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Real-time polymerase chain reaction for diagnosis and quantitation of negative strand of chikungunya virus

2013; Elsevier BV; Volume: 77; Issue: 2 Linguagem: Inglês

10.1016/j.diagmicrobio.2013.06.018

1879-0070

Chun Wei Chiam, Yoke Fun Chan, Shih Keng Loong, Sara Su Jin Yong, Poh Sim Hooi, I‐Ching Sam,

Viral Infections and Outbreaks Research

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 R2 and efficiency and detected more positive samples. Peak viral load of 12.9 log10 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.