A reliable multiplex genotyping assay for HCV using a suspension bead array

Abstract

The genotyping of the hepatitis C virus (HCV) plays an important role in the treatment of HCV because genotype determination has recently been incorporated into the treatment guidelines for HCV infections. Most current genotyping methods are unable to detect mixed genotypes from two or more HCV infections. We therefore developed a multiplex genotyping assay to determine HCV genotypes using a bead array. Synthetic plasmids, genotype panels and standards were used to verify the target-specific primer (TSP) design in the assay, and the results indicated that discrimination efforts using 10 TSPs in a single reaction were extremely successful. Thirty-five specimens were then tested to evaluate the assay performance, and the results were highly consistent with those of direct sequencing, supporting the reliability of the assay. Moreover, the results from samples with mixed HCV genotypes revealed that the method is capable of detecting two different genotypes within a sample. Furthermore, the specificity evaluation results suggested that the assay could correctly identify HCV in HCV/human immunodeficiency virus (HIV) co-infected patients. This genotyping platform enables the simultaneous detection and identification of more than one genotype in a same sample and is able to test 96 samples simultaneously. It could therefore provide a rapid, efficient and reliable method of determining HCV genotypes in the future.

The Distribution of Hepatitis C Virus Genotypes of Patients with Chronic Hepatitis C Infection in the Eskisehir Region of Turkey

“…Interferon-based HCV treatment efficiency is influenced by factors including HCV genotype, initial viral load, and virological response during treatment [8,9]. Several methods have been developed for HCV genotyping such as direct sequencing, restriction fragment length polymor- phism, DNA hybridization, and multiplex real-time reverse transcription polymerase chain reaction (M-Real-Time RT-PCR) [5,6]. The hepatitis Cvirus has high mutation potentialand it displays phenotypically variability in the short term.…”

“…Genotype 1b is also associated with poor prognosis and is more resistant than the other genotypes. Especially in patients with genotypes 1 and 4, treatment protocolsshouldbe reevaluated given that sustained viral response in these patients might be weak [5]. Currently the Ministry of Health in Turkey requires that HCV genotypes be systematically determined beforestarting an HCV treatment regimen, since the determination of HCV genotypes is an important treatment factor.…”

“…This virus is classified into six major genotypes and more than 100 subtypesas shown by sequence analysis; recently a seventh genotype has been identified [3,4].It is known that genotypes are important predictors for epidemiology, clinical courses,and responses to antiviral therapy. Determination of HCV genotype is essential; it is incorporated into the treatment guidelines for HCV infections, including the 2009 American Association for the Study of Liver Diseases guidelines and the 2011 European Association for the Study of the Liver clinical practice guidelines [5]. The present study investigated the distribution pattern of HCV genotypes in patients with chronic hepatitis C infection in the Eskisehir, Turkey region.…”

Nanotechnology: A reality for diagnosis of HCV infectious disease

“…Thus, multiplex assays are required in viral detection in these groups. Yang et al (2014) proposed to develop a multiplex assay by coupling new specific probes against HIV or HBV to their bead array test [22] . Conventional molecular methods can detect HBV, HCV, HIV-1, and HIV-2 in a single test, such as qPCR COBAS TaqScreen MPX test v2.0 (Roche Molecular Systems Inc., Pleasanton CA, US) .…”

Quantum-dot-based suspension microarray for multiplex detection of lung cancer markers: preclinical validation and comparison with the Luminex xMAP® system

“…Suspension (or bead-based) microarrays employing fluorophore-encoded polymeric microspheres are currently of particular interest for multiplexed analysis of biological samples in clinical applications. A wide range of different bead-based microarrays have been developed and successfully applied to detection of specific cancer markers 12 13 and nucleic acids 14 , as well as for multiplexed genotyping 15 16 ,analysis of single nucleotide polymorphisms (SNPs) 17 , allergy testing 18 , identification of pathogens 19 , and the detection of biological warfare agents 20 .The detection principle is based on optical encoding and quantitative analysis of each analyte with an antigen-specific fluorophore-encoded microbead population carrying its unique optical code. Fluorophore-encoded microspheres can be rapidly analyzed by means of classical flow cytometry 21 22 .…”