A reverse transcriptase polymerase chain reaction assay for the detection of thermophilic Campylobacter spp.
A novel method was developed for the detection of thermophilic enteropathogenic campylobacters based on the detection of mRNA using the reverse transcriptase polymerase chain reaction (RT-PCR). The RNA extraction method, DNase treatment and RT-PCR assay were shown to be specific for mRNA. The assay is specific for the thermophilic campylobacters Campylobacter jejuni, Campylobacter coli and Campylobacter upsaliensis and restriction fragment length polymorphism (RFLP) analysis of the 256 bp amplified product with the restriction endonucleases Alu I, Dde I and Dra I revealed distinct species specific patterns. The assay was applied to the detection of C. jejuni cells killed by heating at 72 degreesC for 5 min and mRNA was detected by RT-PCR immediately after heat killing but became undetectable within 4 h when the cells were held at 37 degreesC. The assay therefore can differentiate between viable and dead cells of C. jejuni.
Preston Public Health Laboratory, The Royal Preston Hospital, PO Box 202, Sharoe Green Lane, Fulwood, Preston, Lancashire, PR2 9HG, UK.
A D
AD
Sails
F J
FJ
Bolton
A J
AJ
Fox
D R
DR
Wareing
D L
DL
Greenway
D002167:Campylobacter / Q000235:genetics* / Q000302:isolation & purification*; D017000:Campylobacter coli / Q000235:genetics / Q000302:isolation & purification; D016123:Campylobacter jejuni / Q000235:genetics / Q000302:isolation & purification; D006358:Hot Temperature; D012150:Polymorphism, Restriction Fragment Length; D012329:RNA, Bacterial / Q000302:isolation & purification; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity
D012329:RNA, Bacterial