OPTIMIZATION OF REAL-TIME PCR AIMING AT THE MOLECULAR DIAGNOSIS OF TUBERCULOSIS.

Thiago Milech de Assunção (INCT-TB); Ana Christina de Oliveira Dias (INCT-TB); Luiz Augusto Basso (INCT-TB); Diógenes Santiago Santos (INCT-TB); Eraldo Luis Batista Jr. (INCT-TB)

Mycobacterium tuberculosis (Mt) infection remains a serious public health problem due to its high risk of person-to-person transmission, morbidity and mortality. Currently, there are approximately 9,2 million new infections and 1,7 million deaths attributed to tuberculosis (TB) every year. Progressive increases in TB infections are expected and a worldwide annual incidence of 14,4 million cases was predicted by the World Health Organization. Diagnostic methods of TB, nowadays, promote an unacceptable delay in diagnosis and unfortunately lead to elevated false negative results. The aforementioned is critical since the infected individuals remain untreated, increasing the likelihood of disease transmission and epidemic aggravation. Thus, the rapid detection and identification of Mt in secretions would be highly desirable for proper diagnosis and effective treatment, along with the prevention and control of tuberculosis transmission. Real-time Polymerase Chain Reaction (RT-PCR) using primers and a specific detection probes is a recent approach that offers many advantages such as higher sensitivity, specificity and fast diagnosis. The aims of the present study were to implement a molecular diagnostic test for tuberculosis, as well as to screen and optimize Mt target genes for RT-PCR. Sputum from patients with diagnosed tuberculosis was collected and processed for DNA isolation. RT-PCR was performed on a thermalcycler to amplify and detect the Mt genes. Primers and probes designed to detect Mt single-copy genes conjugated with fluorophores (TaqMan). The construction of synthetic DNA standards for Mt served as a references for absolute quantification of nucleic acids, which reflected absolute numbers of Mt. Those were obtained from a prior amplification and cloning of genomic DNA of Mt, targeting the intergenic sequences as noted above. The results showed that Mycobacterium DNA was detected in DNA isolated from sputum; nevertheless, control individuals failed to show any signs of fluorescence increase, corroborating the absence of cross amplification of host DNA as well as DNA from other pathogens recovered from the patients. The positive results obtained call for further experiments aiming at diagnosis of paucibacilar TB.