McKillip John L, Jaykus Lee-Ann, Drake MaryAnne
School of Biological Sciences, Louisiana Tech University, Ruston, USA.
J Food Prot. 2002 Nov;65(11):1775-9. doi: 10.4315/0362-028x-65.11.1775.
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.
研究了储存时间以及在肉汤培养基和食品培养基中生长对大肠杆菌O157:H7 DNA提取效率和聚合酶链反应（PCR）检测大肠杆菌O157:H7敏感性的影响。通过针对slt-II基因的稀释系列PCR评估检测限。对于大肠杆菌O157:H7的纯培养物，细胞密度与DNA产量之间的关系通常呈对数线性。当细菌以10(6) CFU/ml的密度悬浮于脱脂乳中，在4℃下保存，并每隔24小时取样时，在整个96小时的储存期内，细胞密度、总DNA产量和PCR检测限均保持稳定。然而，当大肠杆菌O157:H7在脱脂乳中生长至最终细胞密度为10(6) CFU/ml时，尽管这些样品的总DNA产量与大肠杆菌O157:H7在4℃下储存96小时生长静止的样品一致，但PCR扩增效率却大幅降低。这一结果很可能是由于DNA纯度不佳，从病原体生长而非储存的食品基质中提取DNA时一直观察到这种情况。本研究结果强调了在食品基质中检测病原体时，多种成分可能会严重影响DNA提取和PCR扩增效率的可能性。显然，在核酸扩增技术广泛应用于食品系统之前，谨慎的做法是在多种食品基质以及细菌群体既静止又活跃生长的条件下测试其功效。
