Microarrays for Genotyping Human Group A Rotavirus by Multiplex Capture and Type-Specific Primer Extension

Abstract

Human group A rotavirus (HRV) is the major cause of severe gastroenteritis in infants worldwide. HRV shares the feature of a high degree of genetic diversity with many other RNA viruses, and therefore, genotyping of this organism is more complicated than genotyping of more stable DNA viruses. We describe a novel microarray-based method that allows high-throughput genotyping of RNA viruses with a high degree of polymorphism by multiplex capture and type-specific extension on microarrays. Denatured reverse transcription (RT)-PCR products derived from two outer capsid genes of clinical isolates of HRV were hybridized to immobilized capture oligonucleotides representing the most commonly occurring P and G genotypes on a microarray. Specific primer extension of the type-specific capture oligonucleotides was applied to incorporate the fluorescent nucleotide analogue cyanine 5-labeled dUTP as a detectable label. Laser scanning and fluorescence detection of the microarrays was followed by visual or computer-assisted interpretation of the fluorescence patterns generated on the microarrays. Initially, the method detected HRV in all 40 samples and correctly determined both the G and the P genotypes of 35 of the 40 strains analyzed. After modification by inclusion of additional capture oligonucleotides specific for the initially unassigned genotypes, all genotypes could be correctly defined. The results of genotyping with the microarray fully agreed with the results obtained by nucleotide sequence analysis and sequence-specific multiplex RT-PCR. Owing to its robustness, simplicity, and general utility, the microarray-based method may gain wide applicability for the genotyping of microorganisms, including highly variable RNA and DNA viruses.

Detection and Genotyping of Human Rotavirus VP4 and VP7 Genes by Reverse Transcriptase PCR and Reverse Hybridization

“…These include type-specific PCR (20), restriction fragment length polymorphism (24), sequence analysis (3,5,10), capture and primer extension (32), and hybridization to oligonucleotide probes (7,43,44). …”

A DNA oligonucleotide microarray for detecting human astrovirus serotypes

“…By using a series of short probes, however, conserved regions of the genomes can still be sufficiently variable for characterization of enteric viruses. For example, Chizhikov, et al (2002) and Lovmar, et al (2003) used short probes (about 20 nucleotides in length) to successfully distinguish different rotavirus isolates.…”

DNA microarrays and their applications in medical microbiology

“…A novel ArrayTube assay, which incorporates oligonucleotide DNA probes representing 24 of the most epidemiologically relevant O antigens and 47 H antigens, has been described for fast DNA serotyping of E. coli . 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.…”