Detection of hepatitis C virus RNA by a combined reverse transcription PCR assay: comparison with nested amplification and antibody testing

Abstract

Many of the current reverse transcription (RT)-PCR assays for the detection of hepatitis C virus (HCV) RNA are multistep processes which use multiple enzymes and buffers. The assays are also often suboptimal, requiring nested amplification to achieve the desired levels of sensitivity. As a result, these assays are cumbersome and prone to false-positive results. The susceptibility to contamination is further aggravated by the lack of carryover controls. We have previously reported the development of a combined RT-PCR assay for HCV RNA detection which is sensitive and simple to perform. We have since successfully integrated dUTPuracil-N-glycosylase carryover prevention into the combined assay. Restriction of as much as 0.5 l of deoxyuridine-containing amplification products has been achieved. The performance of the improved combined assay was compared directly with conventional nested RT-PCR and antibody detection. The combined assay was found to have sensitivity similar to that of nested RT-PCR in detecting HCV RNA from HCV antibody-positive specimens. In an analysis of hepatitis B virus antibody-positive specimens, nested amplification had false-positive rates ranging from 8 to 31%, while no false-positive results were seen with the combined assay. In comparison with serological methods, the combined assay had specificity and sensitivity of 100 and 95%, respectively.

Quantification of hepatitis C virus by TaqMan PCR: Comparison with HCV amplicor monitor assay

“…The Amplicor Monitor assay was performed with 100 l of serum according to the procedure used for quantitation of the human immunodeficiency virus [Mulder et al, 1994]. The principle of this assay has been described previously [Young et al, 1993[Young et al, , 1995Roth et al, 1996]. It involves three steps: sample preparation after adding a constant amount of internal quantitative standard RNA; RT-PCR with rTth DNA polymerase; and detection of amplified products on Amplicor microwell plates coated with albuminconjugated immobilized probes and an internal quantitative standard amplicon-specific DNA probe.…”

“…The quantitation of serum levels of HCV RNA in chronic hepatitis C has been regarded as one of the most important indicators for the outcome of IFN therapy, since a sustained response can be expected in patients with a low virus load [Lau et al, 1993]. Several assays have been developed for evaluation of the HCV RNA load using polymerase chain reaction (PCR) or a hybridization procedure such as competitive reverse transcription-PCR (CRT-PCR) [Kato et al, 1990[Kato et al, , 1993, combined reverse transcription-PCR assay (Amplicor-HCV Monitor; Roche Molecular Systems, Branchburg, NJ) [Young et al, 1993[Young et al, , 1995Roth et al, 1996], and branched chain DNA probe assay (bDNA probe assay; Chiron, Emeryville, CA) [Lau et al, 1993;Detmer et al, 1996].…”

Recombinase polymerase amplification as a promising tool in hepatitis C virus diagnosis

“…In this approach, HCV RNA is extracted from the sample and reverse transcribed into the complementary DNA, which is then amplified into a large number of detectable copies by the polymerase chain reaction (PCR). Unlike antibody detection that could be positive for years after resolving infection, the presence of HCV RNA indicates active infection and it can be detected in 1-2 wk post-infection [27,28] . NAT offers accurate and sensitive diagnosis of HCV without any additional confirmatory test and can be used to diagnose individuals with acute HCV infection.…”

Development and validation of an RT‐qPCR assay for rapid detection and quantification of hepatitis C virus RNA for routine testing in Moroccan clinical specimens

“…Most NATs are based on conventional reverse transcription-polymerase chain reaction (RT-PCR). [11][12][13] Alternatives were based on branched-DNA (bDNA) assay [14][15][16] or in-house competitive PCR (cPCR). [17][18][19][20][21] RT-qPCR is one of the faster and the most sensitive molecular approaches used to detect HCV RNA.…”