Real-time detection system for quantification of hepatitis C virus genome

doi:10.1016/S0016-5085(99)70185-X

Gastroenterology

Volume 116, Issue 3, March 1999, Pages 636-642

https://doi.org/10.1016/S0016-5085(99)70185-X Get rights and content

Abstract

Background & Aims: For diagnosis of hepatitis C virus infection and monitoring of viral load in patients, a highly sensitive and accurate hepatitis C virus quantification system is essential. Methods: Hepatitis C virus genome was detected by real/time detection system using an ABI Prism 7700 sequence detector (Perkin Elmer Corp./Applied Biosystems, Foster City, CA). Results: As few as 10 copies of the genome were detected, and the quantification range was between 10¹ and 10⁸ copies (r > 0.99). This system was 10–100-fold more sensitive than an Amplicor monitor (Roche Diagnostic Systems, Branchburg, NJ). The coefficient of variation values for both intra-assay precision and interassay reproducibility of identifying the genome quantification ranged from 0.37% to 2.00% and 0.88% to 4.66%, respectively. The system could detect the genome in 98% of patients with chronic hepatitis, 95.8% of patients with liver cirrhosis, and 100% of patients with hepatocellular carcinoma who had the antibody to hepatitis C virus, but could not detect the genome in patients without the antibody. Conclusions: The establishment of a real-time detection system enables more accurate diagnosis of infection and monitoring of viral load in interferon-treated patients via quantification of viral genome.

GASTROENTEROLOGY 1999;116:636-642

Section snippets

Construction and in vitro RNA transcription of standard RNA

The plasmids pKI5 (genotype 1b HCV) and pKII5 (genotype 2b HCV)²³ were used as sense templates after digestion with HindIII. RNA was transcribed from the templates using T7 RNA polymerase and MEGAscript in vitro transcription kits according to the manufacturer's protocol (Ambion, Austin, TX).

To purify standard RNA from template DNA perfectly, the synthetic RNA was treated with 50 U of deoxyribonuclease 1 (Boehringer Mannheim GmbH, Mannheim, Germany) at 37°C for 10 minutes, followed by

Results

The sensitivity and quantitativity of RTD-PCR were examined by using synthetic HCV RNA (Figure 1). Set 2 primers and probes enabled detection of as few as 10 copies of genotype 1b and genotype 2b synthetic RNA (Figure 3).

. Amplification plots of synthetic HCV RNA. Tenfold serial dilutions of target HCV-RNA were prepared in triplicate, reverse transcribed and amplified using TaqMan probe, and detected using ABI Prism 7700 sequence detector. For each dilution, Δ normalized reporter (Rn) is plotted

Discussion

We developed a novel HCV-RNA quantification system using an RTD-PCR assay consisting of a dye-labeled oligonucleotide probe, primers complementary to the HCV 5' UTR of HCV genomes, rTth DNA polymerase, and a sequence detector. This technique enables cDNA synthesis and PCR amplification and analysis in a single reaction tube. The sensitivity and specificity of this assay were evaluated using synthetic HCV RNA as the standard and HCV RNA–positive or –negative sera as the control. We also

Acknowledgements

The authors thank Dr. Makoto Yoshiba for his thoughtful comments and suggestions and Atsuko Fujisawa for creating the figures.

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^☆: Address requests for reprints to: Michinori Kohara, Ph.D., Department of Microbiology, The Tokyo Metropolitan Institute of Medical Science, 3-18-22, Honkomagome, Bunkyo-ku, Tokyo 113-8613, Japan. e-mail: [email protected]; fax: (81) 3-3828-8945.

^☆☆: Supported in part by a Grant-in-Aid for Specially Promoted Research on Viral Diseases from the Tokyo Metropolitan Government; a grant from the Ministry of Education, Science, and Culture of Japan; and a grant from the Ministry of Health and Welfare of Japan.

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Liver, Pancreas, and Biliary TractReal-time detection system for quantification of hepatitis C virus genome☆,☆☆

Abstract

Section snippets

Construction and in vitro RNA transcription of standard RNA

Results

Discussion

Acknowledgements

Lancet

Detection of antibody to hepatitis C virus in prospectively followed transfusion recipients with acute and chronic non-A, non-B hepatitis

N Engl J Med

Interrelationship between blood transfusion, non-A, non-B hepatitis and hepatocellular carcinoma

Hepatology

Hepatitis C virus infection is associated with the development of hepatocellular carcinoma

Proc Natl Acad Sci USA

An assay for circulating antibodies to a major etiologic virus of human non-A non-B hepatitis

Science

Genetic organization and diversity of the hepatitis C virus

Proc Natl Acad Sci USA

Enhanced sensitivity of a 2nd generation ELISA for antibody to hepatitis C virus

Vox Sang

Performance of enzyme-linked immunosorbent assay system for antibodies to hepatitis C virus with two new proteins (c11/c7)

Clin Chem

Quantitation of hepatitis C virus RNA by competitive polymerase chain reaction

J Med Virol

Detection of hepatitis C virus by a combined reverse transcription-polymerase chain reaction assay

J Clin Microbiol

Hepatitis C virus genotypes 1 and 2 respond to interferon-α with different virologic kinetics

J Infect Dis

Branched DNA amplification multimers for the sensitive, direct detection of human hepatitis viruses

Nucleic Acids Symp Ser

Liver, Pancreas, and Biliary Tract
Real-time detection system for quantification of hepatitis C virus genome☆,☆☆