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Developing novel species‐specific DNA markers for PCR ‐based species identification of the Lactobacillus sakei group
Author(s) -
Huang C.H.,
Liou J.S.,
Huang L.,
Watanabe K.
Publication year - 2018
Publication title -
letters in applied microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.698
H-Index - 110
eISSN - 1472-765X
pISSN - 0266-8254
DOI - 10.1111/lam.12825
Subject(s) - lactobacillus sakei , biology , primer (cosmetics) , polymerase chain reaction , rapd , 16s ribosomal rna , lactobacillus , genotype , genetics , microbiology and biotechnology , gene , bacteria , population , genetic diversity , chemistry , demography , organic chemistry , sociology
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 , 10 4 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 . 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.