|Sung, K - UGA|
Submitted to: Molecular and Cellular Probes
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: December 8, 2004
Publication Date: N/A
Interpretive Summary: Campylobacter, the most frequent cause of human bacterial diarrhea in the United States, is commonly associated with poultry. To better understand the sources involved in Campylobacter contamination of poultry we developed a molecular method, reverse transcriptase-polymerase chain reaction (RT-PCR), which specifically detects messenger RNA, for the detection of live Campylobacter. The relationship between time and temperature to destroy the mRNA signal of four selected genes was determined by RT-PCR. We observed that the durability of the four mRNA species depended significantly on the individual Campylobacter strain, the condition of heat treatment, post-treatment holding time, and the message targeted. This study suggested that while RT-PCR is generally a good technique to distinguish between live and dead cells, the assay is not useful for Campylobacter spp.
Technical Abstract: Polymerase chain reaction (PCR) is a rapid, specific, and sensitive technique for detecting pathogenic bacteria. However, the technique cannot distinguish between viable and nonviable cells. Bacterial mRNA has been suggested as an indicator for cell viability as it expresses a component of ongoing metabolic activity. Based on this suggestion, our study focused on the development and application of a reverse transcriptase-PCR (RT-PCR), which specifically detects mRNA, for the detection of viable Campylobacter. The expression of four genes, flaA, tkt, porA and a 256 bp amplicon of a putative haem-copper oxidase domain downstream from a previously uncharacterized C. jejuni two-component regulator, in heat-inactivated Campylobacter spp. was explored to determine an optimum target for RT-PCR amplification. Furthermore, the relationship between time and temperature to ablate the mRNA signal was determined by RT-PCR. mRNA half-life was assessed according to post-heating holding time at 37oC. An amplification sensitivity of Campylobacter spp. was estimated by PCR. A diversity of Campylobacter spp. was tested, however, our RT-PCR technique was specific for C. jejuni, C. coli, and C. lari. Messenger RNA from the four genes was detected for varying intervals after the cells had been killed by heat, but gradually the message disappeared when heat-treated cells were held at 37oC. We observed that the durability of mRNA species detected by our RT-PCR technique depends significantly on the individual Campylobacter strain tested, the condition of heat treatment and post-treatment holding time, and the transcript targeted. The intensity of the RT-PCR products decreased as the temperature of heat treatment increased and as the subsequent holding time extended. The 256 bp amplicon was determined to be the most stable mRNA species tested. mRNA of the 256 bp amplicon was detectable even after Campylobacter spp. had been killed at temperatures of 95 to 99oC. Using DNA-based PCR, the four genes could be amplified after 48 h holding time after each heat inactivation, indicating that the chromosomal DNA was minimally influenced by the heat treatment. PCR products from the 256 bp amplicon 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, the assay does not appear to be useful for Campylobacter spp.