RESUMO
The reverse transcription - polymerase chain reaction method (RT-PCR) has leading position on diagnostic infections, caused by RNA-containing viruses. This method presents severe requirements to carrying out of everybody stages of analysis (extraction of nucleic acid, carry out reverse transcription, amplification of DNA). It is necessary to account the possibility of false positive or false negative results appearance. The use on RT-PCR only positive (PCS) and negative (NCS) control samples is insufficient for the control of stages of RNA extraction and reverse transcription. That is way there is necessity the construction of inner control sample (ICS) to control of these stages. The main goal of present is the ground of use genetic engineering constructions (GEC) as control samples (PCS and ICS) on evaluation of diagnostic kits for reveal of RNA of hazard and extremely hazard agents of virus infections by RT-PCR. The vector recombinant plasmids, containing the insertion of cDNA of agent´s genomic RNA are used as PCS, RNA was packed in membrane protein of MS2 bacteriophage, is used as ICS. It is demonstrated that ICS does no influence on sensitivity of RT-PCR both for use of native agents and for use of synthetic nucleic acids of Ebola, Marburg, Lassa, Machupo, Venezuelan encephalitis equine (VEE), Rift Valley fever and rabies viruses. The possibility of use of PCS and ICS for standardization of diagnostic kits is discussed.
Assuntos
RNA Viral/análise , Kit de Reagentes para Diagnóstico/normas , Padrões de Referência , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Animais , Engenharia Genética , Sensibilidade e EspecificidadeRESUMO
AIM: Detection-and identification of Venezuelan equine encephalomyelitis (VEE) virus RNA in biological samples by reverse-transcription polymerase chain reaction (RT-PCR) and RT-PCR in real time (rRT-PCR). MATERIALS AND METHODS: VEE, Sindbis, West Nile, Japanese and tick-borne encephalitis viruses were studied. Cell culture of chicken fibroblasts, outbred mice and rats, Javanese macaques were used in the experiments. Biological activity determination of the running culture of causative agents used in the experiments was carried out by negative colony method in monolayer cell culture under agar coating. and using intra-cerebral infection of mice. Reagent kits developed in the 48th Central Research Institute and Institute of Analytical Instrument Engineering were used during execution of experiments of VEE virus RNA detection by RT-PCR and rRT-PCR. RESULTS: VEE virus was detected in biological samples by various methods. Data from RT-PCR and rRT-PCR are in accordance with the results of virus detection in samples using sensitive animals. CONCLUSION: Use of molecular-diagnostics methods for detection in biological samples of a causative agent of a dangerous infectious disease is important for procuring biological safety of Russian Federation.
Assuntos
Vírus da Encefalite Japonesa (Espécie)/genética , Vírus da Encefalite Equina Venezuelana/genética , Vírus da Encefalite Transmitidos por Carrapatos/genética , RNA Viral/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Sindbis virus/genética , Vírus do Nilo Ocidental/genética , Infecções por Alphavirus/diagnóstico , Infecções por Alphavirus/virologia , Animais , Animais não Endogâmicos , Galinhas , Vírus da Encefalite Japonesa (Espécie)/isolamento & purificação , Vírus da Encefalite Equina Venezuelana/isolamento & purificação , Vírus da Encefalite Transmitidos por Carrapatos/isolamento & purificação , Encefalite Japonesa/diagnóstico , Encefalite Japonesa/virologia , Encefalite Transmitida por Carrapatos/diagnóstico , Encefalite Transmitida por Carrapatos/virologia , Encefalomielite Equina Venezuelana/diagnóstico , Encefalomielite Equina Venezuelana/virologia , Fibroblastos/patologia , Fibroblastos/virologia , Humanos , Macaca mulatta , Camundongos , RNA Viral/isolamento & purificação , Ratos , Kit de Reagentes para Diagnóstico/normas , Sindbis virus/isolamento & purificação , Febre do Nilo Ocidental/diagnóstico , Febre do Nilo Ocidental/virologia , Vírus do Nilo Ocidental/isolamento & purificaçãoRESUMO
Epidemiologic analysis of epidemic outbreaks caused by American equine encephalitis causative agents is carried out in the review. Eastern equine encephalomyelitis (EEE), Western equine encephalomyelitis (WEE) and Venezuela equine encephalomyelitis (VEE) viruses are etiologic agents of dangerous transmissive diseases that are usually accompanied by fever and neurologic symptoms. Among the New World alphaviruses, VEE virus has the most potential danger for humans and domestic animals. Currently, enzootic strains of VEE play an increasing role as etiologic agents of human diseases. Most of the VEE cases in humans in endemic regions during inter-epidemic period are caused by infection with VEE subtype ID virus. A possibility of emergence of novel epidemic outbreaks of VEE is determined by mutations of ID subtype strains into IC subtype, and those currently pose a potential threat as an etiologic agent of the disease. Despite low morbidity, EEE and WEE are a problem for healthcare due to a relatively high frequency of lethal outcomes of the disease.