An improved PCR primer pair based on 16S rDNA for the specific detection of Salmonella serovars in food samples.
Salmonella serovars are some of the major bacterial pathogens that can cause sporadic cases and outbreaks of foodborne illness. Based on the sequence data in the V3 region of the 16S rRNA gene, two PCR primer pairs have been designed for the detection of all serovars of Salmonella. However, none of these primers were specific for Salmonella because complete sequence homology with certain non-Salmonella strains has been found within each of them. Thus, the specificities of these two primer pairs could not rely on only one of the two primers. In this study, we modified our previous 16SFI primer by extending one base at the 5' end and three bases at the 3' end. The modified primer, 16S-Sal, was designed with one or more mismatched bases near the 3' end of the primer annealing to the corresponding sequences of non-Salmonella strains. Such modification eliminates interference from Citrobacter freundii and Enterobacter cloacae as occurs with the 16SFI primer. When 16S-Sal and a degenerate primer, 16S-CCR, were used as a primer pair, detection specificity of Salmonella serovars was achieved. Because this primer pair was used for PCR detection of the salmonellae in food samples, such as whole milk and chicken meat, as low as 1 to 9 CFU/g (ml) of the food sample could be detected when a 8-h preculture step was performed prior to the PCR. For chicken meat, the endogenous microflora did not interfere with the PCR results.
Lin
Chien-Ku
CK
Department of Biotechnology, Hung-Kuang University, Shalu, Taichung County, Taiwan 433, Republic of China.
Hung
Cho-Lien
CL
Hsu
Shu-Chen
SC
Tsai
Cheng-Chih
CC
Tsen
Hau-Yang
HY
D000818:Animals; D001483:Base Sequence; D002645:Chickens; D017931:DNA Primers; D004269:DNA, Bacterial / Q000032:analysis*; D004275:DNA, Ribosomal / Q000032:analysis*; D005516:Food Microbiology; D006801:Humans; D008460:Meat / Q000382:microbiology; D008892:Milk / Q000382:microbiology; D016133:Polymerase Chain Reaction / Q000379:methods*; D012475:Salmonella / Q000235:genetics / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D016415:Sequence Alignment; D012689:Sequence Homology, Nucleic Acid
D017931:DNA Primers; D004269:DNA, Bacterial; D004275:DNA, Ribosomal