A comparative study of the ability of EMA and PMA to distinguish viable from heat killed mixed bacterial flora from fish fillets.
Ethidium bromide monoazide (EMA) and propidium monoazide (PMA) were utilized to selectively allow real-time PCR (Rti-PCR) amplification of target DNA from viable but not heat killed cells from the mixed bacterial flora derived from cod fillets. A linear range of DNA amplification was exhibited from 3.2x10(2) to 1.0x10(5) genomic targets per Rti-PCR. Following the heat treatment of cell suspensions the surviving populations with the EMA and PMA Rti-PCR method were evaluated by comparison with plate counts and MPN assays following different heat exposures (45 to 95 degrees C) for 5 min. The percent of erroneous survival with PMA Rti-PCR was higher than with EMA treatment. Cellular leakage was examined by following the extracellular increase of 260 and 280 nm absorbing materials. Initial leakage of protein and nucleic acids occurred at 50 degrees C, the maximal amount of leakage occurred at 70 degrees C.
Lee
Jung-Lim
JL
Department of Food Science, Massachusetts Agricultural Experiment Station University of Massachusetts, Amherst, MA 01003, USA.
Levin
Robert E
RE
D000818:Animals; D001386:Azides / Q000378:metabolism*; D001419:Bacteria / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification* / Q000378:metabolism; D015169:Colony Count, Microbial / Q000379:methods; D004996:Ethidium / Q000378:metabolism*; D048388:Gadiformes / Q000382:microbiology*; D006358:Hot Temperature; D008460:Meat / Q000382:microbiology; D050296:Microbial Viability; D016133:Polymerase Chain Reaction / Q000379:methods; D011419:Propidium / Q000031:analogs & derivatives* / Q000378:metabolism
D001386:Azides; C533957:propidium monoazide; D011419:Propidium; D004996:Ethidium