Zhao Li-Hong, Ma Yu-Yan, Wang Hong, Zhao Shu-Ping, Zhao Wei-Ming, Li Hua, Wang Lei-Yi
Department of Microbiology, School of Medicine, Shandong University, Jinan 250012, China.
Acta Biochim Biophys Sin (Shanghai). 2006 Oct;38(10):731-6. doi: 10.1111/j.1745-7270.2006.00213.x.
The aim of this study was to establish and apply a real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) for rubella virus (RV) RNA. First, the primer and TaqMan probe concentrations, as well as reaction temperatures were optimized to establish an efficient real-time quantitative RT-PCR assay for RV RNA. Next, an RV-specific PCR amplicon was made as an external standard to estimate the linearity, amplification efficiency, analytical sensitivity and reproducibility of the real time quantitative assay. Finally, the assay was applied to quantify RV RNA in clinical samples for rubella diagnosis. The RV-specific PCR amplicon was prepared for evaluation of the assay at 503 bp, and its original concentration was 2.75x109 copies/mul. The real time quantitative assay was shown to have good linearity (R2=0.9920), high amplification efficiency (E=1.91), high sensitivity (275 copies/ml), and high reproducibility (variation coefficient range, from 1.25% to 3.58%). Compared with the gold standard, the specificity and sensitivity of the assay in clinical samples was 96.4% and 86.4%, respectively. Therefore, the established quantitative RT-PCR method is a simple, rapid, less-labored, quantitative, highly specific and sensitive assay for RV RNA.
本研究的目的是建立并应用一种针对风疹病毒（RV）RNA的实时定量逆转录-聚合酶链反应（RT-PCR）。首先，对引物和TaqMan探针浓度以及反应温度进行优化，以建立一种高效的针对RV RNA的实时定量RT-PCR检测方法。接下来，制备一个RV特异性PCR扩增子作为外部标准，以评估实时定量检测的线性、扩增效率、分析灵敏度和重现性。最后，将该检测方法应用于临床样本中RV RNA的定量分析，以进行风疹诊断。制备了503 bp的RV特异性PCR扩增子用于检测方法的评估，其初始浓度为2.75x109拷贝/微升。实时定量检测显示具有良好的线性（R2 = 0.9920）、高扩增效率（E = 1.91）、高灵敏度（275拷贝/毫升）和高重现性（变异系数范围为1.25%至3.58%）。与金标准相比，该检测方法在临床样本中的特异性和灵敏度分别为96.4%和86.4%。因此，所建立的定量RT-PCR方法是一种用于RV RNA的简单、快速、省力、定量、高度特异且灵敏的检测方法
