Development of a multiplex PCR for detection and identification of aquatic mycobacteria

Objective: The mycobacteria that are most frequently associated with mycobacteriosis in fish are M. marinum, M. fortuitum and M. chelonae. Mycobacterial species are traditionally differentiated on the basis of their phenotypic characteristics which are very slow and labour-intensive procedure and suffer from inaccuracy. Advances in polymerase chain reaction (PCR) have resulted in widespread use of this technique. More recently a method termed multiplex PCR has been developed to include more than one pair of primers in which more than one target sequence is amplified. In this study a multiplex PCR was developed aiming at detection and identification of 3 aforementioned mycobacteria species. Method & Materials: To test the specificity of the multiplex PCR, a total of 59 mycobacterial isolates and type strains were screened. The specificity of the multiplex PCR assay was further tested using 4 reference strains and a number of field isolates of mycobacteria and 23 non-mycobacterial reference and field isolates. Using the Primer Select module in the Lasergene, Version 6.0, (DNASTAR), a series of genus and speciesspecific primers from the 16S rRNA and the hsp65 genes was designed. Results & Conclusion: DNA extracted from different species of mycobacteria reacted with the appropriate set of primers and no cross-reactivity was observed between different mycobaterial species or with any of the non-mycobacterial species. The level of sensitivity obtained for this multiplex PCR is noteworthy. The same sensitivity limit for the assay was achieved for both culture extracted DNA and fish tissues spiked with bacterial suspensions; this indicated that the assay is appropriate for use on field samples, i.e., frozen or fresh tissues. The rapidity and sensitivity of this multiplex PCR strongly supports the use of the assay as an efficient alternative to culture and biochemical tests. This one-step, single-tube multiplex PCR reaction not only minimizes the chance of contamination but also saves time and is cost-effective. The multiplex PCR protocol developed in this study is highly sensitive and unlike other PCR-based methods such as nested-PCR, hybridisation or restriction enzyme analysis does not require extra procedures; hence, it enables rapid detection and identification of the most common aquatic mycobacteria. However, due to sequence similarity of mycobacterial 16S rRNA gene, further analysis of mycobacterial isolates examined in this study such as sequence determination of different genes is required in future.