A Simple and Efficient Multiplex PCR Assay for the Identification of Mycobacterium Genus and Mycobacterium tuberculosis Complex to the Species Level

PURPOSE: The Mycobacterium tuberculosis complex comprises M. tuberculosis, M. bovis, M. bovis bacillus Calmette-Guerin (BCG) and M. africanum, and causes tuberculosis in humans and animals. Identification of Mycobacterium spp. and M. tuberculosis complex to the species level is important for practical use in microbiological laboratories, in addition to optimal treatment and public health. MATERIALS AND METHODS: A novel multiplex PCR assay targeting a conserved rpoB sequence in Mycobacteria spp., as well as regions of difference (RD) 1 and RD8, was developed and evaluated using 37 reference strains and 178 clinical isolates. RESULTS: All mycobacterial strains produced a 518-bp product (rpoB), while other bacteria produced no product. Virulent M. tuberculosis complex strains, M. tuberculosis, M. bovis and M. africanum, produced a 254-bp product (RD1), while M. bovis BCG, M. microti and nontuberculous mycobacteria produced no RD1 region product. Additionally, M. tuberculosis and M. africanum produced a 150-bp product (RD8), while M. bovis and M. bovis BCG produced a 360-bp product (deleted form of RD8). M. microti and nontuberculous mycobacteria produced no RD8 region product. This assay identified all Mycobacterium spp. and all M. tuberculosis complex strains to the species level. CONCLUSION: The multiplex PCR assay of the present study could be implemented as a routine test in microbiology laboratories, and may contribute to more effective treatment and surveillance of tuberculosis stemming from the M. tuberculosis complex.

PCR-reverse Blot Hybridization Assay for Species Identification of Dermatophytes / 대한의진균학회지

BACKGROUND: Dermatophytes (Trichophyton, Microsporum, and Epidermophyton) cause cutaneous mycoses called dermatophytosis. Forproper anti-dermatophytosis therapy, accurate and early diagnosis of dermatophytes is important. Laboratory diagnosis of dermatophytosis for dermatophytes still relies on microscopic and macroscopic examination of in vitro cultures and some physiological tests. These methods (conventional methods) are time-consuming (2~4 weeks) and yet, still have low sensitivity and specificity. Recently, in order to overcome such limitations of conventional methods, molecular-based methods have been developed to identify dermatophytes. The polymerase chain reaction-reverse blot hybridization assay (PCR-REBA) allows sensitive and specific identification of dermatophytes species. OBJECTIVE: This study was aimed to develop a new PCR-REBA with higher sensitivity using less amount of probe concentration, so the assay can be more practical in clinical settings. METHODS: For this, PCR primers and species-specific oligonucleotide probes were designed within the internal transcribed sequences 1 region between 5.8S and 18S rRNA. The species-specific probes designed in this study was to identify 6 species (T. rubrum, T. mentagrophytes, T. tonsurans, M. canis, M. gypseum, and E. floccosum) comprised 99% of dermatophytes isolatedin Korea. RESULTS: The detection efficiency of the PCR-REBA was compared with the microscopic method, and the results showed that the sensitivity of the PCR-REBA developed in this study is 100 times higher than previously developed one. Subsequently, the results of PCR-REBA were evaluated using clinical isolates. DNAs from a total of 68 clinical isolates were analyzed by PCR-REBA, and the inconsistent results between PCR-REBA and conventional microscopic identification results were confirmed by sequence analysis. CONCLUSION: In brief, the results showed that results of sequence analysis were identical with PCR-REBA implying newly developed PCR-REBA is very useful method for accurate and rapid identification of dermatophytes and would provide higher simplicity, specificity, sensitivity than conventional method.