Application of real time RT-PCR method for rapid detection of yellow fever.

[Objective] To establish real time RT-PCR method for yellow fever virus and apply it to rapidly detect yellow fever at frontier ports. [Methods] 5-UTR sequence was transcribed in vitro as positive control template. The optimized reaction system was obtained by adjusting different reaction condition and primers / probe final concentration. The established real time RT-PCR method was used to detect yellow fever vaccine, mosquitoes and serum specimen of entry FUO persons. [Results] The optimized reaction system invloved 750 nM 5UTR-FP, 750 nM 5UTR-RP and 500 nM 5UTR-Probe. Real time RT-PCR conditions were reverse transcription at 45℃ for 10min and denaturation at 95℃ for 10min followed by 45 cycles of 95℃ 5 s, 56℃ 10 s and 72℃ 15 s. Fluorescence was obtained at 56℃ anneal-stage. This method for yellow fever virus had no cross-reaction with dengue virus, west nile virus, Japanese encephalitis virus and chikungunya virus. The detection limit was twenty copies virus per reaction. Real time RT-PCR results with yellow fever vaccine, mosquitoes and serum specimen of entry FUO persons showed that yellow fever vaccine was proved to be positive and other clinical specimen were negative. [Conclusion] The established real time RT-PCR method has high sensitivity and strong specificity, which is suitable for rapid detection of yellow fever virus, and could prevent this infectious disease from spreading into China.