Evaluation of a nested-pcr for Mycobacterium tuberculosis detection in blood and urine samples

The polymerase chain reaction (PCR) and its variations, such as the nested-PCR, have been described as promising techniques for rapid diagnosis of tuberculosis (TB). With the aim of evaluating the usefulness of a nested-PCR method on samples of blood and urine of patients suspected of tuberculosis we analyzed 192 clinical samples, using as a molecular target the insertion element IS6110 specific of M. tuberculosis genome. Nested-PCR method showed higher sensitivity in patients with extrapulmonary tuberculosis (47.8 percent and 52 percent in blood and urine) when compared to patients with the pulmonary form of the disease (sensitivity of 29 percent and 26.9 percent in blood and urine), regardless of the type of biological sample used. The nested-PCR is a rapid technique that, even if not showing a good sensitivity, should be considered as a helpful tool especially in the extrapulmonary cases or in cases where confirmatory diagnosis is quite difficult to be achieved by routine methods. The performance of PCR-based techniques should be considered and tested in future works on other types of biological specimens besides sputum, like blood and urine, readily obtainable in most cases. The improving of M. tuberculosis nested-PCR detection in TB affected patients will give the possibility of an earlier detection of bacilli thus interrupting the transmission chain of the disease.

Diferenciação de micobactérias por PCR multiplex / Differentiation of micobacteria by multiplex PCR

O trabalho visou à otimização de um método baseado na reação em cadeia da polimerase multiplex - para diferenciação de micobactérias de interesse para a saúde pública. A PCR Multiplex baseou-se na amplificação simultânea do genehsp65, presente em todo gênero Mycobacterium, do gene dnaJ, presente apenas em Mycobacterium tuberculosis e Mycobacterium avium e da sequência de inserção IS6110 presente no complexo Mycobacterium tuberculosis, gerando amplicons de 165pb, 365pb e 541pb, respectivamente. O limite de detecção foi de 1fg para o alvo hsp65, 100pg para o dnaJ e 0,1fg para o IS6110. A PCR multiplex detectou até 100pg de DNA de Mycobacterium tuberculosis. O sistema demonstrou ser específico e sensível na detecção de Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium avium e Mycobacterium smegmatis. Os resultados obtidos utilizando cepas de referência demonstraram que a PCR multiplex pode ser uma ferramenta rápida, sensível e específica na diferenciação de micobactérias.

This study aimed to optimize a method based on the polymerase chain reaction - multiplex PCR - for differentiation of mycobacteria species of interest for public health. The multiplex PCR was based on simultaneous amplification of the hsp65 gene, which is present in all species of the Mycobacterium genus, the dnaJ gene, which is present only in Mycobacterium tuberculosis and Mycobacterium avium and the IS6110 insertion sequence, which is present in the Mycobacterium tuberculosis complex, generating amplicons of 165 bp, 365 bp and 541 bp, respectively. The detection limit was 1 fg for the hsp65 target, 100 pg for dnaJ and 0.1 fg for IS6110. The multiplex PCR detected down to 100 pg of DNA of Mycobacterium tuberculosis. The system was shown to be specific and sensitive for detection of Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium avium and Mycobacterium smegmatis. The results obtained using reference strains of mycobacteria showed that multiplex PCR may be a fast, sensitive and specific tool for differentiation of mycobacteria.

[Differentiation of micobacteria by multiplex PCR]. / Diferenciação de micobactérias por PCR multiplex.

This study aimed to optimize a method based on the polymerase chain reaction - multiplex PCR - for differentiation of mycobacteria species of interest for public health. The multiplex PCR was based on simultaneous amplification of the hsp65 gene, which is present in all species of the Mycobacterium genus, the dnaJ gene, which is present only in Mycobacterium tuberculosis and Mycobacterium avium and the IS6110 insertion sequence, which is present in the Mycobacterium tuberculosis complex, generating amplicons of 165 bp, 365 bp and 541 bp, respectively. The detection limit was 1 fg for the hsp65 target, 100 pg for dnaJ and 0.1 fg for IS6110. The multiplex PCR detected down to 100 pg of DNA of Mycobacterium tuberculosis. The system was shown to be specific and sensitive for detection of Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium avium and Mycobacterium smegmatis. The results obtained using reference strains of mycobacteria showed that multiplex PCR may be a fast, sensitive and specific tool for differentiation of mycobacteria.