Preliminary development and validation of a real-time reverse transcription PCR assay for the semi-quantification of viable Campylobacter jejuni in water samples.
A TaqMan-based real-time reverse transcription PCR (RT-PCR) assay was developed for semi-quantification of viable Campylobacter jejuni in water samples. This preliminary assay is based on measuring the heat-shock induction of groEL messenger RNA (mRNA); the logic being that only viable cells can synthesise new mRNA. A 128-bp C. jejuni groEL DNA fragment was cloned and used as a positive control standard in TaqMan runs. The assay could detect as few as 13 genome equivalent copies of groEL plasmid DNA and 1-2 colony forming unit (CFU) of viable C. jejuni. A multi-step concentration technique was developed for collecting C. jejuni from large volumes of water samples. An average recovery of 20% (range: 8-47%) was obtained at spiking levels of 750-6,500 CFU per 10-litre of river water. Starting from concentration, the enumeration of viable C. jejuni in river water samples was achieved in approximately 12 hours. Quantification of viable C. jejuni in natural river water samples by this method showed similar trends to a culture-based double enrichment most probable number (MPN) -PCR method. There is potential to apply this fast, specific and sensitive semi-quantitative method to monitor a range of water samples for viable C. jejuni.
Lin
S
S
Institute of Environmental Science and Research (ESR) Ltd., PO Box 29-181, Christchurch, New Zealand. susan.lin@esr.cri.nz
Gilpin
B
B
Scholes
P
P
Podivinsky
E
E
Klena
J
J
Savill
M
M
D016123:Campylobacter jejuni / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification* / Q000378:metabolism; D018834:Chaperonin 60 / Q000096:biosynthesis / Q000235:genetics*; D004269:DNA, Bacterial / Q000235:genetics; D010957:Plasmids; D012329:RNA, Bacterial / Q000096:biosynthesis / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D012015:Reference Standards; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D045483:Rivers / Q000382:microbiology*; D013997:Time Factors; D014871:Water Microbiology / Q000592:standards*
D018834:Chaperonin 60; D004269:DNA, Bacterial; D012329:RNA, Bacterial; D012333:RNA, Messenger