Microplate hybridization assay for detection and differentiation of mycobacterium tuberculosis from non-tuberculous mycobacteria

Abstract

[한글]

[영문]Tuberculosis caused by Mycobacterium tuberculosis (MTB) still remains to be the most dreadful infectious disease affecting almost every country. In addition, with the recent increase in the frequency of non-tuberculous mycobacterial infections, it is becoming increasingly necessary to differentiate MTB from non-tuberculous mycobacteria (NTM) infections because the remedies for diseases resulting from NTM should be different from those for MTB infection.In the present study, a simple, rapid, accurate and sensitive microplate hybridization assay for detecting MTB was developed. For this, a region of the rpoB gene was used to design PCR primers, MTB probe and Mycobacterium genus-specific probe molecules. The specificity of the assay was confirmed using DNA extracted from fifteen different mycobacterial reference strains and twelve different non-mycobacterial reference strains, and the sensitivity was determined to be 100 fg using genomic DNA of MTB reference strain, H37Rv. Subsequently, DNAs extracted from 62 sputum samples with diverse smear scores and culture positive results were used to evaluate the assay performance. In brief, the sensitivity of the assay were 98.4% (61/62).Next, a microplate hybridization assay for identifying NTM species was developed. For this, the species-specific probes for 13 different NTM species which are frequently detected in clinical practice were designed based on a region of the rpoB gene. The specificity of the individually probes was confirmed using DNA extracted from 15 mycobacterial reference strains including MTB, and 12 non-mycobacterial reference strains. The findings from the experiment above suggest that microplate hybridization assay is capable of isolating and differentiating MTB from NTM, as well as molecular diagnostic method with good sensitivity identification of NTM. From this, it is expected that the microplate hybridization assay developed in this study may be a useful.