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United States Department of Agriculture

Agricultural Research Service

Research Project: MOLECULAR CHARACTERIZATION OF PATHOGENS AND THEIR RESPONSES TO ENVIRONMENTAL FACTORS

Location: Molecular Characterization of Foodborne Pathogens

Title: Detection of live/dead Campylobacter jejuni cells by real-time PCR

Authors
item Chen, Chin-Yi
item He, Yiping

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: October 10, 2006
Publication Date: October 13, 2006
Citation: Chen, C., He, Y. 2006. Detection of live/dead Campylobacter jejuni cells by real-time PCR. Abstract. p.3-1.

Technical Abstract: Campylobacter is the leading cause of food-borne illness and is frequently associated with undercooked chicken. Campylobacters are difficult to isolate and cultivate in the laboratory due to their preference for oxygen levels below that of the air. With the high prevalence of Campylobacter in foods, sensitive detection methods such as conventional PCR that detects both live and dead bacteria may result in a high number of samples that test positive but that only contain dead bacteria. We evaluated PCR-methods methods that are able to quantitatively differentiate live from dead Campylobacter cells, which can be used for detection, as well as for physiological studies of Campylobacter. Two PCR primers and a TaqMan probe were designed for a real-time PCR assay using the luxS gene as a target for detection of Campylobacter in the presence or absence of the chemical ethidium monoazide (EMA). EMA enters cells with damaged membranes and binds to DNA preventing amplification of DNA from dead cells. Campylobacter were subjected to different treatments, and then the DNA was extracted, and PCR was performed with and without EMA. Bacteria collected from various growth stages or after long-term storage (1 to 4 weeks) under different conditions were also tested. The real-time PCR results were compared to two other methods: plate counting and examination of cells stained by a commercial bacterial viability staining kit by confocal microscopy. The three different methods showed differences in their ability to identify/classify live and dead Campylobacter cells, particularly under long-term storage conditions. The plate counting method was the least sensitive of all, showing no counts under most adverse conditions. Thus caution needs to be taken when conclusions are drawn from only from one method.

Last Modified: 7/30/2014
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