Development of a high resolution melting analysis for detection and differentiation of human astroviruses

• Published in: Journal of virological methods 2014-05, Vol.200, p.29-34
• Main Authors: Hata, Akihiko, Kitajima, Masaaki, Tajiri-Utagawa, Etsuko, Katayama, Hiroyuki
• Format: Article
• Language: English
• Subjects: Child, Preschool; DNA Primers - genetics; Genotyping Techniques - methods; High-Throughput Screening Assays; HRM; Human astroviruses; Humans; Infant; Mamastrovirus - classification; Mamastrovirus - genetics; Mamastrovirus - isolation & purification; Phylogenetic analysis; RNA, Viral - genetics; RT-PCR; Sensitivity and Specificity; Time Factors; Transition Temperature; Viral Load - methods
• ISSN: 0166-0934; 1879-0984
• DOI: 10.1016/j.jviromet.2014.01.023

•An HRM assay to detect and differentiate human astrovirus sequences was developed.•Primer-pairs were designed based on a nucleotide sequence alignment of astroviruses in a database.•RT-PCR using the primer-pairs successfully amplified all known genogroups of astrovirus genes.•Astrovirus sequences in fecal specimens were successfully differentiated by the HRM assay. Human astroviruses (AstVs), the common causes of viral gastroenteritis, consist of 8 different sero- or genotypes in which a variety of subtypes have been found. In the present study, a rapid and high-throughput method for detection and sequence-discrimination of AstVs by high resolution melting (HRM) analysis was developed. A newly designed primer set for the assay targeting ORF1b–ORF2 junction region of AstVs successfully reacted with all 8 serotypes of AstVs and allowed genotyping using their amplicons. The HRM assay consists of intercalating dye based real time quantitative PCR (qPCR) and melting curve analysis. The qPCR assay was sensitive enough to detect 1.0×101copies/reaction of AstV serotypes. However, 1.0×103copies/reaction of AstVs gene was required to obtain a sequence-specific difference curve, indicating that pre-amplification is necessary to apply the assay to samples containing low numbers of AstVs. AstVs in clinical specimens were subjected to the HRM assay after pre-amplification. The strains possessing same nucleotide sequences at the target region showed an identical difference curve and those possessing different nucleotide sequences showed a distinguishable difference curve. The newly developed HRM assay is an effective technique for screening of AstVs to quantify and discriminate the strains.