Detection and Differentiation of Mycobacterium Species in Clinical Specimens by Real-Time Polymerase Chain Reaction

ABSTRACT

Mycobacterium tuberculosis complex and Mycobacterium avium complex are causative agents of tuberculosis which is a major cause of human morbidity and mortality. Rapid diagnosis and species differentiation of mycobactria in specimens are important to control the disease and use appropriate antimicrobial therapy. In this study, primers and probe were design based on 16S-23S rDNA spacer sequence by using LightCycler 2.0 software. One set of primer, Rank 9, was used to perform real-time PCR and conventional PCR for detection and differentiation of Mycobacterium spp. Sybr Green dye for signal detection, successfully amplified all extracted DNA of mycobacteria. Its amplicon can be visualized by gel electrophoresis with the corresponding length of 214 bp. The nonspecific products of about 300 bp, were found in conventional PCR. The result suggested that these primers are not appropriate for conventional PCR. A hybridization probe for strain differentiation and verification of the protocol in clinical specimens is needed to complete the protocol.