A reverse-transcription competitive PCR assay based on chemiluminescence hybridization for detection and quantification of hepatitis C virus RNA.
A reverse-transcription competitive PCR (RT-cPCR) combined with chemiluminescence hybridization was designed for the detection and quantitative determination of serum hepatitis C virus (HCV) RNA. The concentration of HCV RNA was calculated based on an external standard curve that was generated by coamplification of internal competitor and target sequences in serial dilutions. The detection limit of the chemiluminescence RT-cPCR was 100 copies/ml (94 IU/ml). Meanwhile, the linear range for quantitation extended from 850 copies/ml (795 IU/ml) to 4.95x10(7) copies/ml. The performance of the current assay for measuring circulating HCV levels from 26 anti-HCV-antibody positive patients was compared with that of branched-chain DNA (bDNA) and nested RT-PCR assays. Eighteen patients had HCV RNA levels that exceeded the quantitation limit by the chemiluminescence RT-cPCR, but only 11 patients were quantitation-positive by the bDNA. A significant correlation of the quantitation values was found between the chemiluminescence RT-cPCR and the bDNA (R2=0.8391). Among the eight patients with HCV RNA titers below the quantitation limit, four remained positive by the chemiluminescence cRT-PCR, demonstrating the results in agreement with those using the nested RT-PCR. Furthermore, good linearity was revealed for the HCV genotypes 1b, 2a, 2b in 3-order magnitude diluted serum samples. In conclusion, the proposed chemiluminescence RT-cPCR method can detect quantitatively HCV RNA as accurately as the bDNA method and has sensitivity as high as nested RT-PCR.
Young
Kung-Chia
KC
Department of Medical Technology, Medical College, National Cheng Kung University, 70101, Tainan, Taiwan, ROC. t7908077@mail.edu.tw
Chang
Ting-Tsung
TT
Hsiao
Wei-Chiang
WC
Cheng
Pin-Nan
PN
Chen
Shu-Hui
SH
Jen
Chung-Min
CM
D021121:Branched DNA Signal Amplification Assay; D002138:Calibration; D004076:Digoxigenin; D016174:Hepacivirus / Q000302:isolation & purification*; D006526:Hepatitis C / Q000097:blood / Q000175:diagnosis* / Q000821:virology; D018937:Hepatitis C Antibodies / Q000097:blood; D006801:Humans; D008163:Luminescent Measurements; D012367:RNA, Viral / Q000097:blood*; D012015:Reference Standards; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D014766:Viremia / Q000175:diagnosis*
D018937:Hepatitis C Antibodies; D012367:RNA, Viral; D004076:Digoxigenin