[Detection of Banna virus-specific nucleic acid with TaqMan RT-PCR assay].
BACKGROUND
To develop a rapid and more sensitive TaqMan RT-PCR assay specific for Banna virus.


METHODS
Total RNA of strains of Banna virus and other arboviruses were extracted and reverse transcribed to cDNAs. With the cDNAs as templates, the TaqMan RT-PCR assay was developed and optimized on ABI 7300 apparatus for specific-detection of Banna virus. The sensitivity, specificity and the possibility of this assay to detect Banna virus in pools of mosquitoes. Meanwhile, human sera samples added with different dilutions of Banna virus culture medium supernatant were evaluated.


RESULTS
All the collected 12 Banna virus strains in our country were detected as positive, while the results of other arboviruses such as Japanese encephalitis virus (JEV), Batai virus, Sindbis virus and Orbiviruses were negative. This assay was more than 100 times sensitive than that of traditional PCR. About 0.5 CCID-50 of Banna virus in 50 microl samples could be detected with this assay. The sensitivity decreased by about 10 times when the dsRNA was denatured in 65 degrees C instead of 99 degrees C, and also when the virus seeds were kept at room temperature for 1 day or 1 week at 4 degrees C. There was no significant difference in the results of Banna virus detection between artificial human serum samples and positive control virus. Six were detected as positive for Banna virus-specific nucleic acids in 112 pools of mosquitoes, while 3 was positive with virus isolation.


CONCLUSIONS
The Banna virus-specific TaqMan RT-PCR assay was proved to be highly sensitive, specific and showed a good reproducibility; the assay may be applied for surveillance of this virus in clinical samples and pools of mosquitoes screening.
Xu
Li-hong
LH
Institute for Viral Disease Control and Prevention, China CDC, Beijing 100052, China. lhxu8804@yahoo.com.cn
Cao
Yu-xi
YX
He
Li-fang
LF
Wang
Huan-qin
HQ
He
Ying
Y
Fu
Shi-hong
SH
Sun
Xiao-hong
XH
Wang
Huan-yu
HY
Liu
Wei-bin
WB
Wang
Li-hua
LH
Liang
Guo-dong
GD
D000818:Animals; D018162:Coltivirus / Q000235:genetics* / Q000302:isolation & purification; D009033:Culicidae / Q000821:virology; D006801:Humans; D012367:RNA, Viral / Q000235:genetics* / Q000302:isolation & purification; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D019914:Taq Polymerase / Q000378:metabolism*
D012367:RNA, Viral; D019914:Taq Polymerase