Influence of growth in a food medium on the detection of Escherichia coli O157:H7 by polymerase chain reaction.
The effects of storage time and growth in broth culture and in a food medium on the efficiency of Escherichia coli O157: H7 DNA extraction and on the sensitivity of polymerase chain reaction (PCR) detection of E. coli O157:H7 were investigated. Detection limits were evaluated with dilution series PCR targeting the slt-II gene. The relationship between cell density and DNA yield was generally log-linear for pure cultures of E coli O157:H7. When the bacteria were suspended in skim milk at a density of 10(6) CFU/ml. held at 4 degrees C, and sampled at 24-h intervals, cell density, total DNA yield, and PCR detection limits remained stable throughout the 96-h storage period. However, when E coli O157:H7 was grown in skim milk to a final cell density of 10(6) CFU/ml, PCR amplification efficiency was drastically reduced, although overall DNA yields from these samples were consistent with those for the samples in which E. coli O157:H7 growth was static over 96 h of storage at 4 degrees C. This result is most likely due to poor DNA purity, which was consistently observed when DNA was extracted from food matrices in which the pathogen was grown rather than stored. The results of this investigation underscore the likelihood that multiple components may drastically affect DNA extraction and PCR amplification efficiency in the detection of pathogens in the food matrix. It is clear that before nucleic acid amplification technologies are widely applied to food systems, it would be prudent to test their efficacy in multiple food matrices and under conditions in which the bacterial population is both static and actively growing.
McKillip
John L
JL
School of Biological Sciences, Louisiana Tech University, Ruston, USA.
Jaykus
Lee-Ann
LA
Drake
MaryAnne
M
D015169:Colony Count, Microbial; D003470:Culture Media; D004269:DNA, Bacterial / Q000032:analysis*; D019453:Escherichia coli O157 / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification*; D005506:Food Contamination / Q000032:analysis*; D005516:Food Microbiology; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013997:Time Factors
D003470:Culture Media; D004269:DNA, Bacterial