Developing novel species-specific DNA markers for PCR-based species identification of the Lactobacillus sakei group

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Abstract

Identification of members of the Lactobacillus sakei group (LSG) by common phenotypic and genotypic methods is generally inadequate and time-consuming. The objective of this study was to develop novel species-specific primers based on sequence-characterized amplified region (SCAR) markers using random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) analysis. Three species-specific fragments were gel-purified, cloned and sequenced after preliminary screening of 80 random primers. Accordingly, three pairs of primers Lcur-F/R, Lgram-F/R and Lsakei-F/R were designed based on single species-specific bands (281, 278 and 472 bp) that were obtained from Lactobacillus curvatus, Lactobacillus graminis and L. sakei, respectively. The specificities of these primer pairs were confirmed in 21 LSG strains and 31 nontarget Lactobacillus strains. In addition, the detection limits for each primer pair were approx. 105, 104 and 106 cells per gram of meat samples spiked with L. curvatus, L. graminis and L. sakei, respectively. In conclusion, we have successfully developed a rapid, accurate and effective PCR-based method for identification of species in the LSG.

Significance and Impact of the Study:

Neither phenotypic nor the most commonly used genotypic method (16S rRNA gene sequencing) provides sufficient resolution for accurate identification of the Lactobacillus sakei group. A sequence-characterized amplified region method developed in this study provides a rapid, cost-effective way to detect the member of the L. sakei group in meat sample.

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