Department of Bacteriology, Faculty of Medical Sciences, Kyushu University, Higashi-ku, Fukuoka 812-8582, Japan.
Anal Biochem. 2011 Nov 15;418(2):286-94. doi: 10.1016/j.ab.2011.06.033. Epub 2011 Jun 30.
In assays to determine whether viable cells of Enterobacteriaceae are present in pasteurized milk, the typical ethidium monoazide (EMA) polymerase chain reaction (PCR) targets a short stretch of DNA. This process often triggers false-positive results owing to the high level of dead cells of Enterobacteriaceae that had initially contaminated the sample. We have developed a novel, direct, real-time PCR that does not require DNA isolation (DQ-PCR) to detect low levels of cells of Enterobacteriaceae regardless of live and dead cells first. We confirmed that the DQ-PCR targeting a long DNA (the 16S ribosomal RNA [rRNA] gene, amplified length of 1514 bp) following EMA treatment is a promising tool to detect live bacteria of all genera owing to the complete suppression of background signal from high levels of dead bacteria in pasteurized milk. However, when identifying viable bacteria in pasteurized milk, commercial PCR primers designed for detecting long stretches of DNA are generally not available. Thus, we treated samples with EMA and then carried out an initial round of PCR of a long stretch of DNA (16S gene, 1514 bp). We then performed another round of PCR, a novel nested PCR to generate short products using commercial primers. This procedure resulted in the rapid detection of low levels of viable cells of Enterobacteriaceae.
在检测巴氏杀菌奶中是否存在肠杆菌科活细胞的测定中，典型的吖啶单（EMA）聚合酶链反应（PCR）针对一小段 DNA。由于最初污染样品的肠杆菌科死细胞水平较高，该过程经常会导致假阳性结果。我们开发了一种新颖的、直接的、实时 PCR，不需要 DNA 分离（DQ-PCR），无论初始是活细胞还是死细胞，都可以检测到低水平的肠杆菌科细胞。我们证实，在 EMA 处理后靶向长 DNA（16S 核糖体 RNA [rRNA] 基因，扩增长度为 1514bp）的 DQ-PCR 是一种有前途的工具，可以检测所有属的活细菌，因为它完全抑制了巴氏杀菌奶中高水平死细菌的背景信号。然而，在鉴定巴氏杀菌奶中的活菌时，通常无法使用用于检测长片段 DNA 的商业 PCR 引物。因此，我们用 EMA 处理样品，然后进行长片段 DNA（16S 基因，1514bp）的初始一轮 PCR。然后，我们进行了另一轮 PCR，即使用商业引物生成短产物的新型嵌套 PCR。该程序可快速检测到低水平的肠杆菌科活细胞。
