McKillip J L, Jaykus L A, Drake M
Department of Food Science and Technology, Southeast Dairy Foods Research Center, Mississippi State University, Mississippi 39762, USA.
J Food Prot. 1999 Aug;62(8):839-44. doi: 10.4315/0362-028x-62.8.839.
Polymerase chain reaction (PCR) and reverse transcriptase (RT)-PCR using primers targeting 16S rRNA sequences in Escherichia coli O157:H7 were applied to monitor the stability of rDNA and rRNA in cells killed by mild heat treatment (60 degrees C) in skim milk. Serial dilutions of purified RNA and DNA from E. coli O157:H7 in skim milk were amplified by RT-PCR or PCR, respectively, before heat treatment and at time points 0, 6, 12, 24, and 48 h after heating. In general, DNA-PCR provided stronger amplification signals compared to RT-PCR at the corresponding time points with the same PCR primer set, indicating a lower efficiency of RNA amplification compared to that of DNA. Ribosomal RNA and rDNA could be amplified by RT-PCR or PCR from both viable and dead cells throughout the 48-h posttreatment holding period. For RT-PCR, amplification signals decreased in intensity with increased holding time, while the efficiency of amplification of DNA sequences from dead cells remained fairly stable throughout the study. DNA persistence was greater than that of rRNA following cell death by mild heat treatment in skim milk. Skim milk did not appear to accelerate nucleic acid degradation. While rRNA was less stable than DNA, its detection by RT-PCR may not be appropriate as an exclusive indicator of cell viability in minimally processed foods.
使用靶向大肠杆菌O157:H7中16S rRNA序列的引物进行聚合酶链反应（PCR）和逆转录酶（RT）-PCR，以监测脱脂乳中经温和热处理（60℃）杀死的细胞中rDNA和rRNA的稳定性。在热处理前以及加热后0、6、12、24和48小时的时间点，分别通过RT-PCR或PCR对脱脂乳中大肠杆菌O157:H7的纯化RNA和DNA的系列稀释液进行扩增。一般来说，在相同PCR引物组的相应时间点，与RT-PCR相比，DNA-PCR提供更强的扩增信号，表明RNA扩增效率低于DNA。在整个48小时的处理后保存期内，核糖体RNA和rDNA均可通过RT-PCR或PCR从活细胞和死细胞中扩增出来。对于RT-PCR，扩增信号强度随保存时间延长而降低，而在整个研究过程中，死细胞DNA序列的扩增效率保持相当稳定。在脱脂乳中经温和热处理导致细胞死亡后，DNA的持久性大于rRNA。脱脂乳似乎不会加速核酸降解。虽然rRNA比DNA更不稳定，但通过RT-PCR检测rRNA可能不适宜作为最低限度加工食品中细胞活力的唯一指标。
