Heat-treated Campylobacter spp. and mRNA stability as determined by reverse transcriptase-polymerase chain reaction.
The detection method of reverse transcriptase-polymerase chain reaction (RT-PCR), which specifically targets mRNA, was developed and tested for detection of cultivable 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. was 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 95-99 degrees 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 10(2) to 10(3) C. jejuni CFU per mL, exhibiting the highest level of sensitivity among the genes tested. The results in the present study indicate that mRNA from Campylobacter spp. may persist in a form that is detectable by RT-PCR amplification for an extended period after heat treatment, demonstrating a poor correlation between mRNA detection and cell cultivability. With these results in mind, further investigations are necessary to determine the correlation between RT-PCR amplification and viability.
Sung
Kidon
K
Department of Food Science and Technology, University of Georgia, Athens, GA, USA.
Hiett
Kelli L
KL
Stern
Norman J
NJ
D001426:Bacterial Proteins; D002167:Campylobacter / Q000235:genetics* / Q000254:growth & development / Q000302:isolation & purification; D002470:Cell Survival; D005408:Flagellin / Q000032:analysis; D005506:Food Contamination / Q000032:analysis; D005784:Gene Amplification; D005798:Genes, Bacterial; D006358:Hot Temperature; D012329:RNA, Bacterial / Q000032:analysis; D012333:RNA, Messenger / Q000032:analysis*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013045:Species Specificity
D001426:Bacterial Proteins; D012329:RNA, Bacterial; D012333:RNA, Messenger; D005408:Flagellin; C067138:flaA protein, bacteria