Department of Bacteriology, Faculty of Medical Sciences, Kyushu University, Higashi-ku, Fukuoka 812-8582, Japan.
Anal Biochem. 2011 Nov 1;418(1):37-43. doi: 10.1016/j.ab.2011.06.027. Epub 2011 Jun 29.
The polymerase chain reaction (PCR) can confirm the presence of bacteria, but it is unable to differentiate between live and dead bacteria. Although ethidium monoazide (EMA)- and propidium monoazide (PMA)-based PCR have been evaluated, a quantity of ≥ 10(3)cells/ml dead cells produces a false-positive reading at 40 to 50 cycles (K. Rudi et al., Appl. Environ. Microbiol. 71 (2005) 1018-1024). After confirming the precision of real-time PCR of a long DNA target (16S or 23S ribosomal RNA [rRNA] gene, 1490 or 2840 bp), we evaluated the degree of suppression of an EMA treatment on the 16S/23S PCR using various amplification lengths (110-2840 bp) with heat-killed cells of Enterobacteriaceae (e.g., Salmonella enteritidis). We found that the inhibition rate was proportional to the PCR amplification length; short DNA (110 bp) amplification slightly delayed the threshold cycle (C(T)) of heat-killed cells of Enterobacteriaceae when compared with no EMA treatment. Regardless of the amplification length, the C(T) delay using live cells of Enterobacteriaceae with EMA was negligible. Thus, our real-time PCR of a long DNA (16S or 23S) template following EMA treatment is a rapid viable bacterial assay, which can potentially target all genera, for testing pasteurized milk that may have originally been contaminated with high levels of dead bacteria.
聚合酶链反应（PCR）可以确认细菌的存在，但无法区分活细菌和死细菌。尽管已经评估了基于吖啶橙（EMA）和吖啶丙啶（PMA）的 PCR，但在 40 到 50 个循环时，数量≥10(3)个细胞/ml 的死细胞会产生假阳性读数（K. Rudi 等人，应用环境微生物学。71 (2005) 1018-1024）。在确认实时 PCR 对长 DNA 靶标（16S 或 23S 核糖体 RNA [rRNA] 基因，1490 或 2840 bp）的精度后，我们评估了 EMA 处理对 16S/23S PCR 的抑制程度，使用不同的扩增长度（110-2840 bp）对肠杆菌科（例如，肠炎沙门氏菌）的热失活细胞进行 EMA 处理。我们发现抑制率与 PCR 扩增长度成正比；与未经 EMA 处理相比，短 DNA（110 bp）扩增略微延迟了肠杆菌科热失活细胞的阈值循环（C(T)）。无论扩增长度如何，使用 EMA 处理的肠杆菌科活细胞的 C(T)延迟都可以忽略不计。因此，我们对 EMA 处理后的长 DNA（16S 或 23S）模板进行实时 PCR 是一种快速的活菌检测方法，该方法可能适用于所有属，用于检测可能最初受到高水平死菌污染的巴氏杀菌牛奶。
