[Development and clinical trial of fluorescence real time PCR to detect rubella virus].
To establish a novel rapid, convenient, sensitive and specific method applicable to quantitative analysis of the rubella virus extensively, RV total RNA was extracted with Trizol. The envelope glycoprotein E1 gene was amplified from rubella virus by PCR, and the PCR products were cloned into the pMD18-T cloning vector and transfected into DH5alpha. After Amp selection and analysis of restriction enzyme, the clones carrying the E1 gene were identified. After quantitation and serial dilution, the quantitative analysis of E1 gene was made by real-time PCR with the use of FAM as indicator. Standard curve of the real-time PCR was plotted with starting cDNA concentration versus threshold cycle. Then the new method was used to measure 50 cases with suspectable RV infection. The results were compared with those obtained by ELISA assay. TaqMan(r)MGB real-time PCR could help evaluate the level of virus reliably. The correlation coefficient of the standard curve is 0.998, and the linear range of the system is from 10(3) copies/microl to 10(9) copies/microl in clinical samples. The CV value is 0.94% in batch assay and 3.36% in day to day assay. The new method is more sensitive and specific than ELISA assay. For its simplicity, sensitivity, specificity and digitized results, the real-time PCR for quantification of RV cDNA in clinical samples is available.
Pu
Xiaorong
X
Department of Laboratorial Medicine, Chongqing University of Medical Sciences, Chongqing 400016, China.
Zhang
Dechun
D
D004279:DNA, Viral / Q000032:analysis; D005453:Fluorescence; D006801:Humans; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012412:Rubella virus / Q000302:isolation & purification*; D012680:Sensitivity and Specificity
D004279:DNA, Viral