[Optimization of the REP-PCR molecular classification method of Salmonella and its application in drug-resistant strains].

OBJECTIVE

To optimize the reaction conditions of REP-PCR molecular classification method of Salmonella; primarily apply it in drug-resistant strains, and supply data to a system of Salmonella homology tracing.

METHODS

Genomic DNA of Salmonella enteritidis 510041 was used as the template for PCR. Target sequences in 510041 genomic DNA were amplified with the primers designed according to the references. To optimize template concentration, Mg2+ concentration, primers concentration of PCR, the factor to be optimized was designed in different concentration grads and other factors were fixed. The REP-PCR fingerprint maps of 24 strains of Salmonella epidemic drug-resistant isolates were analyzed with the optimum reaction conditions. The 24 strains of Salmonella epidemic drug-resistant isolates were classified according to their fingerprint maps, and the classification results were compared to classification results form drug-resistant spectrum.

RESULTS

The fingerprint map bands were most clear when 25 microl of the reaction system contained 100 ng template, 2.0 mmol/L Mg2+ and 0.4 micromol/L each primer. The REP-PCR fingerprint maps of different serotypes and serum clusters of Salmonella were different. The amplification products contained 2 to 6 bands, whose length were between 0.5 kb to 2.5 kb. The 24 strains of Salmonella isolates were classified in 15 types according to fingerprint maps and 7 types according to drug-resistant spectrum.

CONCLUSION

The optimum REP-PCR molecular classification method of Salmonella was established fingerprint maps classification method was more sensitive than drug-resistant spectrum classification method.