A comparison of sample preparation methods for PCR detection of pathogenic Yersinia enterocolitica from ground pork using swabbing and slurry homogenate techniques.

摘要

Two sample preparation methods for multiplex polymerase chain reaction (PCR) for detection of plasmid-bearing virulent Yersinia enterocolitica (YEP(+)) from ground pork were compared. Two sets of ground pork samples were inoculated with 10, 1, and 0.5 CFU/cm(2) of a YEP(+) strain, one set was swabbed and the second set was dispersed into a slurry homogenate. Both swab and slurry homogenate samples were enriched in sterile Whirl Pak bags containing modified trypticase soy broth for 48 h at 12 degrees C. From the enriched swab samples, the bacterial cells were pelleted, washed, boiled in sterile distilled water, and treated with proteinase K to prepare cell lysates to use as a DNA template. Since slurry homogenate samples contained food material, DNA extraction was performed using a commercial kit. The DNA from cell lysates and from extracted slurry homogenate samples were evaluated as templates for multiplex PCR employing primers for the chromosomal ail and plasmid virF genes. The enrichment of the YEP(+) strain was more efficient using the sponge-swabbed samples than the slurry homogenate samples at all three inoculum levels tested. It was necessary to dilute the DNA extracted from slurry homogenate to determine the optimal concentration of each sample for PCR amplification. No amplification signal was detected using undiluted DNA, possibly due to DNA inhibitors present in the slurry homogenate that were not removed in the process of extraction. However, DNA could be detected in undiluted cell lysates from swab samples. Thus, the cell lysates from swab samples are more advantageous than DNA extracted from ground pork slurry homogenate samples for the PCR assay.