Comparison of Reverse Hybridization, Microarray, and Sequence Analysis for Genotyping Hepatitis B Virus

Pas, Suzan D.; Tran, Nathalie; Man, Robert A. de; Burghoorn-Maas, Chantal; Vernet, Guy; Niesters, Hubert G. M.

doi:10.1128/jcm.01519-07

Cited by 42 publications

(25 citation statements)

References 27 publications

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“…It is an improved version of an assay that was described previously (24,34) and was designed to improve nucleotide identification and to detect an extended list of mutants and the recently identified H genotype.…”

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Microarray for Hepatitis B Virus Genotyping and Detection of 994 Mutations along the Genome

et al. 2010

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Genome analysis of hepatitis B virus (HBV) in patient sera is helpful for monitoring treatment. We developed an improved version of a DNA microarray to identify HBV genotypes and to detect mutations of interest in the S, Pol, Core, and X genes. It includes an automated software analysis of fluorescence values for simpler, more robust data interpretation. In this version, probes were added to identify genotype H, to analyze 155 additional positions, and to detect 561 additional polymorphisms. Sequences were added to the alignments to resolve hybridization problems due to natural polymorphisms in the vicinity of important codons. The duplex PCR protocol allowed whole-genome analysis in a single tube. An alternative nested-PCR protocol allowed genotyping and mutations in S and reverse transcriptase (rt) genes in patients with low viral loads, as demonstrated in patients with less than 400 HBV genome copies/ml. Reproducibility was high, with variation coefficients lower than 3%. Only 0.57% of 20,771 codons from 253 samples could not be identified. The concordance with Sanger sequencing for the identification of codons improved from 92.8% to 95.7% with the improved version. Concordance was higher than 91% for codons associated with resistance to lamivudine, emtricitabine, telbivudine, famciclovir, entecavir, and tenofovir with vaccine escape and for pre-Core mutants. Concordance was lower for adefovir resistance mutations (68.6%) and mutations in the basal core promoter (60.3%), probably because hybridization efficiency was affected by the low GC content of the probes. A concordance of 93.7% with sequencing for genotype identification was observed in 190 specimens, lower than that obtained with the first version, possibly due to mixed virus populations.

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Microarray for Hepatitis B Virus Genotyping and Detection of 994 Mutations along the Genome

et al. 2010

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“…These findings are consistent with those of other studies that have reported that the INNO-LiPA DR assay is more sensitive than sequencing for the detection of nucleotide variations, allowing the detection of mixtures of wild-type isolates and ETV-resistant mutants (3,7). Along these lines, a recent study by Pas et al demonstrated the practicability and usefulness of HBV genotyping by the INNOLiPA DR assay and reported that they achieved a higher sensitivity and a higher specificity with this technique than with sequencing and DNA chip technologies (8). However, one disadvantage of the INNO-LiPA DR assay is the limited scope of mutations represented in the assay, based on current knowledge of ETV resistance-associated mutations.…”

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Use of the Novel INNO-LiPA Line Probe Assay for Detection of Hepatitis B Virus Variants That Confer Resistance to Entecavir Therapy

et al. 2009

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A line probe assay (INNO-LiPA DR, version 3) for the detection of hepatitis B virus mutations that confer resistance to entecavir therapy was evaluated. The INNO-LiPA DR assay is a highly sensitive assay that is easily applicable for the detection and monitoring of entecavir resistance-conferring mutations and is more sensitive than sequencing for the detection of mixed sequences.Hepatitis B virus (HBV) infection is a common cause of chronic liver disease, and the risk of disease progression is associated with elevated HBV DNA levels. With the introduction of new HBV polymerase inhibitors, such as entecavir (ETV) and tenefovir (TDF), major advances have been made in the antiviral management of chronic hepatitis B (CHB) (13). However, the widespread use of these agents had led to the increasing emergence of drug-resistant mutations in HBV.ETV is a potent deoxyguanosine analog, although it is less effective against lamivudine (LVD)-resistant HBV variants than against wild-type HBV (1). ETV resistance requires substitutions at residues T184, S202, and M250 in reverse transcriptase (rtT184, rtS202, and rtM250, respectively) in HBV isolates containing preexisting, LVD resistance-conferring substitutions (10-12).This study assesses a prototype line probe assay (LiPA), the INNO-LiPA DR (version 3) assay (LiPA Innogenetics, Ghent, Belgium), for the detection and characterization of the HBV polymerase mutations associated with ETV resistance. A practical feature of this assay is that the single-stage PCR product can be used for combined hybridization of strips from the INNOLiPA DR assay (version 3) and INNO-LiPA DR assay (version 2), the latter of which is used to detect the main variants resistant to LVD and adefovir-dipivoxil (ADV) (7), because both assays use the same set of primers. The INNO-LiPA DR assay was performed according to the manufacturer's instructions. Briefly, HBV DNA extracted from 200 l of serum with a QIAamp DNA blood kit (Qiagen, Venlo, The Netherlands) was amplified by a single round of PCR with the kit-supplied primer mix: sense primer (nucleotides 255 to 278; 5Ј-CGTGGTGGACTTCTCTC AATTTTC-3Ј) and antisense primer (nucleotides 1122 to 1099; 5Ј-AGAAAGGCCTTGTAAGTTGGCGA-3Ј), both of which included domains A to F of the HBV polymerase. The amplified product was used for reverse hybridization with specific oligonucleotide probes immobilized on nitrocellulose strips. After hybridization, the strips were incubated with streptavidin conjugate. Colored lines were interpreted for the presence of mutations in the HBV polymerase. The probes fixed in the INNO-LiPA DR assay strips cover known HBV polymerase variations associated with ETV resistance (codons rt184, rt202, and rt250). The strips contain 2 control lines and 22 HBV probe lines representing wild-type or mutant variants: for codon rt184, 1 line reactive to the normal variant (amino acid T), 3 lines reactive (indistinctly) to mutated amino acid S, C, G, or A (marked as SCGA lines), and 2 lines to mutated amino acid I, L, F, or M (marked as ILFM lines); f...

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“…Microarrays have also been used to characterize and type other gastroenteritis-causing viral pathogens including rotavirus, norovirus, and astrovirus (Chizhikov et al, 2002;Honma et al, 2007;Jaaskelainen and Maunula, 2006;Lovmar et al, 2003). Beyond diarrheal illnesses, Pas et al (2008) reported the comparison of reverse hybridization, microarray, and sequence analysis for hepatitis B virus (HBV) genotyping, suggesting that the InnoLipa HBV genotyping strip assay, a microarray-based system, detected dual infections and was an easy and quick tool for HBV genotyping.…”

Section: Comparative Genomics and Microbial Typingmentioning

confidence: 99%

DNA microarrays and their applications in medical microbiology

Nsofor¹

2014

Biotechnol. Mol. Biol. Rev.

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Rapid diagnosis and treatment of disease is often based on the identification and characterization of causative agents derived from phenotypic characteristics. This can be laborious and time consuming, often requiring many skilled personnel and a large amount of lab space. However, the introduction of nucleic acid amplification techniques into molecular biology has transformed the laboratory detection of pathogens. The progression of the molecular diagnostic revolution currently relies on the ability to efficiently and accurately offer multiplex detection and characterization for a variety of infectious disease pathogens. DNA microarray analysis has the capability to offer robust multiplex detection. Multiple microarray platforms exist, including printed double-stranded DNA and oligonucleotide arrays, in situ-synthesized arrays, high-density bead arrays, electronic microarrays, and suspension bead arrays. The aim of this paper was to review DNA microarray technology, highlighting two major types: the oligonucleotide-based array and the PCR product-based array. Although, the use of microarrays to generate gene expression data has become routine, applications pertinent to microbiology continue to rapidly expand. This review highlights uses of microarray technology that impact diagnostic microbiology, including the detection and identification of pathogens, determination of antimicrobial resistance, epidemiological strain typing, and determination of virulence factors.

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Comparison of Reverse Hybridization, Microarray, and Sequence Analysis for Genotyping Hepatitis B Virus

Cited by 42 publications

References 27 publications

Microarray for Hepatitis B Virus Genotyping and Detection of 994 Mutations along the Genome

Microarray for Hepatitis B Virus Genotyping and Detection of 994 Mutations along the Genome

Use of the Novel INNO-LiPA Line Probe Assay for Detection of Hepatitis B Virus Variants That Confer Resistance to Entecavir Therapy

DNA microarrays and their applications in medical microbiology

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