Use of ethidium monoazide and PCR in combination for quantification of viable and dead cells in complex samples.
The distinction between viable and dead cells is a major issue in many aspects of biological research. The current technologies for determining viable versus dead cells cannot readily be used for quantitative differentiation of specific cells in mixed populations. This is a serious limitation. We have solved this problem by developing a new concept with the viable/dead stain ethidium monoazide (EMA) in combination with real-time PCR (EMA-PCR). A dynamic range of approximately 4 log(10) was obtained for the EMA-PCR viable/dead assay. Viable/dead differentiation is obtained by covalent binding of EMA to DNA in dead cells by photoactivation. EMA penetrates only dead cells with compromised membrane/cell wall systems. DNA covalently bound to EMA cannot be PCR amplified. Thus, only DNA from viable cells can be detected. We evaluated EMA-PCR with the major food-borne bacterium Campylobacter jejuni as an example. Traditional diagnosis of this bacterium is very difficult due to its specific growth requirements and because it may enter a state where it is viable but not cultivable. The conditions analyzed included detection in mixed and natural samples, survival in food, and survival after disinfection or antibiotic treatment. We obtained reliable viable/dead quantifications for all conditions tested. Comparison with standard fluorescence-based viable/dead techniques showed that the EMA-PCR has a broader dynamic range and enables quantification in mixed and complex samples. In conclusion, EMA-PCR offers a novel real-time PCR method for quantitative distinction between viable and dead cells with potentially very wide application.
Rudi
Knut
K
Matforsk, Norwegian Institute for Food Research, Osloveien 1, 1430 As, Norway. knut.rudi@matforsk.no
Moen
Birgitte
B
Drømtorp
Signe Marie
SM
Holck
Askild L
AL
D000345:Affinity Labels; D000818:Animals; D001386:Azides; D002169:Campylobacter Infections / Q000382:microbiology / Q000662:veterinary; D016123:Campylobacter jejuni / Q000254:growth & development* / Q000302:isolation & purification*; D002645:Chickens / Q000382:microbiology*; D015169:Colony Count, Microbial; D004269:DNA, Bacterial / Q000032:analysis; D008461:Meat Products / Q000382:microbiology; D016133:Polymerase Chain Reaction / Q000379:methods*; D011201:Poultry Diseases / Q000382:microbiology*
D000345:Affinity Labels; D001386:Azides; D004269:DNA, Bacterial; C014535:8-azidoethidium