Zhao Lihong, Li Ruiying, Liu Aihua, Zhao Shuping
Department of Laboratory, Tai'an Central Hospital, Tai'an 271000, China.
Department of Reproductive Genetics, Tai'an Central Hospital, Tai'an 271000, China.
J Virol Methods. 2015 Jul;219:84-89. doi: 10.1016/j.jviromet.2015.03.023. Epub 2015 Apr 7.
The objective of this study was to build and apply a duplex real time quantitative reverse transcription-polymerase chain reaction (RT-PCR) for rubella virus. Firstly, a 60-bp-long armored RV RNA was constructed in the laboratory. Secondly, a duplex real time RT-PCR assay was established. Thirdly, the 60-bp-long armored RV RNA was used as an internal positive control (IPC) for the duplex real time RT-PCR. And finally the duplex real time RT-PCR assay was applied to detect RV RNA in clinical specimens. The in-house assay has a high amplification efficiency (0.99), a high analytical sensitivity (200 copies/mL), and a good reproducibility. The diagnostic specificity and sensitivity of the in-house assay were both 100%, due to the monitoring of the armored RV RNA IPC. Therefore, the in-house duplex real time quantitative RT-PCR assay is a specific, sensitive, reproducible and accurate assay for quantitation of RV RNA in clinical specimens. And noncompetitive armored RV RNA IPC can monitor RT-PCR inhibition and prevent false-negative and inaccurate results in the real time detection system.
本研究的目的是构建并应用一种用于风疹病毒的双重实时定量逆转录-聚合酶链反应（RT-PCR）。首先，在实验室构建了一条60个碱基对长的装甲风疹病毒RNA。其次，建立了一种双重实时RT-PCR检测方法。第三，将60个碱基对长的装甲风疹病毒RNA用作双重实时RT-PCR的内部阳性对照（IPC）。最后，将双重实时RT-PCR检测方法应用于检测临床标本中的风疹病毒RNA。该内部检测方法具有高扩增效率（0.99）、高分析灵敏度（200拷贝/毫升）和良好的重复性。由于对装甲风疹病毒RNA IPC进行了监测，该内部检测方法的诊断特异性和灵敏度均为100%。因此，该内部双重实时定量RT-PCR检测方法是一种用于定量临床标本中风疹病毒RNA的特异性、灵敏性、可重复性和准确性均很高的检测方法。并且非竞争性装甲风疹病毒RNA IPC可以监测RT-PCR抑制情况，并防止实时检测系统中出现假阴性和不准确的结果。
