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Project ID: ICA4-CT-2001-10028
Finanziato nell'ambito di: FP5-INCO 2
Paese: Malaysia

Genotyping of isolates using (GTG) 5 repetitive PCR

Molecular Characterization of aquaculture-associated bacteria by (GTG)5 repetitive PCR

Epidemiological typing of bacteria or pathogens has become more and more important in public health control. PCR is a powerful technique that has revolutionized the molecular biology. Repetitive-PCR (rep-PCR) is a commonly used method in PCR. In this study, rep-PCR was used to genotype 140 of the aquaculture-associated bacteria isolates, which yield negative results for Cat genes detection. Rep-PCR entails the use of primers complementary to highly conserved, repetitive sequences present in multiple copies on the genome.

Because the distance between the repetitive elements varies among strains, PCR amplification of the DNA sequences found between them results in generation of a distinct fingerprint. Prior to amplification, crude genomic DNA of isolates was harvested using boiling method. For the detection and confirmation of PCR products by gel electrophoresis, the amplification product mixture was subjected to electrophoresis. Subsequently, amplified DNA fragments of specific sizes were visualized by UV fluorescence after being stained with ethidium bromide. The (GTG)5 rep-PCR fingerprinting profiles obtained were analyzed with the GelCompare software (version 4.1).

The overall achievable patterns were used to construct dendrograms using the UPGMA (unweighted pair group average method) clustering algorithms. To determine the reproducibility of the (GTG)5 rep-PCR analysis procedure, (GTG)5 rep-PCR was repeated at least three times for DNA from each strain, so that the amplification profiles for each attempt could be compared. We found that profiles for a given strain to be highly reproducible, with very little variation from one (GTG)5 rep-PCR analysis to another. (GTG)5 rep-PCR was used as a mean to determine the clonal relatedness of the isolates in this study by their chromosomal polymorphism. Our (GTG)5 rep-PCR analysis revealed homogeneous isolates and of match those of UoS's profile.

In summary, results from this study demonstrated that genotyping aquaculture-associated bacteria isolates by using (GTG)5 rep-PCR is feasible for differentiation of various strains. The classical and microbiological methods, based on the identification of phenotypic markers, while perfectly adequate to identify microorganisms on the level of species, are often not reliable enough when it comes to differentiating further into individual strains. Molecular typing procedures like (GTG)5 rep-PCR are applied to show clonal and close relationship between isolates of one species.

Thus, it is possible to identify pathogen reservoirs and to follow up the regional and global spread of pathogens in aquaculture. In addition, it gives an insight into the evolutionary dynamics of the bacterial genome. (GTG)5 rep-PCR has shown to be rapid, sensitive, discriminative and cost effective in genotyping the aquaculture-associated bacteria isolates from Malaysia.

Informazioni correlate

Reported by

Aquatic Animal Health Unit
Faculty of Veterinary Medicine, Universiti Putra Malaysia
43400 Serdang