A reverse transcriptase-polymerase chain reaction assay for detection of viable Campylobacter spp.
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The study focused on the development and application of a reverse transcriptase-PCR (RT-PCR), which specifically detects mRNA, for the detection of viable Campylobacter. 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 heattreated 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 The main goal of the second study was to determine whether the ability of coccoid and VBNC cells of Campylobacter spp. were infectious after passage through day-old chicks. Further, this study was conducted to confirm the non-recovery of heat-killed Campylobacter spp. correlated with mRNA message detection through chicken challenge. The levels of four Campylobacter spp., previously isolated from poultry feces, declined progressively over time and loss of culturability occurred after 6 to 7 weeks incubation in phosphate buffed saline (PBS) at 4oC. Cold-stored, nonculturable and heat-inactivated (60oC for 10 min) Campylobacter spp. produced inconsistent amplified products from RT-PCR assay depending on the target genes and strains used, though all fresh cultures showed mRNA signals. Mostly, signals of mRNA species from viable but nonculturable (VBNC) and heat-killed Campylobacter spp. AH-1, AH-2 and CH-3 persisted. RT-PCR amplification of tkt, porA, and a 256 bp amplicon from a previously described putative haem-copper oxidase provided consistent signal while flaA did not. Presumed VBNC and heat-inactivated Campylobacter spp., which produced positive mRNA signal but was not culturable by conventional culture-based methods, did not establish colonization in the intestine of chicks seven days after challenge. 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. Furthermore, the results question the correlationship between mRNA durability as assayed by RT-PCR and cell viability, as well as the significance of the VBNC cells in environmental transmission of Campylobacter spp.