Direct identification of Mycobacterium tuberculosis from sputum on Ziehl-Neelsen acid fast stained slides by use of silica-based filter combined with polymerase chain reaction assay.

Abstract

This paper describes a method for isolation of deoxyribonucleic acid (DNA) from Ziehl-Neelsen stained sputum smears on glass slides; and isolated DNA was used for the IS6110 polymerase chain reaction (PCR)-based identification of M. tuberculosis. A total of 221 samples from newly diagnosed suspected tuberculosis cases were first examined by microscopic examination. For DNA extraction by silica-based filter, a home-made modified spin column gave the efficacy as did the nucleospin tissue reagent kit and therefore was selected for PCR template preparation. The extracted DNA was amplified by the IS6110 PCR using a primer pair that amplifies a 377-bp target, and the product was analyzed by agarose gel electrophoresis with confirmation by Southern blot hybridization. In comparison with culture, PCR with template prepared by the silica based filter showed overall sensitivity and specificity of 91.7 and 100 per cent, respectively. This study used the over one year and less than one year slides samples to study the effect of storage time. In the more than one year storage group, PCR assay gave a sensitivity and specificity of 83.3 and 100 per cent, respectively. In conclusion, the applicability of the PCR directly to DNA extracted from Ziehl-Neelsen stained smears could become a valuable alternative approach for rapid identification of M. tuberculosis, and could be used to evaluate quality of the control of local laboratories in tuberculosis (TB) screening and solve the problem of specimen transportation. In addition, the method could be used in retrospective studies involving a wide range of PCR-based analyses, such as detection of rifampicin resistant gene in multidrug-resistant tuberculosis (MDR-TB) study.

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