病毒2型(RHDV2)SC 2020/04株VP60基因序列为参考,设计出1对特异性引物和TaqMan探针,建立了一种快速、灵敏且特异的用于检测新型病毒的荧光定量RT-PCR检测方法。试验结果显示:本研究建立的方法,标准曲线线性关系良好,R^(2)值达到0.999;其特异性较好,与兔出血症病毒1型(RHDV1)、仙台病毒(SV)和轮状病毒(RRV)病原均无交叉反应;灵敏性高,最低检出量为1μl 1×10拷贝;且重复性良好,批内和批间试验的变异系数平均值均小于2%。临床检测结果表明,利用该方法对108份临床病料进行检测,检出RHDV2阳性样品72份,检出率为66.7%,明显高于常规的RT-PCR方法(62.0%)。结果证明,新建立的TaqMan荧光定量RT-PCR方法适合于RHDV2感染的特异性诊断。
A rapid,sensitive and specific fluorescence quantitative real-time PCR(RT-qPCR)method for the detection of rabbit hemorrhagic disease virus type 2(RHDV2)was established.A pair of specific primers and TaqMan probes were designed in the conserved region of VP60 gene of the new variant strain SC 2020/04.The results showed that the standard curve had a good linear relationship,the R^(2) was 0.999.The TaqMan-based RT-qPCR method had good specificity and no cross reactions with rabbit hemorrhagic disease virus type1(RHDV1),sendai virus(SV)and rotavirus(RRV).The method established in this study had high sensitivity,and the minimum detectable amount was 10 copies per microlitre.Moreover,the repeatability was good,and the mean coefficient of variation was less than 2%.The TaqMan-based RT-qPCR method was used to detect 108 clinical samples.The detection rate was 66.7%,which was higher than that of conventional RT-PCR.To sum up,the newly established TaqMan-based RT-qPCR method is suitable for the specific diagnosis of RHDV2 infection.
作者
陈萌萌
仇汝龙
范志宇
胡波
宋艳华
魏后军
朱伟峰
徐为中
王芳
CHEN Meng-meng;QIU Ru-long;FAN Zhi-yu;HU Bo;SONG Yan-hua;WEI Hou-jun;ZHU Wei-feng;XU Wei-zhong;WANG Fang(Institute of Veterinary Medicine,Jiangsu Academy of Agricultural Sciences/Key Laboratory for Veterinary Bio-Product Engineering,Ministry of Agriculture,Nanjing 210014,China)
出处
《江苏农业学报》
CSCD
北大核心
2021年第6期1476-1480,共5页
Jiangsu Journal of Agricultural Sciences
基金
国家自然科学基金项目(31702274)
现代农业产业技术体系建设专项资金项目(CARS-43-C-1)。
