Evaluating the sensitivity of three primers using PCR-restriction fragment length polymorphism analysis for rapid identification of Mycobacterium simiae isolated from pulmonary tuberculosis patients

ABSTRACT

Nowadays the molecular methods widely use for rapid identification of Mycobacterium other than tuberculosis [MOTT]. The Mycobacterium simiae isolates are cause of majority of human pulmonary diseases compared with other atypical mycobacteria. As sensitivity of primers and digestion patterns for diversified fragments is different, this survey evaluated the three various fragments using the PCR- restriction fragment length polymorphism analysis [PRA] for rapid diagnostic of M simiae isolates. Strains that were identified as M. simiae [1.7 isolates] by phenotypic [photochromogen and positive niacin] methods were selected for this study. The fragments of the 16S-23S rRNA gene spacer and hsp65 gene were amplified by PCR. Subsequently the amplicons were digested with three restriction enzyme namely Avail, Hphl and Hpall for a 644bp region of hsp65 DNAs, BstEll and Haelll endonucleases for 439bp region of hsp65 gene [TB11 and TB12 fragment] and Haelll digestion for 225bp region of 16S-23S rRNA gene spacer. Of 962 culture positive specimens, 17 [1.7%] were identified as M. simiae species; majority of them were multidrug-resistance [12; 70.5%]. The overall detection rate by Tbll, Tbl2 and SP primers were 82.3% whereas hsp65 primer was 100% [p>0.005]. We also found out that the Hpall and Hphl enzymes were more specific to distinguish M simiae species than other restriction enzyme used in this study. The high discriminative power of hsp65 pattern particularly Hpall digestion, provide an exact and cost-effective method for rapid identification of M. simiae strains among registered pulmonary cases