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United States Department of Agriculture

Agricultural Research Service

Title: A Comparison of Sample Preparation Methods for Pcr-Based Detection of Pathogenic Yersinia Enterocolitica in Ground Pork

Author
item Bhaduri, Saumya

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: June 12, 2002
Publication Date: August 13, 2002
Citation: BHADURI, S. A COMPARISON OF SAMPLE PREPARATION METHODS FOR PCR-BASED DETECTION OF PATHOGENIC YERSINIA ENTEROCOLITICA IN GROUND PORK. MEETING ABSTRACT. 2002.

Technical Abstract: Sample preparation methods for multiplex PCR for detection of plasmid-bearing virulent Yersinia enterocolitica (YEP+) from ground pork (GP) were compared. The GP samples were first inoculated with 10, 1 and 0.5 CFU/cm2 of YEP+, one set was swabbed and the second set was dispersed into a slurry using the homogenate technique (HT). Both swab and HT samples were enriched in sterile Whirl Pak bags containing MTSB broth for 48 h at 12oC until the level of YEP+ were 105 cells/ml. Cell lysates were prepared from cell pellets of enriched swab samples. Since HT samples contained food material, DNA extraction was performed using a commercial kit. The DNA from cell lysates and from extracted samples was evaluated as templates for multiplex PCR employing primers for chromosomal ail and plasmid virF genes. Dilutions of the DNA extracted from HT were necessary to determine the optimal concentration of sample for use in the PCR assay. No amplification signal was detected using undiluted DNA, possibly due to inhibitors present in HT that were not removed by DNA extraction. However, YEP+ were detected in undiluted cell lysates from swab samples. Thus, use of cell lysates from swab sample enrichments is more advantageous than use of DNA extracted from HT samples for the PCR assay since the DNA extraction step, the dilutions of the DNA, and PCR inhibitors are eliminated.

Last Modified: 10/1/2014
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