摘要
目的应用基于TaqMan探针的实时定量RT-PCR检测轮状病毒(Rotavirus,RV)G4型VP7基因。方法从G1、G2、G3和G4型轮状病毒Vero细胞培养物中提取病毒总RNA,分别进行RT-PCR和实时定量RT-PCR,检测引物及探针的特异性;构建含轮状病毒G4型VP7基因的质粒,制备RNA参考品,绘制实时定量RT-PCR标准曲线;验证实时定量RT-PCR的灵敏度和精密性;对5个轮状病毒G4型培养物及G1、G2和G3型各3个病毒培养物进行检测,分析其实际应用性。结果以轮状病毒G4型VP7基因引物和探针只能从G4型轮状病毒总RNA中扩增出目的基因或检测到荧光信号的扩增,未在G1、G2、G3型轮状病毒总RNA中扩增出目的基因或检测到荧光信号的扩增;在2.34×107～2.34×103拷贝/μl范围内,反应扩增效率大于90%,R2大于0.98;该方法可检出100数量级的RNA样品,且试验内及试验间变异系数分别小于2.5%和4%;用建立的实时定量RT-PCR只检出5个轮状病毒G4型样品,而G1、G2、G3型样品均未检出。结论 TaqMan实时定量RT-PCR是一种灵敏度较高、特异性和精密性良好的定量检测轮状病毒G4型的方法。
Objective To detectthe VP7 gene of rotavirus serotype G4 by Taq Manprobe-basedreal-timequantitative RT-PCR.Methods Total RNAs were extracted from the rotavirus serotypes G1,G2,G3 and G4 cultured in Vero cells for test for the specificity of primers and probes by RT-PCR and real-time quantitative RT-PCR respectively.The plasmid carrying the VP7 gene of rotavirus serotype G4 was constructed,and RNA reference was prepared,based on which a standard curve of real-time quantitative RTPCR was plotted,and the sensitivity and precision of the method were verified.Five cultures of rotavirus serotype G4 and three cultures of each of serotypes G1,G2 and G3 were detected by TaqMan probe-based real-time quantitative RT-PCR to evaluate the practicality.Results Only from the total RNA of rotavirus serotype G4,the target gene or the fluorescent signal was amplified by using the primers and probes for VP7 gene of rotavirus serotype G4.No target gene or fluorescent signal was amplified from the total RNA of rotavirus serotype G1,G2 or G3.Within a RNA concentration range of 2.34 × 103 ～ 2.34 × 107 copies/μl,the amplification efficacy was more than 90%,and the R2 was more than 0.98.The RNA samples at a concentration level of 100 copies/μl was detected by the method,and the intra-and inter-coefficients of variations（CVs）were less than 2.5% and less than 4% respectively.By the developed method,only the detection results of five rotavirus serotype G4 samples were positive,while those of serotype G1,G2 and G3 were negative.Conclusions TaqMan probe-based real-time quantitative RT-PCR is a sensitive,specific and precise method for detection of rotavirus serotype G4.
出处
《中国生物制品学杂志》
CAS
CSCD
2010年第11期1260-1263,共4页
Chinese Journal of Biologicals
