| RRC ID |
34502
|
| 著者 |
Soejima T, Minami J, Yaeshima T, Iwatsuki K.
|
| タイトル |
An advanced PCR method for the specific detection of viable total coliform bacteria in pasteurized milk.
|
| ジャーナル |
Appl Microbiol Biotechnol
|
| Abstract |
Pasteurized milk is a complex food that contains various inhibitors of polymerase chain reaction (PCR) and may contain a large number of dead bacteria, depending on the milking conditions and environment. Ethidium monoazide bromide (EMA)-PCR is occasionally used to distinguish between viable and dead bacteria in foods other than pasteurized milk. EMA is a DNA-intercalating dye that selectively permeates the compromised cell membranes of dead bacteria and cleaves DNA. Usually, EMA-PCR techniques reduce the detection of dead bacteria by up to 3.5 logs compared with techniques that do not use EMA. However, this difference may still be insufficient to suppress the amplification of DNA from dead Gram-negative bacteria (e.g., total coliform bacteria) if they are present in pasteurized milk in large numbers. Thus, false positives may result. We developed a new method that uses real-time PCR targeting of a long DNA template (16S-23S rRNA gene, principally 2,451 bp) following EMA treatment to completely suppress the amplification of DNA of up to 7 logs (10(7) cells) of dead total coliforms. Furthermore, we found that a low dose of proteinase K (25 U/ml) removed PCR inhibitors and simultaneously increased the signal from viable coliform bacteria. In conclusion, our simple protocol specifically detects viable total coliforms in pasteurized milk at an initial count of ≥1 colony forming unit (CFU)/2.22 ml within 7.5 h of total testing time. This detection limit for viable cells complies with the requirements for the analysis of total coliforms in pasteurized milk set by the Japanese Sanitation Act (which specifies <1 CFU/2.22 ml).
|
| 巻・号 |
95(2)
|
| ページ |
485-97
|
| 公開日 |
2012-7-1
|
| DOI |
10.1007/s00253-012-4086-0
|
| PMID |
22644523
|
| MeSH |
Animals
Azides / metabolism
Colony Count, Microbial / methods*
DNA, Bacterial / genetics
DNA, Ribosomal / genetics
Enterobacteriaceae / genetics
Enterobacteriaceae / isolation & purification*
Enzyme Inhibitors / metabolism
Ethidium / metabolism
Microbial Viability
Milk / microbiology*
Polymerase Chain Reaction / methods*
Sensitivity and Specificity
|
| IF |
3.53
|
| 引用数 |
16
|
|
WOS 分野
|
BIOTECHNOLOGY & APPLIED MICROBIOLOGY
|
| リソース情報 |
| 一般微生物 |
JCM 1236
JCM 1242
JCM 1658
JCM 1666
JCM 1667
JCM 1685
JCM 2152
JCM 2403
JCM 21070 |
