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Improved PCR detection of Campylobacter jejuni from chicken rinses by a simple sample preparation procedure
1Microbiology Research Division, Bureau of Microbial Hazards, Food Directorate, Health Protection Branch, Health Canada, Ottawa, Ont. haiyan_wang@hc-sc.gc.ca
Abstract:
Many food samples and enrichment media are inhibitory to the PCR, thereby lowering its detection capacity. A simple sample preparation method based on buoyant density centrifugation was examined for its application in PCR detection of Campylobacter jejuni from chicken rinse samples. Bacterial cells were spiked at different levels in a mixture of Preston broth and chicken rinse (4:1 ratio) and 0.9 ml of these mixtures were layered over 0.6 ml of gradient medium made from Percoll. PCR sensitivity for bacterial samples treated with this procedure was approximately 10-100 times higher than for samples without treatment. This sample preparation method allowed for the detection of C. jejuni from 26 of 31 naturally contaminated chicken samples after a 20-24-h enrichment period in Preston broth, compared with only 14 positives for untreated samples. In addition, the effect of Oxyrase on the growth and PCR detection of C. jejuni was examined. While Oxyrase significantly enhanced the growth and the PCR signals of C. jejuni in pure culture, it appeared not to improve the PCR detection of C. jejuni in naturally contaminated chickens.
Insights
A new sample preparation method significantly enhances PCR detection of Campylobacter jejuni in chicken. This buoyant density centrifugation technique improves sensitivity and accuracy for identifying this foodborne pathogen.
Area of Science:
- Food Microbiology
- Molecular Biology
- Diagnostic Techniques
Background:
- Polymerase Chain Reaction (PCR) is crucial for detecting foodborne pathogens.
- Food matrices and enrichment media can inhibit PCR efficiency, reducing detection capacity.
- Campylobacter jejuni is a common cause of foodborne illness, necessitating reliable detection methods.
Purpose of the Study:
- To evaluate a simple sample preparation method using buoyant density centrifugation for improved PCR detection of Campylobacter jejuni.
- To assess the impact of this method on PCR sensitivity in chicken rinse samples.
- To investigate the effect of Oxyrase on Campylobacter jejuni growth and PCR detection.
Main Methods:
- Buoyant density centrifugation using Percoll gradient medium was employed for sample preparation.
- Chicken rinse samples were spiked with known levels of Campylobacter jejuni.
- PCR sensitivity was compared between treated and untreated samples.
- The influence of Oxyrase on bacterial growth and PCR detection was analyzed.
Main Results:
- The buoyant density centrifugation method increased PCR sensitivity by 10-100 times compared to untreated samples.
- Detection of C. jejuni in naturally contaminated chicken samples improved from 14 to 26 positives using the new method.
- Oxyrase enhanced C. jejuni growth and PCR signals in pure culture but did not improve detection in naturally contaminated samples.
Conclusions:
- Buoyant density centrifugation is an effective sample preparation technique for enhancing PCR-based detection of Campylobacter jejuni in chicken.
- This method significantly improves the ability to detect low levels of C. jejuni in complex food matrices.
- Further investigation is needed to understand Oxyrase's limited benefit in naturally contaminated samples.