Rapid quantitative detection of Lactobacillus sakei in meat and fermented sausages by real-time PCR.
A quick and simple method for quantitative detection of Lactobacillus sakei in fermented sausages was successfully developed. It is based on Chelex-100-based DNA purification and real-time PCR enumeration using a TaqMan fluorescence probe. Primers and probes were designed in the L. sakei 16S-23S rRNA intergenic transcribed spacer region, and the assay was evaluated using L. sakei genomic DNA and an artificially inoculated sausage model. The detection limit of this technique was approximately 3 cells per reaction mixture using both purified DNA and the inoculated sausage model. The quantification limit was established at 30 cells per reaction mixture in both models. The assay was then applied to enumerate L. sakei in real samples, and the results were compared to the MRS agar count method followed by confirmation of the percentage of L. sakei colonies. The results obtained by real-time PCR were not statistically significantly different than those obtained by plate count on MRS agar (P > 0.05), showing a satisfactory agreement between both methods. Therefore, the real-time PCR assay developed can be considered a promising rapid alternative method for the quantification of L. sakei and evaluation of the implantation of starter strains of L. sakei in fermented sausages.
Martín
Belén
B
Institute for Food and Agricultural Research and Technology (IRTA), Meat Technology Center, Granja Camps i Armet, E-17121 Monells, Spain.
Jofré
Anna
A
Garriga
Margarita
M
Pla
Maria
M
Aymerich
Teresa
T
D000818:Animals; D001483:Base Sequence; D015169:Colony Count, Microbial; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D021903:DNA, Ribosomal Spacer / Q000235:genetics; D005285:Fermentation; D005516:Food Microbiology; D007778:Lactobacillus / Q000235:genetics* / Q000302:isolation & purification*; D008460:Meat / Q000382:microbiology*; D008461:Meat Products / Q000382:microbiology*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000706:statistics & numerical data; D012680:Sensitivity and Specificity; D012689:Sequence Homology, Nucleic Acid
D017931:DNA Primers; D004269:DNA, Bacterial; D021903:DNA, Ribosomal Spacer