Análise in silicode iniciadores de genes referência utilizados como normalizadores em estudos utilizando qPCR para avaliação da expressão de isolados de Klebsiella pneumoniae

Abstract

Klebsiella pneumoniaeis a pathogenic bacterium considered to be an "urgent threat to human health"becausethe number of antibiotic-resistant bactéria is increasing, especially those considered to be the last line for its treatment, such as colistin.Forthis it is necessary to understand their mechanisms of resistance to know the best forms of treatment and to develop new drugs to treat infections. For this purpose, real-time quantitative PCR in relative expression studies has become one of the most effective tools to understand bacterial functioning at the transcriptional level, but for the results to be reliable and real, it is necessary to perform the normalization step, which among the possible the most common is through the useof reference genes. However, the choice of the genes to be used as normalizers among the genes pointed out in the literature has been controversial and, in many cases, with little reliability. This difficulty would be eliminated if there were a robust database for various types of studies for species other than humans and rats. Thus, there was a need to evaluate amongthe bacterial gene expression studies using qPCR, as normalizing genes and the primers used to amplify them.In a literature review, available in Pubmed, the 16Sand rpoBgenes were most commonly used as normalizers. Through in silico analysis after literature analysis it was possible to observe that the sequences of shared primer pairs were only smaller series within the ideal standard and the best ones in the future usedin later experiments, among others only one referring to one of the genes most commonly used, the 16S.