Amplificação da sequência de inserção IS6110 diretamente do esfregaço das lâminas coradas para bacilo álcool- ácido resistente

Abstract

The obtainment of DNA directly from smear slides is a valuable option to resolve the problem of specimen transportation, as it avoids the risk of bacilli transmission; besides, this characteristic is particularly opportune in places where the geography itself hampers the access to samples and the PCR has emerged as a promising molecular technique for rapid diagnosis of tuberculosis. Two hundred eighty seven convenient samples from the laboratory of the Faculty of Medicine/Laboratory of Mycobacteria/Hospital of Clinics of University Federal of Minas Gerais were selected. The DNA was extracted by the Chelex® + NP40 method and amplified by IS6110-PCR under the following conditions: 7.0 µL of Buffer (10x), 3.0 µL MgCl 2 (50 mM), 0.2 µL dNTP (25 mM), 25pmol of each oligonucleotide (IS1- 5 CCT GCG AGC GTA GGC GTC GG 3 and IS2- 5 CTC GTC CAG CGC CGC TTC GG 3) and 0.5 µL (500 U) of Taq DNA Polymerase Invitrogen ® , in a final volume of 50 L. The initial DNA denaturation was at 94°C for 2 min, following a total of 40 cycles of 94ºC for 30 sec, with every annealing temperature for 2 min, 71ºC for 1 min, and a final extension of 72°C for 10 min. The amplicons were revealed in 2% agarose gel stained by ethidium bromide. The PCR result was then compared with in Lowenstein-Jensen medium. The sensitivity and specificity as compared with those of culture were 37.5 e 100%, respectively. The positive predictive value was of 100%, the negative predictive value was of 80.8%, and the accuracy was of 82.8%. The high specificity of in house DNA amplification becomes this method, a quick and simple procedure for tuberculosis diagnosis; it could also assure the utilization of this method in places in which the bacilloscopy is positive, with a higher prevalence of Nontuberculous Mycobacteria. Besides to offer biosafety in the transportation, the in house DNA amplification could also be utilized in situations where the amount of bacterial cells is insufficient. Therefore, this method is an important tool when associated to clinical criteria, speeds up the treatment andcontributes to interrupt the disease transmission chain.