Establishment and application of a TaqMan real-time quantitative reverse transcription-polymerase chain reaction assay for rubella virus RNA.
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.
Zhao
Li-Hong
LH
Department of Microbiology, School of Medicine, Shandong University, Jinan 250012, China. zhaolihong7097@yahoo.com.cn
Ma
Yu-Yan
YY
Wang
Hong
H
Zhao
Shu-Ping
SP
Zhao
Wei-Ming
WM
Li
Hua
H
Wang
Lei-Yi
LY
D006801:Humans; D012367:RNA, Viral / Q000032:analysis*; D012015:Reference Standards; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000592:standards; D012409:Rubella / Q000175:diagnosis*; D012412:Rubella virus / Q000737:chemistry / Q000235:genetics*
D012367:RNA, Viral