Rapid detection of viable bacteria by nested polymerase chain reaction via long DNA amplification after ethidium monoazide treatment

被引:22
作者
Soejima, Takashi [1 ,2 ]
Schlitt-Dittrich, Frank [1 ]
Yoshida, Shin-ichi [2 ]
机构
[1] Morinaga Milk Ind Co, Biol Funct Res Dept, Food Sci & Technol Inst, Kanagawa 2528583, Japan
[2] Kyushu Univ, Dept Bacteriol, Fac Med Sci, Higashi Ku, Fukuoka 8128582, Japan
关键词
EMA; PMA; Real-time PCR; Pasteurized milk; Cells of Enterobacteriaceae; E; sakazakii; Nested PCR; DEAD BACTERIA; PCR; QUANTIFICATION; SAMPLES; CELLS; LIVE;
D O I
10.1016/j.ab.2011.06.033
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
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. (C) 2011 Elsevier Inc. All rights reserved.
引用
收藏
页码:286 / 294
页数:9
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