title: Performance of a Taqman Assay for Improved Detection and Quantification of Human Rhinovirus Viral Load.
abstract: Human rhinovirus (HRV) is the major aetiology of respiratory tract infections. HRV viral load assays are available but limitations that affect accurate quantification exist. We developed a one-step Taqman assay using oligonucleotides designed based on a comprehensive list of global HRV sequences. The new oligonucleotides targeting the 5'-UTR region showed high PCR efficiency (E = 99.6%, R2= 0.996), with quantifiable viral load as low as 2 viral copies/μl. Assay evaluation using an External Quality Assessment (EQA) panel yielded a detection rate of 90%. When tested on 315 human enterovirus-positive specimens comprising at least 84 genetically distinct HRV types/serotypes (determined by the VP4/VP2 gene phylogenetic analysis), the assay detected all HRV species and types, as well as other non-polio enteroviruses. A commercial quantification kit, which failed to detect any of the EQA specimens, produced a detection rate of 13.3% (42/315) among the clinical specimens. Using the improved assay, we showed that HRV sheds in the upper respiratory tract for more than a week following acute infection. We also showed that HRV-C had a significantly higher viral load at 2-7 days after the onset of symptoms (p = 0.001). The availability of such assay is important to facilitate disease management, antiviral development, and infection control.
authors.affiliation: Department of Medicine, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia.
authors.forename: Kim Tien
authors.initials: KT
authors.lastname: Ng
authors.affiliation: Department of Medicine, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia.
authors.forename: Jack Bee
authors.initials: JB
authors.lastname: Chook
authors.affiliation: Department of Medicine, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia.
authors.forename: Xiang Yong
authors.initials: XY
authors.lastname: Oong
authors.affiliation: Department of Medical Microbiology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia.
authors.forename: Yoke Fun
authors.initials: YF
authors.lastname: Chan
authors.affiliation: Division of Genetics and Molecular Biology, Institute of Biological Sciences, Faculty of Science, University of Malaya, Kuala Lumpur, Malaysia.
authors.forename: Kok Gan
authors.initials: KG
authors.lastname: Chan
authors.affiliation: Department of Primary Care Medicine, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia.
authors.forename: Nik Sherina
authors.initials: NS
authors.lastname: Hanafi
authors.affiliation: Department of Medicine, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia.
authors.forename: Yong Kek
authors.initials: YK
authors.lastname: Pang
authors.affiliation: Department of Medicine, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia.
authors.forename: Adeeba
authors.initials: A
authors.lastname: Kamarulzaman
authors.affiliation: Department of Medical Microbiology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia.
authors.forename: Kok Keng
authors.initials: KK
authors.lastname: Tee
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D006801:Humans; D008875:Middle Aged; D010802:Phylogeny; D010850:Picornaviridae Infections / Q000821:virology*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012141:Respiratory Tract Infections / Q000821:virology; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D012229:Rhinovirus / Q000235:genetics* / Q000472:pathogenicity; D019562:Viral Load