Improved molecular typing assay for rhinovirus species A, B, and C.
Human rhinoviruses (RVs), comprising three species (A, B, and C) of the genus Enterovirus, are responsible for the majority of upper respiratory tract infections and are associated with severe lower respiratory tract illnesses such as pneumonia and asthma exacerbations. High genetic diversity and continuous identification of new types necessitate regular updating of the diagnostic assays for the accurate and comprehensive detection of circulating RVs. Methods for molecular typing based on phylogenetic comparisons of a variable fragment in the 5' untranslated region were improved to increase assay sensitivity and to eliminate nonspecific amplification of human sequences, which are observed occasionally in clinical samples. A modified set of primers based on new sequence information and improved buffers and enzymes for seminested PCR assays provided higher specificity and sensitivity for virus detection. In addition, new diagnostic primers were designed for unequivocal species and type assignments for RV-C isolates, based on phylogenetic analysis of partial VP4/VP2 coding sequences. The improved assay was evaluated by typing RVs in >3,800 clinical samples. RVs were successfully detected and typed in 99% of the samples that were RV positive in multiplex diagnostic assays.
Department of Pediatrics, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin, USA yabochkov@wisc.edu.
Yury A
YA
Bochkov
Department of Pediatrics, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin, USA.
Kristine
K
Grindle
Department of Pediatrics, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin, USA.
Fue
F
Vang
Department of Bioinformatics, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin, USA.
Michael D
MD
Evans
Department of Pediatrics, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin, USA Department of Medicine, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin, USA.
James E
JE
Gern
D020121:5' Untranslated Regions; D036022:Capsid Proteins / Q000235:genetics; D002648:Child; D002675:Child, Preschool; D017931:DNA Primers / Q000235:genetics; D005260:Female; D005838:Genotype; D060005:Genotyping Techniques / Q000379:methods*; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012367:RNA, Viral / Q000235:genetics; D012229:Rhinovirus / Q000145:classification* / Q000235:genetics*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA
D020121:5' Untranslated Regions; D036022:Capsid Proteins; D017931:DNA Primers; D012367:RNA, Viral; C442544:VP2 protein, Rhinovirus