The development of a qualitative real-time RT-PCR assay for the detection of hepatitis C virus

Clancy, A.; Crowley, Brendan; Niesters, Hubert G. M.; Herra, C. M.

doi:10.1007/s10096-008-0556-9

Cited by 35 publications

(23 citation statements)

References 10 publications

(13 reference statements)

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“…Unlike serological assays, those based on real-time RT-PCR can be used for the diagnosis of acute hepatitis before seroconversion and in the case of some seronegative patients with immune deficiency. Detection based on real-time RT-PCR is also useful for confirming indeterminate serological results and monitoring response to treatment [13]. …”

Section: Introductionmentioning

confidence: 99%

A novel duplex real-time reverse transcriptase-polymerase chain reaction assay for the detection of hepatitis C viral RNA with armored RNA as internal control

Shao

2010

Virol J

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BackgroundThe hepatitis C virus (HCV) genome is extremely heterogeneous. Several HCV infections can not be detected using currently available commercial assays, probably because of mismatches between the template and primers/probes. By aligning the HCV sequences, we developed a duplex real-time reverse transcriptase-polymerase chain reaction (RT-PCR) assay using 2 sets of primers/probes and a specific armored RNA as internal control. The 2 detection probes were labelled with the same fluorophore, namely, 6-carboxyfluorescein (FAM), at the 5' end; these probes could mutually combine, improving the power of the test.ResultsThe limit of detection of the duplex primer/probe assay was 38.99 IU/ml. The sensitivity of the assay improved significantly, while the specificity was not affected. All HCV genotypes in the HCV RNA Genotype Panel for Nucleic Acid Amplification Techniques could be detected. In the testing of 109 serum samples, the performance of the duplex real-time RT-PCR assay was identical to that of the COBAS AmpliPrep (CAP)/COBAS TaqMan (CTM) assay and superior to 2 commercial HCV assay kits.ConclusionsThe duplex real-time RT-PCR assay is an efficient and effective viral assay. It is comparable with the CAP/CTM assay with regard to the power of the test and is appropriate for blood-donor screening and laboratory diagnosis of HCV infection.

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Section: Introductionmentioning

confidence: 99%

A novel duplex real-time reverse transcriptase-polymerase chain reaction assay for the detection of hepatitis C viral RNA with armored RNA as internal control

Shao

2010

Virol J

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“…The observed false positive results, hence, higher HCV antibody seroprevalence (1.67%) by Diaspot rapid enzyme immunoassay compared to that of Biorad Genscreen HCV Ag-Ab Monolisa assay (0.33%) based on 'sandwich' ELISA technique in this study contrast the outcomes of studies by other researchers who found false negative results using Diaspot rapid one-step enzyme immunoassay [18] . Current trends in transfusion service demand that initial screening of blood and blood products be based on ELISA technique using kit that both screen for the HCV core antigen and anti-HCV antibodies that improves early detection of HCV infection during the window period and subsequent confirmation of HCV status with recombinant immunoblotting assay (RIBA) or nucleic acid testing (NAT) by real-time PCR [40][41][42] . Literatures have showed that continued development of newer testing techniques have helped to significantly improve early detection and reduce the window period from 82 days to 66 days and even lower [43] .…”

Section: Discussionmentioning

confidence: 99%

Hepatitis C Virus Diagnosis in Prospective Blood Donors: Epidemiology, Optimal Testing Approach and Treatment Cut-offs

Ifechukwudebelu¹,

Kolawole²,

Ositadima³

2017

Am. J. Biomed. Sci.

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Hepatitis C virus (HCV) infection has been noted a major public health problem with no vaccines available currently. This study aims at optimizing 'safe blood' practice by advancing HCV testing beyond Diaspot rapid enzyme immunoassay in current use in Nigerian Health Institutions. Between August, 2014 and November, 2015, a total of 300 blood donors' plasma samples were screened with both Diaspot and HCV Ag-Ab enzyme-linked immunosorbent assay techniques. HCV-RNA confirmation and quantification were performed with real-time polymerase chain reaction (PCR). Alanine aminotransferase (ALT) was performed on confirmed positive blood donor for treatment purpose. The overall gender ratio and mean age of blood donors screened for HCV were 1.5:1 and 27.67 ± 7.77 years respectively. Of the 300 blood donors screened, 5 (1.67%) and 1 (0.33%) were seropositive for HCV on the basis of Diaspot and enzyme-linked immunosorbent assay (ELISA) techniques respectively. Diagnostic odds ratio showed that ELISA is nearly 9-fold a better diagnostic tool compared to Diaspot technique. Real-time PCR assay confirmed positivity of 1 (0.33%) of the blood donor for hepatitis C. HCV-RNA viral load and plasma ALT of the lone sample were 133, 209 IU/mL and 11.1 IU/L respectively. HCV prevalence among blood donors in Ekiti state is low, 0.33%. Enzyme-linked immunosorbent assay technique should be the starting point of HCV serologic screening and a surrogate technique for real-time PCR. The development of workable algorithm to reduce risks associated with blood transfusion and enhances both blood donors' and recipients' safety is highly imperative.

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“…Total nucleic acid (TNA) is extracted from nasopharyngeal swabs in Universal Transport Medium (UTM, COPAN, Brescia, Italy) using NucliSens extraction on easyMAG (BioMérieux, Lyon, France). Ten microliters of Phocine Distemper Virus (PDV) internal control (IC) (Clancy et al, 2008) (PDV stock kindly supplied by Groningen Medical Center, Groningen, The Netherlands) is added to 200 μL UTM sample to extract and elute 110 μL of TNA. Five microliters of TNA extract is mixed with 1× TaqMan Fast Virus 1-Step Master Mix (Thermo Fisher Scientific, Waltham, MA), 0.5 μM of each primer, and 0.2 μM of each probe in a total volume of 20 μL.…”

Section: Mers-cov Ldtmentioning

confidence: 99%

Migrating a lab-developed MERS-CoV real-time PCR to 3 “Sample to Result” systems: experiences on optimization and validation

Frans

Beuselinck

Peeters

et al. 2019

Diagnostic Microbiology and Infectious Disease

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The goal of the study was to adapt our Middle East respiratory syndrome coronavirus (MERS-CoV) lab-developed test (LDT) to 3 "Sample to Result" (S2R) systems: BD MAX (BD), ELITe InGenius (ELITechGroup), and ARIES (Luminex). The BD MAX and InGenius system allowed use of lab-developed primers and TaqMan probes, while ARIES required conversion to MultiCode primers for melting curve analysis. Each device required ≤1 day of training and assay optimization. No discordant results were noted after analysis of 32 External Quality Control (EQC) samples. On a 10-fold dilution series of a MERS-CoV-positive EQC sample, InGenius obtained the highest detection rate. Laboratory technicians rated the ARIES as the user-friendliest. It also required the least hands-on time. BD MAX had the lowest turnaround time and highest throughput. While each device had distinguishing system properties with associated (dis)advantages, the 3 S2R systems were comparable in terms of assay development and validation.

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The development of a qualitative real-time RT-PCR assay for the detection of hepatitis C virus

Cited by 35 publications

References 10 publications

A novel duplex real-time reverse transcriptase-polymerase chain reaction assay for the detection of hepatitis C viral RNA with armored RNA as internal control

A novel duplex real-time reverse transcriptase-polymerase chain reaction assay for the detection of hepatitis C viral RNA with armored RNA as internal control

Hepatitis C Virus Diagnosis in Prospective Blood Donors: Epidemiology, Optimal Testing Approach and Treatment Cut-offs

Migrating a lab-developed MERS-CoV real-time PCR to 3 “Sample to Result” systems: experiences on optimization and validation

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