Diagnosis of Barmah Forest virus infection by a nested real-time SYBR green RT-PCR assay.
Barmah Forest virus (BFV) is a mosquito borne (+) ssRNA alphavirus found only in Australia. It causes rash, myalgia and arthralgia in humans and is usually diagnosed serologically. We developed a real-time PCR assay to detect BFV in an effort to improve diagnosis early in the course of infection. The limit of detection was 16 genome equivalents with a specificity of 100%. Fifty five serum samples from BFV-infected patients were tested by the PCR. 52 of 53 antibody-positive samples were PCR negative. Two culture-positive (neutralizing antibody negative) samples were positive on first round PCR, while one sample (IgM and neutralizing antibody strongly positive, IgG negative) was positive on second round PCR, suggesting that viral RNA is detectable and transiently present in early infection. PCR can provide results faster than culture, is capable of high throughput and by sequencing the PCR product strain variants can be characterized.
Centre for Infectious Diseases and Microbiology Laboratory Services, Westmead, NSW, Australia. linda.hueston@swahs.health.nsw.gov.au
Linda
L
Hueston
Cheryl S
CS
Toi
Neisha
N
Jeoffreys
Tania
T
Sorrell
Gwendolyn
G
Gilbert
D000524:Alphavirus / Q000235:genetics* / Q000302:isolation & purification*; D018354:Alphavirus Infections / Q000175:diagnosis* / Q000821:virology*; D000818:Animals; D052160:Benzothiazoles; D002460:Cell Line; D003959:Diamines; D006801:Humans; D057230:Limit of Detection; D009930:Organic Chemicals; D016133:Polymerase Chain Reaction / Q000379:methods*; D011804:Quinolines; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
D052160:Benzothiazoles; D003959:Diamines; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I