A reverse-transcription competitive PCR assay based on chemiluminescence hybridization for detection and quantification of hepatitis C virus RNA

Kung-Chia Young, Ting-Tsung Chang, Wei Chiang Hsiao, Pin-Nan Cheng, Shu-Hui Chen, Chung Min Jen

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Abstract

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 4.95×107 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.

Original languageEnglish
Pages (from-to)27-39
Number of pages13
JournalJournal of Virological Methods
Volume103
Issue number1
DOIs
Publication statusPublished - 2002 Apr 3

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Luminescence
Hepacivirus
Reverse Transcription
RNA
Polymerase Chain Reaction
DNA
Hepatitis C Antibodies
Viral Load
Serum
Limit of Detection
Genotype

All Science Journal Classification (ASJC) codes

  • Virology

Cite this

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title = "A reverse-transcription competitive PCR assay based on chemiluminescence hybridization for detection and quantification of hepatitis C virus RNA",
abstract = "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 4.95×107 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.",
author = "Kung-Chia Young and Ting-Tsung Chang and Hsiao, {Wei Chiang} and Pin-Nan Cheng and Shu-Hui Chen and Jen, {Chung Min}",
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AU - Young, Kung-Chia

AU - Chang, Ting-Tsung

AU - Hsiao, Wei Chiang

AU - Cheng, Pin-Nan

AU - Chen, Shu-Hui

AU - Jen, Chung Min

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N2 - 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 4.95×107 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.

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