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Title: HEAT-TREATED CAMPYLOBACTER SPP. AND MRNA STABILITY AS DETERMINED BY REVERSE-TRANSCRIPTASE POLYMERASE CHAIN REACTION (RT-PCR)

Author
item SUNG, KIDON - NCTR FDA
item Hiett, Kelli
item Stern, Norman

Submitted to: Foodborne Pathogens and Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/8/2004
Publication Date: 7/10/2005
Citation: Sung, K., Hiett, K.L., Stern, N.J. 2005. Heat-treated campylobacter spp. and mrna stability as determined by reverse-transcriptase polymerase chain reaction (rt-pcr). Foodborne Pathogens and Disease. 2:130-137.

Interpretive Summary: The Centers for Disease Control and Prevention estimates that Campylobacter enteritis is a multi billion dollar disease and that the consumption of poultry is a primary source for clinical infections in humans. Therefore, the development and application of new technologies for the detection of Campylobacter in poultry flocks is essential for the development of intervention strategies and the reduction of Campylobacter in poultry for human consumption. Additionally, a method that distinguishes between viable and non-viable cells would be of great benefit. We investigated four targets for use in a reverse-transcriptase PCR (RT-PCR) detection assay. The technique of RT-PCR specifically targets mRNA which is generally a good indicator of cell viability. One goal of the investigation was to determine if a distinction could be made between viable Campylobacter cells and Campylobacter cells made non-viable by heat treatment. Our results demonstrated that the RT-PCR technique was highly dependent upon the individual Campylobacter strains tested, the temperature of heat-treatments coupled with the duration of holding times, and the transcript targeted. Further optimization is needed for application of RT-PCR in real-world settings.

Technical Abstract: The detection method of reverse-transcriptase polymerase chain reaction (RT-PCR), which specifically targets mRNA, was developed and tested for detection of viable Campylobacter spp. The expression of four DNA targets, flaA, tkt, porA and a putative haem-copper oxidase domain, were assayed in heat-inactivated Campylobacter spp. to determine an optimum target for RT-PCR amplification. A diversity of Campylobacter spp. were tested, however, the presented RT-PCR technique was specific for C. jejuni, C. coli, and C. lari. The durability of mRNA species detected by our RT-PCR technique was dependent upon the individual Campylobacter spp. examined, the condition of heat treatment and post-treatment holding time, as well as the transcript targeted. The putative oxidase was determined to be the most stable mRNA species for this assay. The mRNA of the putative oxidase gene was detectable even after Campylobacter spp. had been treated at temperatures of 95 to 99°C. Using DNA-based PCR, the four DNA targets could be amplified after heat inactivation followed by a 48 h holding time, indicating that the chromosomal DNA was not substantially degraded by the heat treatment. PCR products from the putative oxidase gene were detected at 102 to 103 C. jejuni CFU per ml, exhibiting the highest level of sensitivity among the genes tested. This study suggests that while RT-PCR is generally a good technique to distinguish between viable and nonviable cells, targets should be carefully characterized when used to determine cell viability of Campylobacter spp.