A real-time Taqman method for hepatitis C virus genotyping

Rolfe, Kathryn J.; Alexander, Graeme; Wreghitt, Tim; Parmar, Surendra; Jalal, Hamid; Curran, Martin D.

doi:10.1016/j.jcv.2005.02.011

Cited by 22 publications

(9 citation statements)

References 21 publications

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“…These methods include PCR based assays, [13,14] Line probe assays, [12] RFLP assays [9,10] and sequence analysis. In all these assays, sequence analysis was considered to be more reliable and used as the gold standard method.…”

Section: Discussionmentioning

confidence: 99%

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A single-step multiplex quantitative real time polymerase chain reaction assay for hepatitis C virus genotypes

Singh

Mankotia

Irshad

2017

Journal of Translational Internal Medicine

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Background and Objectives: The variable response of hepatitis C virus (HCV) genotypes towards anti-viral treatment requires prior information on the genotype status before planning a therapeutic strategy. Although assays for typing or subtyping of HCV are available, however, a fast and reliable assay system is still needed. The present study was planned to develop a single-step multiplex quantitative real time polymerase chain reaction (qPCR) assay to determine HCV genotypes in patients' sera. Methods: The conserved sequences from 5′ UTR, core and NS5b regions of HCV genome were used to design primers and hydrolysis probes labeled with fluorophores. Starting with the standardization of singleplex (qPCR) for each individual HCV-genotype, the experimental conditions were finally optimized for the development of multiplex assay. The sensitivity and specificity were assessed both for singleplex and multiplex assays. Using the template concentration of 10 2 copies per microliter, the value of quantification cycle (Cq) and the limit of detection (LOD) were also compared for both singleplex and multiplex assays. Similarly, the merit of multiplex assay was also compared with sequence analysis and restriction fragment length polymorphism (RFLP) techniques used for HCV genotyping. In order to find the application of multiplex qPCR assay, it was used for genotyping in a panel of 98 sera positive for HCV RNA after screening a total number of 239 patients with various liver diseases. Results: The results demonstrated the presence of genotype 1 in 26 of 98 (26.53%) sera, genotype 3 in 65 (66.32%) and genotype 4 in 2 (2.04%) sera samples, respectively. One sample showed mixed infection of genotype 1 and 3. Five samples could not show the presence of any genotype. Genotypes 2, 5 and 6 could not be detected in these sera samples. The analysis of sera by singleplex and RFLP indicated the results of multiplex to be comparable with singleplex and with clear merit of multiplex over RFLP. In addition, the results of multiplex assay were also found to be comparable with those from sequence analysis. The sensitivity, specificity, Cq values and LOD values were compared and found to be closely associated both for singleplex and multiplex assays. Conclusion: The multiplex qPCR assay was found to be a fast, specific and sensitive method that can be used as a technique of choice for HCV genotyping in all routine laboratories.

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Section: Discussionmentioning

confidence: 99%

“…[12] Recently, some methods involved the use of single-step real-time reverse transcription-PCR (RT-PCR) with target-specific TaqMan probe. [13,14] However, all these existing techniques for HCV genotyping are time consuming and quite expensive. Therefore, it appears worth to attempt other regions for standardization of new assays.…”

Section: Introductionmentioning

confidence: 99%

A single-step multiplex quantitative real time polymerase chain reaction assay for hepatitis C virus genotypes

Singh

Mankotia

Irshad

2017

Journal of Translational Internal Medicine

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“…For viraemic patients, HCV genotype was identified using an improved Taqman HCV genotyping assay (Rolfe et al, 2005(Rolfe et al, , 2009). For non-viraemic patients, the infecting viral type was ascertained by serotyping using the Murex HCV Serotyping kit (Abbott Diagnostics, Berkshire), following manufacturers' guidelines.…”

Section: Hcv Typingmentioning

confidence: 99%

Spontaneous loss of hepatitis C virus RNA from serum is associated with genotype 1 and younger age at exposure

Rolfe

Curran

Alexander

et al. 2011

Journal of Medical Virology

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A variety of factors have been associated with spontaneous loss of hepatitis C virus (HCV)‐RNA from serum, including infecting HCV type, although results are conflicting. This study aimed to investigate further whether infecting HCV type was linked to spontaneous loss of HCV‐RNA. Serum samples from 321 untreated HCV antibody positive patients presenting at the Hepatology clinic at Addenbrooke's Hospital, Cambridge between 2004 and 2007 were tested. These individuals were classified either as HCV antibody and HCV‐RNA positive (viremic, n = 219) or HCV antibody positive and repeatedly HCV‐RNA negative (non‐viremic, n = 102). Infecting HCV type was identified by genotyping (viremic) or serotyping (non‐viremic). Binomial regression analysis investigated the independent effect of HCV type on spontaneous loss of HCV‐RNA from serum by comparing the two groups. Ninety‐one percent of patients were found to be either genotype 1 or genotype 3. The prevalence of type 1 infection was greater among non‐viremic (64.5%) than viremic individuals (45%). After controlling for the effects of potential confounding factors, multivariable analyses showed that individuals with type 1 infections were more likely to be non‐viremic than genotype 3 infections (RR = 2.07; 95% CI: 1.25, 3.43; P = 0.005). Individuals infected at an older age were also less likely to become HCV‐RNA negative spontaneously (RR = 0.42 comparing those infected at ≥20 years of age against those infected at <20 years of age, 95% CI: 0.25, 0.72; P = 0.002). In conclusion, the results suggest that HCV genotype 1 infections are more likely than genotype 3 infections to become spontaneously non‐viremic, as are infections acquired at younger age. J. Med. Virol. 83:1338–1344, 2011. © 2011 Wiley‐Liss, Inc.

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“…Diagnosis of Hepatitis A and C viruses (HAV, HCV) employs rRT-PCR assays for rapidity and accuracy. In a one-step rRT-PCR Taqman assay to quantify the 5' non-coding region of HAV [3] , for routine genotyping of HCV [29,30] and a multiplex quantitative rRT-PCR assay for simultaneous detection, identification and quantification of hepatitis B virus (HBV-DNA virus), HCV and human immunodeficiency virus type 1 (HIV-1) in plasma or serum samples using flourogenic probes showed increased sensitivity and accuracy [31,32] , which proves its potentiality to be used for large-scale nucleic acid testing of blood donations [30] .…”

Section: Applications Of Rrt-pcr As Diagnosticmentioning

confidence: 99%

Applications of Real-time Reverse Transcription Polymerase Chain Reaction in Clinical Virology Laboratories for the Diagnosis of Human Diseases

Manojkumar¹,

Mrudula²

2006

American J. of Infectious Diseases

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Early diagnosis that prevents further expansion of the causative agent and thereby aids control and eradication of infectious agents is critical for countries trying to obtain a particular disease free status. In these diagnostics speed is paramount, including the assay's applicability, sensitivity, specificity, cost effectiveness and patentability. Real-time Reverse Transcription PCR (rRT-PCR) has revolutionized the field of molecular biology and is being utilized increasingly in novel clinical diagnostic assays. This technique has been applied for rapid detection of point mutation, drug resistant variant and genotyping in several viruses. The combination of excellent sensitivity, specificity, low contamination risk, and speed has made this technique an appealing alternative to cell culture or immunoassay-based testing methods for diagnosing infectious diseases. rRT-PCR assays are most established for the detection of viral load and therapy monitoring. In this review, the usefulness or applications of rRT-PCR assays in the diagnosis of some of the important human viral infections have been summarized.

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A real-time Taqman method for hepatitis C virus genotyping

Cited by 22 publications

References 21 publications

A single-step multiplex quantitative real time polymerase chain reaction assay for hepatitis C virus genotypes

A single-step multiplex quantitative real time polymerase chain reaction assay for hepatitis C virus genotypes

Spontaneous loss of hepatitis C virus RNA from serum is associated with genotype 1 and younger age at exposure

Applications of Real-time Reverse Transcription Polymerase Chain Reaction in Clinical Virology Laboratories for the Diagnosis of Human Diseases

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