PCR em tempo real para detecção do vírus da doença de Aujeszky / Real time PCR for detection of Aujeszky's disease virus

O objetivo deste trabalho foi desenvolver uma PCR em tempo real (qPCR) para o diagnóstico rápido e sensível da doença de Aujeszky. Os iniciadores amplificaram um fragmento de 123 pares de base do gene codificante da glicoproteína D. A qPCR foi testada em 25 amostras de cérebro de suíno positivas e 85 amostras negativas para DA no isolamento viral e na soroneutralização. A sensibilidade analítica foi calculada com acréscimo de um isolado brasileiro do SuHV-1 titulado em amostras de cérebro de suíno negativas na soroneutralização e na PCR. A técnica apresentou sensibilidade analítica de 10-0,5 TCID50/50µL. A qPCR foi capaz de distinguir reações inespecíficas devido a dímero de oligonucleotídeos iniciadores ou amplificações, além do alvo designado (evitando, assim, os falso-positivos), e de obter resultados rápidos.

The aim of this study was to validate a low-cost real-time PCR for a quick and sensitive diagnosis of the disease. The fluorofore used was a DNA intercalating agent, one of the cheaper detection systems. Primers amplified a 123 base pairs fragment of the gene coding for glycoprotein D. PCR was tested on 25 samples of pig brain positive for AD and 85 samples negative in viral isolation and serum neutralization. The detection limit was calculated on samples of pig brain contaminated with a Brazilian isolate of SuHV-1. The technique had a detection limit of 10-0,5 TCID50/50µL. PCR was able to distinguish nonspecific reactions due to primer dimers (thus avoiding false positives) and get faster results.

Detecção dos genes de Staphylococcus aureus, enterotoxinas e de resistência à meticilina em leite / Detection of genes of Staphylococcus aureus, enterotoxins and methicillin resistance in milk

Realizou-se a detecção do gene de Staphylococcus aureus, de enterotoxinas e de resistência à meticilina com extração de DNA feita diretamente de amostras de leite. Das 200 amostras estudadas, 145 (72,5%) amplificaram o gene femA, e estas foram analisadas quanto à presença dos genes sea, seb, sec e mecA. Os genes das enterotoxinas mais prevalentes foram: sea (60%), seb (37,9%) e sec (6,9%). Foram encontradas 18 amostras de leite (11,0 %) com S. aureus portadores do gene mecA. A detecção de S. aureus diretamente do leite, sem a necessidade de isolamento bacteriano e a caracterização do potencial enterotoxigênico, demonstra que a técnica de PCR é muito útil para estudos epidemiológicos das infecções estafilocócicas da glândula mamária. O alto percentual (72,5%) de amostras de leite positivas para a presença do gene femA sugere que S. aureus constitui um dos principais agentes causadores de infecções intramamárias na microrregião de Sete Lagoas-MG e que seu potencial enterotoxigênico e presença do gene mecA, que identifica o S. aureus resistente à meticlina, representa um risco potencial à saúde pública.

This work was performed to detect the Staphylococcus aureus gene, enterotoxins resistance to methicillin with the extraction of DNA directly from milk samples. Of the 200 samples studied 145 (72.5%) amplified the femA gene, which were analyzed regarding the presence of sea, seb, sec and mecA genes. The most prevalent enterotoxins genes were: sea (60%), seb (37.9%) and sec (6.9%). 18 milk samples (11 %) had S. aureus carrying the mecA gene. The detection of S. aureus directly from the milk, with no need for bacterial isolation and the characterization of the enterotoxigenic potential demonstrate that the PCR technique is very useful for epidemiological studies of staphylococcal infections of the mammary gland. The high percentage (72.5%) of positive milk samples for the presence of the femA gene suggests that S. aureus constitutes of the main agents which cause intramammary infections in the micro region of Sete Lagoas-MG and that its enterotoxigenic potential and the presence of the mecA gene, which identifies the S. aureus resistant to methicillin, represent a potential risk to public health.

PCR duplex para diferenciação de amostras vacinais e selvagens do vírus da doença de Aujeszky / A duplex PCR for differentiation of wild and vaccine virus of Aujeszkyïs disease

A duplex PCR was developed to differentiate the wild-type virus from the attenuated virus used in vaccinations. The PCR was able to amplify fragments of 493bp for glycoprotein E (gE) gene and 207bp for glycoprotein B (gB) gene. The analytical sensitivity was determined by addition of a virus field sample titled in the brain samples of pigs. The standard virus strain Shope, the vaccine strain Bartha, and ten other field isolates were subjected to PCR. The PCR was able to amplify fragments of gE and gB in all field samples and only fragments of gB were amplified in the attenuated virus, as expected. The technique was able to detect up to 100.5 TCID50/50mL virus in samples of brain. Duplex PCR proved to be an important tool for differentiation of naturally-infected animals and animals vaccinated with the virus deleted for gE.