Development of a PCR-compatible enrichment medium for Yersinia enterocolitica: amplification precision and dynamic detection range during cultivation.
A Yersinia PCR-Compatible Enrichment (YPCE) medium was developed, which removes the necessity for sample pretreatment before PCR-based detection of Yersinia enterocolitica. The medium was designed through a sequence of independent screening and factorial design experiments to study the PCR inhibition and growth characteristics of medium components. The compatibility of the YPCE medium was evaluated using real-time PCR. The real-time PCR assay, based on the fluorescent double-stranded DNA binding dye SYBR green, generated approximately a 4-log linear range of amplification and in the range of 10(5)-10(8) (CFU/ml), the coefficient of variation <5%. When a background flora was present at concentrations > or = 10(6) (CFU/ml), the DNA amplification was influenced and a change in the log-linear slope leading to a lower amplification efficiency was observed. To study the dynamic detection range and relative amplification precision during enrichment. Y. enterocolitica and background flora were inoculated at various concentrations. It was possible to detect inoculation concentrations of 10(1) (CFU/ml) Y. enterocolitica in the presence of at least an inoculation concentration of 10(3) (CFU/ml) of an undefined background flora and the optimal conditions for sample withdrawal was in the range of 9 to 18 h enrichment. The YPCE medium can, especially for swab samples, form part of a simple analysis procedure allowing high throughput PCR.
Knutsson
Rickard
R
Center for Chemistry and Chemical Engineering, Lund Institute of Technology, Lund University, Sweden.
Fontanesi
Massimo
M
Grage
Halfdan
H
Rådström
Peter
P
D002021:Buffers; D015169:Colony Count, Microbial; D003470:Culture Media; D004269:DNA, Bacterial / Q000032:analysis*; D005516:Food Microbiology; D021141:Nucleic Acid Amplification Techniques; D016133:Polymerase Chain Reaction / Q000379:methods*; D015008:Yersinia enterocolitica / Q000235:genetics* / Q000254:growth & development / Q000302:isolation & purification
D002021:Buffers; D003470:Culture Media; D004269:DNA, Bacterial