期刊
LETTERS IN APPLIED MICROBIOLOGY
卷 66, 期 2, 页码 138-144出版社
WILEY
DOI: 10.1111/lam.12825
关键词
Lactobacillus sakei group; RAPD fingerprinting; rapid identification; sequence-characterized amplified region marker; species-specific PCR primers
资金
- Ministry of Economic Affairs, Taiwan [106-EC-17-A-22-0525]
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. 10 5, 104 and 10 6 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.
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