Traditional identification of mycobacteria based on cultural and biochemical tests can take several weeks and may fail to provide a precise identification. Polymerase Chain Reactionrestriction analysis (PRA) of the gene encoding heat shock protein 65 kDa (hsp65) gene has been proposed as a rapid and inexpensive alternative approach. Despite being widely used for differentiation of mammalian mycobacteria, this method has only been applied in the identification of a small number of aquatic mycobacteria. The present study aimed to evaluate the potential use of PRA of hsp65 for the identification of aquatic mycobacteria compared with sequence analysis. Seventy one mycobacterial isolates including, 10 type/ reference strains and the remainder field isolates, were subjected to PRA of a 441 bp fragment of this gene. For 68 representative isolates, sequence analysis was performed. All rapidly and slowly growing mycobacteria had best matches with 99.3% to 100% similarity with their corresponding species in the databanks.PRA proved to be a simple and rapid method for identifying aquatic mycobacteria.However, the incidence of similar or identical restriction patterns for some species of mycobacteria, and in particular, identification of new species of mycobacteria is a major problem using such a method. In contrast, the nucleic acid sequencing of the hsp65 gene yielded unambiguous results. Mycobacteriosis (fish tuberculosis) is a progressive disease of a wide range of wild and captive marine and freshwater fish species (Chinabut, 1999). The zoonotic nature of the organism, the massive economical losses which have occurred to the aquaculture industry due to this disease and the lack of effective treatment highlight the need for mycobacteria to be rapidly detected and identified to species level.
IntroductionTraditional identification of mycobacteria based on cultural and biochemical tests may take several weeks, and sometimes even then fail to provide a precise identification. In addition, the procedures of these tests are time consuming and complicated. Therefore, various molecular based methods have been developed for the rapid detection and identification of mycobacteria species. There are currently several commercially available methods, including the AccuProbe system (Genprobe, San Diego, Calif), INNO-LiPA (Innogenetics, Ghent, Belgium), or GenoType Mycobacterium assay (Hain, Lifescience, Germany). Although such tests provide desirable sensitivity and specificity, they are limited to the identification of a small number of mycobacteria.
Among other molecular methods, Polymerase Chain Reaction (PCR) -RestrictionEnzyme Analysis (PRA), and sequence analysis have been applied to several mycobacteria genes. In this regard, PCR of the 441 bp of heat shock protein 65 kD gene (hsp65), followed by restriction fragment pattern analysis by BstEII and HaeIII enzymes proposed by Telenti et al. (1993), has provided a rapid method for identifying mycobacteria and closely related species (Brunello et al., 2001; Devallois et ...