title: Real-time polymerase chain reaction for diagnosis and quantitation of negative strand of chikungunya virus.
abstract: Quantitative real-time polymerase chain reaction (qRT-PCR) is useful for diagnosis and studying virus replication. We developed positive- and negative-strand qRT-PCR assays to detect nsP3 of chikungunya virus (CHIKV), a positive-strand RNA alphavirus that causes epidemic fever, rash, and arthritis. The positive- and negative-strand qRT-PCR assays had limits of quantification of 1 and 3 log10 RNA copies/reaction, respectively. Compared to a published E1 diagnostic assay using 30 laboratory-confirmed clinical samples, the positive-strand nsP3 qRT-PCR assay had higher R(2) and efficiency and detected more positive samples. Peak viral load of 12.9 log(10) RNA copies/mL was reached on day 2 of illness, and RNA was detectable up to day 9, even in the presence of anti-CHIKV IgM. There was no correlation between viral load and persistent arthralgia. The positive-strand nsP3 assay is suitable for diagnosis, while the negative-strand nsP3 assay, which uses tagged primers to increase specificity, is useful for study of active viral replication kinetics.
authors.affiliation: Department of Medical Microbiology, Faculty of Medicine, University of Malaya, 50603, Kuala Lumpur, Malaysia.
authors.forename: Chun Wei
authors.initials: CW
authors.lastname: Chiam
authors.forename: Yoke Fun
authors.initials: YF
authors.lastname: Chan
authors.forename: Shih Keng
authors.initials: SK
authors.lastname: Loong
authors.forename: Sara Su Jin
authors.initials: SS
authors.lastname: Yong
authors.forename: Poh Sim
authors.initials: PS
authors.lastname: Hooi
authors.forename: I-Ching
authors.initials: IC
authors.lastname: Sam
mesh_terms: D018354:Alphavirus Infections / Q000175:diagnosis* / Q000821:virology; D000818:Animals; D018771:Arthralgia / Q000821:virology; D002646:Chikungunya virus / Q000235:genetics / Q000302:isolation & purification*; D002522:Chlorocebus aethiops; D006801:Humans; D012367:RNA, Viral / Q000097:blood; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D018709:Statistics, Nonparametric; D014709:Vero Cells; D019562:Viral Load; D014773:Virology / Q000379:methods
chemical_list: D012367:RNA, Viral
keywords: Arthralgia; Chikungunya virus; Molecular diagnosis; Real-time PCR; Viral load