EVALUATION OF PCR AND DNA HYBRIDIZATION PROTOCOLS FOR DETECTION OF VIABLE ENTEROTOXIGENIC CLOSTRIDIUM PERFRINGENS IN IRRADIATED BEEF

Abstract

The sensitivity of DNA hybridization and polymerase chain reaction (PCR), was evaluated in irradiated cooked and raw beef samples. A membrane‐based colony hybridization assay and a PCR protocol, both with specificity for the enterotoxin A gene of Clostridium perfringens, were compared with viable plate counts. The results of the colony hybridization procedure were in agreement with viable plate counts for detection and enumeration of enterotoxigenic C. perfringens. The PCR procedure combined a 4 h enrichment followed by a nucleic acid extraction step and assessed the amplification of 183 and 750 base pair enterotoxin gene targets. Detection of C. perfringens by PCR did not show a reliable correlation with viable plate counts or the colony hybridization assay. C. perfringens killed by irradiation were not detected by the plate count or colony hybridization methods; however, killed cells were detected with the PCR technique. By relying on the growth of viable cells for detection and/or enumeration, the colony hybridization and plate count methods provided a direct correlation with the presence of viable bacteria.; Accepted for Publication August 6, 1997

Sensitive detection of viable Listeria monocytogenes by reverse transcription-PCR

“…Although a short enrichment incubation (ca. 2 h) was necessary to increase the sensitivity of the RT-PCR assay, our results and those of others (1,34) indicate that nonviable cells would be detected following this short enrichment period if DNA-based PCR detection systems were used. Thus, the present assay incorporating RT-PCR technology for the detection of bacterial mRNA would yield fewer false-positive results.…”

“…Many of these rapid methods are based on PCR amplification of specific L. monocytogenes gene sequences combined with ethidium bromide staining of agarose gels (11,17,23,25,33,38), Southern (21,46) or dot blot (4, 7) hybridization with labeled probes, or, more recently, with a fluorogenic probe incorporated into the PCR assay itself (Taq-Man PCR) (3). Although these PCR-based detection methods have enhanced the sensitivity and specificity for rapid identification of L. monocytogenes, they can yield false-positive results due to the amplification of DNA present in nonviable cells (1,34). It has been demonstrated that cells of Escherichia coli and L. monocytogenes that have been rendered nonviable, as determined by viable plate counts, by starvation, desiccation, or heat are still detectable by gene probe-PCR techniques, indicating the lack of a clear relationship between the loss of viability and detectability by PCR (34) (Fig.…”

“…In addition, Baez et al (1) observed false-positive PCR results when assessing the presence of Clostridium perfringens in beef following irradiation. Hence, under many circumstances which render bacteria nonviable, cellular DNA may persist for hours or even days, suggesting that detection of pathogens in contaminated foods by PCR may require additional evidence of cell viability before risk can be assessed (1,34).…”

“…Similarly, PCR amplification techniques show a high degree of specificity and have the added advantage of extreme sensitivity (for a review, see reference 44). However, a disadvantage of conventional PCR techniques is that both viable and nonviable cells may be detected (1,34). This is overcome in many cases by inclusion of an enrichment step to dilute out any nonviable cells that may be present (23,38,40).…”