Genotyping of Hepatitis C Virus by Sequence Analysis of the Amplicon from the Roche Cobas AmpliPrep/Cobas TaqMan Viral Load Assay

Abstract

ᰔHepatitis C virus (HCV), a positive single-stranded RNA virus, is one of the major causes of end-stage liver disease worldwide (5). The nucleotide sequence of the HCV genome is an important predictor of response to antiviral therapy (6). Genotypic analysis is used together with measurement of viral load (VL) to help in the management of patients with HCV infection (1). The 5Ј untranslated region (UTR) is often used for monitoring VL because it is less variable than other regions of the genome and consequently less likely to suffer PCR failures due to sequence variation at the primer binding sites (3,6,8). Although this region is not ideal for determination of the genotype, it is often used for this purpose because of the availability of the template after a VL assay and also because the genotypic information needed for clinical use can be obtained from it (2, 4, 9, 10).Our initial attempt to sequence the HCV 5Ј UTR using the reverse transcription-PCR (RT-PCR) amplicon from the recently introduced Roche Diagnostics (Basel, Switzerland) Cobas AmpliPrep/Cobas TaqMan HCV test (HCV-T) (8) employed a set of sequencing primers originally described by Gargiulo et al. (2) for analysis of the RT-PCR amplicon from the Roche Cobas Amplicor HCV monitor test (HCV-M). This attempt failed because the sequence obtained was often inconsistent with the genome of HCV (data not shown). We suspected that this failure was due to the presence of an abundant amplicon from the internal quantitation standard (QS). The QS is a small nucleic acid that is added to the specimen in the VL assay as an internal control. It is amplified using the same primers used to amplify the HCV target but contains a different internal sequence so that it can be detected using a TaqMan probe with a different fluorophore than the one used to detect the HCV amplicon. To troubleshoot this problem, we sequenced the amplicon from a clinical specimen that was negative for HCV, using the sequencing primers KY78 and KY80 (2), to obtain the sequence of the QS amplicon that is generated by the Roche HCV-T. As shown in Fig. 1, the primers designed by Gargiulo et al. (2) to sequence the amplicon from the HCV-M assay (labeled MF and MR in Fig. 1) are no longer specific for HCV, but hybridize equally well to the QS. Presumably the QS in the HCV-T assay contains additional nucleotides from the HCV genome compared with the QS from the HCV-M assay. We designed new sequencing primers to regain specificity for HCV and avoid sequencing the QS to obtain an acceptable HCV sequence (data not shown). The upstream primer is 5Ј-AGCCATGGCGTTAGTATGAG-3Ј, and the downstream primer is 5Ј-GCAGTACCACAAGG CCTTTC-3Ј. As illustrated in Fig. 1, the upstream primer from Gargiulo et al. (MF) is moved downstream by 3 bp to produce the TF primer, and the downstream primer (MR) is moved upstream by 3 bp to produce the TR primer. Both of the present primers have three nucleotides at the 3Ј end to create specificity for the HCV 5Ј-UTR sequence and avoid amplification of the QS but still avoid the va...

Real-time polymerase chain reaction optimised for hepatitis C virus detection in dried blood spots from HIV-exposed infants, KwaZulu-Natal, South Africa

“…This could be useful for detecting acute HCV infections, which generally have viral loads less than 10 5 IU/mL 33 . The detection of amplified nucleic acid using SYBR Green I is inexpensive and is an easier technical approach in comparison to other real-time PCR formats such as TaqMan and molecular beacon detection methods 14 . It is possible that HCV RNA viral load in our infant DBS specimens was below the level of detection of the assay, since HCV RNA viral loads amongst infants in the first year of infection may be too low for detection 3 …”

“…Numerous studies have reported on the use of real-time PCR for HCV diagnosis using plasma and serum, 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 although few have looked at DBS specimens 21 , 22 , 23 , 24 , 25 . However, a few recent studies have found good correlations for adult serum and plasma compared with DBS specimens analysed using commercial HCV PCR assays 26 , 27 , 28 , 29 …”