Toward an international standard for PCR-based detection of food-borne thermotolerant Campylobacters: assay development and analytical validation.
As part of a European research project (FOOD-PCR), we developed a standardized and robust PCR detection assay specific for the three most frequently reported food-borne pathogenic Campylobacter species, C. jejuni, C. coli, and C. lari. Fifteen published and unpublished PCR primers targeting the 16S rRNA gene were tested in all possible pairwise combinations, as well as two published primers targeting the 23S rRNA gene. A panel of 150 strains including target and nontarget strains was used in an in-house validation. Only one primer pair, OT1559 plus 18-1, was found to be selective. The inclusivity and exclusivity were 100 and 97%, respectively. In an attempt to find a thermostable DNA polymerase more resistant than Taq to PCR inhibitors present in chicken samples, three DNA polymerases were evaluated. The DNA polymerase Tth was not inhibited at a concentration of 2% (vol/vol) chicken carcass rinse, unlike both Taq DNA polymerase and DyNAzyme. Based on these results, Tth was selected as the most suitable enzyme for the assay. The standardized PCR test described shows potential for use in large-scale screening programs for food-borne Campylobacter species under the assay conditions specified.
Lübeck
P S
PS
Danish Veterinary Institute, DK-1790 Copenhagen, Denmark.
Wolffs
P
P
On
S L W
SL
Ahrens
P
P
Rådström
P
P
Hoorfar
J
J
D001483:Base Sequence; D002167:Campylobacter / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D017000:Campylobacter coli / Q000302:isolation & purification; D016123:Campylobacter jejuni / Q000302:isolation & purification; D044863:Campylobacter lari / Q000302:isolation & purification; D017931:DNA Primers; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism; D004795:Enzyme Stability; D005516:Food Microbiology; D006358:Hot Temperature; D016133:Polymerase Chain Reaction / Q000379:methods*; D012336:RNA, Ribosomal, 16S / Q000235:genetics / Q000302:isolation & purification; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013816:Thermodynamics
D017931:DNA Primers; D012336:RNA, Ribosomal, 16S; D004259:DNA-Directed DNA Polymerase