Molecular diagnosis and analysis of Chikungunya virus.
BACKGROUND
In March 2005 a Chikungunya fever outbreak began on the islands of the Indian Ocean. The number of cases of this disease dramatically rose amongst these islands before affecting over a million people in India. Travellers to these regions have returned to the UK with the disease leading to a greater than 15-fold increase in the annual number of Chikungunya virus (CHIKV) sero-positive samples in 2006.


OBJECTIVE
A real-time RT-PCR test was developed for CHIKV and designed to detect currently circulating strains of virus as well as other genotypes. Its sensitivity was compared with an existing standard RT-PCR assay and a previously published real-time assay.


METHODS
A real-time RT-PCR assay was optimised and evaluated using a panel of 55 clinical serum samples and a synthetic RNA transcript as a positive control. Nucleotide sequencing of part of the E1 gene of CHIKV was used to investigate the relatedness of the samples.


RESULTS
The real-time RT-PCR was 10-fold more sensitive than a conventional block-based RT-PCR and could detect as low as 20 copies of RNA transcript. The assay also had 10-fold improved sensitivity in detecting the outbreak strain of virus when compared to another published TaqMan assay. Analysis of sequences from patients that had travelled to India, Mauritius or the Seychelles showed high similarity with published sequences from the Indian Ocean island of Réunion.


CONCLUSIONS
A sensitive and rapid real-time RT-PCR assay has been developed for CHIKV and tested against current isolates.
Edwards
Carolyn J
CJ
Novel and Dangerous Pathogens Department, Centre for Emergency Preparedness and Response, Health Protection Agency, Porton Down, Salisbury, Wiltshire SP4 0JG, United Kingdom. carolyn.edwards@hpa.org.uk
Welch
Stephen R
SR
Chamberlain
John
J
Hewson
Roger
R
Tolley
Howard
H
Cane
Patricia A
PA
Lloyd
Graham
G
D018354:Alphavirus Infections / Q000175:diagnosis* / Q000821:virology*; D002646:Chikungunya virus / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D019156:Indian Ocean; D012367:RNA, Viral / Q000032:analysis / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D019914:Taq Polymerase / Q000378:metabolism; D014195:Travel; D006113:United Kingdom
D012367:RNA, Viral; D019914:Taq Polymerase