STANDARDIZATION OF A ONE-STEP REAL-TIME RT-PCR FOR RAPID DIAGNOSIS OF YELLOW FEVER VIRUS INFECTION

AC34

Felipe Gonçalves Motta Maia and Luiz Tadeu Moraes Figueiredo.

Virology Research Center, School of Medicine of Ribeirão Preto, University of São Paulo, Brazil.

Yellow fever, the prototype viral haemorragic fever, is an acute infection characterized by hepatitis, renal failure, cardiovascular collapse and bleeding. The causative agent, a mosquito-borne RNA virus belonging to the family Flaviviridae, as dengue and Japanese encephalitis viruses. Yellow fever was a severe public health problem until the early 20th century, and today it remains endemic and epidemic in tropical regions of South America and Africa. In Brazil, from December of 2007 until July of 2008, 75 suspicious cases of jungle yellow fever were notified. Among 45 yellow fever confirmed cases, 25 evolved to death (55.6% of case fatality rate). A rapid and reliable detection method is crucial for the patient’s appropriate treatment and to monitor and to control the virus dissemination. We have standardized a Real-Time RT-PCR technique, using Yellow Fever Virus (YFV) specific designed primers. The YFV strain 17D (vaccine) was propagated in C6/36 Aedes albopictus cell culture. Viral RNA was extracted using QIAamp Viral RNA Mini Kit (QIAGEN, Inc.). Real-Time RT-PCR was performed by using SuperScript III Platinum SYBR Green One-Step qRT-PCR Kit (Invitrogen Corp.). The test took 2 hours to be performed. The temperature of melting (Tm), 85.7ºC, was determined increasing the temperature from 60ºC to 95ºC at 0,2 degrees per second. The method showed a high sensitivity.