Development & evaluation of biotinylated DNA probe for clinical diagnosis of chikungunya infection in patients' acute phase serum & CSF samples.
OBJECTIVE
The resurgence of chikungunya virus (CHIKV) in the Indian Ocean Islands and India has drawn worldwide attention due to its explosive nature, high morbidity and complex clinico-pathological manifestations. The early confirmatory diagnosis of CHIKV is essential for management as well as control of unprecedented epidemics. The present study describes the development and evaluation of a highly sensitive and specific E1 structural gene specific biotinylated DNA probe for detection of chikungunya virus in clinical samples using a dot blot format.


METHODS
The complementary DNA (cDNA) of CHIKV was spotted on to nylon membrane. The membrane was subjected to prehybridization and hybridization and developed using a colour development solution containing DAB chromogen.


RESULTS
The CHIKV E1 specific DNA probe was highly sensitive detecting picogram levels of target nucleic acid. The comparative evaluation with SYBR Green I based real-time RT-PCR revealed 99 per cent accordance with a sensitivity and specificity of 99 and 98 per cent, respectively. The specificity of this assay was further confirmed through cross-reaction studies with confirmed dengue and Japanese encephalitis (JE) patient serum samples along with infected culture supernatant of Ross River and Saint Louis encephalitis and plasmid DNA of O'Nyong Nyong, Semlinki forest and Sindbis viruses.


CONCLUSIONS
The DNA probe reported in this study may be useful for specific, sensitive and confirmatory clinical diagnosis of chikungunya infection in acute phase human patient serum and CSF samples. This assay can also be used in the laboratory for quantification of viral antigen in cell culture supernatant for research purpose.
Division of Virology, Defence Research & Development Establishment (DRDO), Gwalior, India.
Jyoti S
JS
Kumar
Manmohan
M
Parida
P V
PV
Lakshmana Rao
D018354:Alphavirus Infections / Q000097:blood / Q000134:cerebrospinal fluid / Q000175:diagnosis*; D000818:Animals; D001710:Biotin / Q000737:chemistry*; D002460:Cell Line; D065632:Chikungunya Fever; D002522:Chlorocebus aethiops; D015342:DNA Probes; D004587:Electrophoresis, Agar Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D009693:Nucleic Acid Hybridization; D060888:Real-Time Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D014709:Vero Cells
D015342:DNA Probes; D001710:Biotin