Padronização de PCR quantitativo e desenvolvimento de metodologia de detecção de norovírus em alimentos. / Quantitative standardization of PCR and development of methodology of detention of norovírus in foods.
AUTOR(ES)
Tulio Machado Fumian
DATA DE PUBLICAÇÃO
2008
RESUMO
Food-borne gastroenteritis is an important problem of public health worldwide. The investigation for virus presence in food becomes a requirement, mainly due to the increasing emergence of outbreaks of gastroenteritis related to the consumption of different foods, in particular fruits, vegetables, milk, oysters and contaminated water. Currently, the Noroviruses (NV) are recognized as the most important etiological agent of outbreaks of food-borne gastroenteritis in developed countries, although little is known about the role of these viruses in developing countries. This study aimed to establish a quantitative amplification method (qPCR) to detect NV genogroups (G) I and II, and to develop a methodology to recover and detect virus in food samples such as cheese minas type and lettuce. The qPCR standardized that target the junction of the open reading framing (ORF) -1 and 2 of NV GI and GII, presented a limit of detection of five viral genome copies by reaction to both tests. The method of recovery and detection was based on the viral elution followed by clarification and a concentration step by using negatively charged membrane with elution in inorganic solvents. On the surface of both samples of food were seeded 50μL of fecal suspension containing approximately 106/μL particles of NV GII or serial dilutions in the base 10 of this suspension. The NV recovery efficiency ranged from 5.2 to 72.3% for lettuce samples and 6.0 to 56.3% for the cheese minas type sample in the different dilutions. The results showed that the method developed was efficient for recovery and detection of NV, and may be applied to viral research direct on food matrices. The standardization of molecular methods for detecting NV in food has been recommended as laboratory basis for epidemiological surveillance of these infections, representing a breakthrough in understanding the route of gastroenteritis infection by NV and for possible interventions in order to reduce or eliminate the health risks.
ASSUNTO(S)
norovirus reação em cadeia da polimerase via transcriptase reversa gastroenterite contaminação de alimentos vigilância epidemiológica gastroenteritis norovirus food contamination epidemiologic surveillance virologia reverse transcriptase polymerase chain reaction
