RESUMO
In diagnostic amplification protocols contamination by previously amplified nucleic acids is considered the major source of false positive results. Substituting dUTP for dTTP in the PCR and initial treatment with uracil-DNA glycosylase (UNG) virtually eliminates these carryover contaminations. Subsequent procedures to visualize the amplicons or to optimize sensitivity and specificity of the test are not always fully compatible with UNG-treated PCR products. Here we describe the more pronounced influence of residual UNG activity on the migration of PCR amplification products in polyacrylamide as compared to agarose gels.
Assuntos
DNA Glicosilases , Nucleotídeos de Desoxiuracil/isolamento & purificação , Eletroforese em Gel de Ágar , Eletroforese em Gel de Poliacrilamida , N-Glicosil Hidrolases/química , Reação em Cadeia da Polimerase , Nucleotídeos de Desoxiuracil/química , Ativação Enzimática , Estabilidade Enzimática , N-Glicosil Hidrolases/metabolismo , Valor Preditivo dos Testes , Temperatura , Fatores de Tempo , Uracila-DNA GlicosidaseRESUMO
1579 stool samples from patients with travel-associated diarrhea were examined by conventional culture methods to detect Shigella spp. (48 positive samples or 3.2%) and by PCR to detect shigellae and enteroinvasive Escherichia coli (EIEC) (87 positive samplers or 5.8%). The numerical relation of shigellae to EIEC in PCR-positive samples was about 60 to 40%. However, the lack of discrimination between shigellae and EIEC is not important for the physician because the choice of antibiotics remains the same.
Assuntos
Diarreia/microbiologia , Escherichia coli/isolamento & purificação , Fezes/microbiologia , Shigella/isolamento & purificação , Escherichia coli/efeitos dos fármacos , Humanos , Testes de Sensibilidade Microbiana , Reação em Cadeia da Polimerase , Estudos Prospectivos , Shigella/efeitos dos fármacos , ViagemRESUMO
Bartonella (Rochalimaea) henselae and/or B. quintana are the causative agents of a variety of infections such as trench fever, bacillary angiomatosis, septicemia, peliosis hepatis and endocarditis. Recently, B. henselae has been identified as a major cause of cat scratch disease. Diagnosis of such infections is based on clinical information, histopathology, culture and serology. However, none of these methods alone is sufficiently sensitive or specific. We have used the PCR to search for DNA specific for B. henselae/B. quintana in 33 clinical samples and in 6 controls. In comparison with clinical data and histopathology, PCR was extremely specific (100%) and reasonably sensitive (61%). Possible explanations for the limited sensitivity of PCR are discussed. We conclude that PCR provides a useful adjunct for the diagnosis of infections caused by B. henselae and B. quintana.
