[Detection of Sindbis virus-specific nucleic acid with SYBR GREEN I real time PCR assay].
BACKGROUND
To develop a rapid, specific and sensitive method for detecting Sindbis virus (SINV) with SYBR GREEN I real time PCR.


METHODS
Total RNA of strains of Sindbis virus and a related virus were extracted and reverse transcribed to cDNAs. With the cDNAs as template, the SYBR GREEN I real time PCR assay was developed and optimized on ABI 7300 apparatus for specific-detection of Sindbis virus, and the sensitivity, specificity and reproducibility were evaluated.


RESULTS
For the PCR, 55 degrees C was chosen as the optimal anneal temperature and 0.5 micromol/L as the optimal primer concentration. Using this method, all the selected SINV were detected as positive, while the results of control arboviruses such as Geta virus, Japanese encephalitis virus (JEV), Batai virus, Seadornavirus Orbiviruses and synthesized WEEV cDNA were negative. With this system, 0.1 PFU/ml SINV cDNA could be detected; The sensitivity of this assay was about 100 times higher than standard RT-PCR. All the results were reproducible within two compatible tests, and the stability of the detection system was very good. The test results of simulated infection human serum samples showed that human serum had no obvious interference with this system. With this system, 6 of 151 clinical samples with unknown fever or encephalitis were determined as positive.


CONCLUSIONS
The developed SYBR GREEN I real time PCR assay for detecting Sindbis virus was highly sensitive, specific and showed a good reproducibility and stability. It is our belief that the present method can be further used in clinic sample to verify its stringency.
He
Li-fang
LF
Yunnan Center for Disease Control and Prevention, Kunming 650022, China.
Xu
Li-hong
LH
Cao
Yu-xi
YX
Wang
Li-hua
LH
Liu
Wei-bin
WB
Fu
Shi-hong
SH
Liang
Guo-dong
GD
D018354:Alphavirus Infections / Q000175:diagnosis / Q000821:virology; D052160:Benzothiazoles; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D003959:Diamines; D009930:Organic Chemicals / Q000737:chemistry; D011804:Quinolines; D012367:RNA, Viral / Q000235:genetics* / Q000302:isolation & purification; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D012845:Sindbis Virus / Q000235:genetics*
D052160:Benzothiazoles; D018076:DNA, Complementary; D003959:Diamines; D009930:Organic Chemicals; D011804:Quinolines; D012367:RNA, Viral; C098022:SYBR Green I