A Novel Hepatitis C Virus Genotyping Method Based on Liquid Microarray

Duarte, Cesar A. B.; Foti, Leonardo; Nakatani, Sueli M.; Riediger, Irina Nastassja; Poersch, Celina O.; Pavoni, Daniela Parada; Krieger, Marco Aurélio

doi:10.1371/journal.pone.0012822

Cited by 10 publications

(8 citation statements)

References 26 publications

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“…Duarte et al 63 developed xMAP Luminex assay system, a liquid microarray-based HCV genotyping method. The complementary sequences against most variable region NS5B and highly conserved 5’UTR were used to determine HCV genotypes.…”

Section: Hcv Genotyping Methodsmentioning

confidence: 99%

Genotyping & diagnostic methods for hepatitis C virus: A need of low-resource countries

Kumar¹,

Rajput²,

Paliwal³

et al. 2018

Indian J Med Res

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Hepatitis C virus (HCV) infection is a blood borne and transfusion-transmitted infection (TTI). It has emerged as one of the major health challenges worldwide. In India, around 12-18 million peoples are infected with HCV, but in terms of prevalence percentage, its looks moderate due to large population. The burden of the HCV infection increases due to lack of foolproof screening of blood and blood products before transfusion. The qualified screening and quantification of HCV play an important role in diagnosis and treatment of HCV-related diseases. If identified early, HCV infection can be managed and treated by recently available antiviral therapies with fewer side effects. However, its identification at chronic phase makes its treatment very challenging and sometimes ineffective. The drugs therapy for HCV infection treatment is also dependent on its genotype. Different genotypes of HCV differ from each other at genomic level. The RNA viruses (such as HCV) are evolving perpetually due to interaction and integration among people from different regions and countries which lead to varying therapeutic response in HCV-infected patients in different geographical regions. Therefore, proper diagnosis for infecting virus and then exact determination of genotype become important for targeted treatment. This review summarizes the general information on HCV, and methods used for its diagnosis and genotyping.

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Section: Hcv Genotyping Methodsmentioning

confidence: 99%

Genotyping & diagnostic methods for hepatitis C virus: A need of low-resource countries

Kumar¹,

Rajput²,

Paliwal³

et al. 2018

Indian J Med Res

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“…The low cost of our assay will facilitate its widespread use, especially in developing countries. An HCV genotyping method based on a suspension bead array has been previously reported (Duarte et al ., ); however, this method has been demonstrated to identify only HCV genotypes 1, 2 and 3, and it does not satisfy global requirements, especially in the Middle East, South Africa and Southeast Asia. Moreover, our assay, which we developed on the Luminex platform, can be easily ported to other platforms, such as the VeraCode platform (Illumina, USA) (data not shown) or other commercially available platforms.…”

Section: Discussionmentioning

confidence: 99%

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

Yang

Wang

Cheng

et al. 2014

Microbial Biotechnology

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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.

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“…All the reactions were performed in a 25 mL reaction with 10 mL of RNA, 1× PCR amplification buffer with Taq DNA polymerase and dNTPs (IBMP, Curitiba, Brazil) and 0.1 U of reverse transcriptase (IBMP, Curitiba, Brazil). All the oligonucleotide sequences are shown in Table I and in reference (Duarte et al 2010). Fig.…”

Section: Methodsmentioning

confidence: 99%

Internal control for real-time polymerase chain reaction based on MS2 bacteriophage for RNA viruses diagnostics

Zambenedetti

Pavoni

Dallabona

et al. 2017

Mem. Inst. Oswaldo Cruz

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BACKGROUND Real-time reverse transcription polymerase chain reaction (RT-PCR) is routinely used to detect viral infections. In Brazil, it is mandatory the use of nucleic acid tests to detect hepatitis C virus (HCV), hepatitis B virus and human immunodeficiency virus in blood banks because of the immunological window. The use of an internal control (IC) is necessary to differentiate the true negative results from those consequent from a failure in some step of the nucleic acid test.OBJECTIVES The aim of this study was the construction of virus-modified particles, based on MS2 bacteriophage, to be used as IC for the diagnosis of RNA viruses.METHODS The MS2 genome was cloned into the pET47b(+) plasmid, generating pET47b(+)-MS2. MS2-like particles were produced through the synthesis of MS2 RNA genome by T7 RNA polymerase. These particles were used as non-competitive IC in assays for RNA virus diagnostics. In addition, a competitive control for HCV diagnosis was developed by cloning a mutated HCV sequence into the MS2 replicase gene of pET47b(+)-MS2, which produces a non-propagating MS2 particle. The utility of MS2-like particles as IC was evaluated in a one-step format multiplex real-time RT-PCR for HCV detection.FINDINGS We demonstrated that both competitive and non-competitive IC could be successfully used to monitor the HCV amplification performance, including the extraction, reverse transcription, amplification and detection steps, without compromising the detection of samples with low target concentrations. In conclusion, MS2-like particles generated by this strategy proved to be useful IC for RNA virus diagnosis, with advantage that they are produced by a low cost protocol. An attractive feature of this system is that it allows the construction of a multicontrol by the insertion of sequences from more than one pathogen, increasing its applicability for diagnosing different RNA viruses.

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A Novel Hepatitis C Virus Genotyping Method Based on Liquid Microarray

Cited by 10 publications

References 26 publications

Genotyping & diagnostic methods for hepatitis C virus: A need of low-resource countries

Genotyping & diagnostic methods for hepatitis C virus: A need of low-resource countries

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

Internal control for real-time polymerase chain reaction based on MS2 bacteriophage for RNA viruses diagnostics

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