| 于小龙1, 2，徐进2，张昊2，许景升2，黄雯2，胡晓梅2，冯洁2*，王学利1*.PMA-PCR方法快速检测VBNC状态青枯菌[J].植物保护,2016,42(1):144-149. |
| PMA-PCR方法快速检测VBNC状态青枯菌 |
| Rapid detection of viable but non-culturable (VBNC) Ralstonia solanacearum by PMA-PCR method |
| 投稿时间：2014-12-31 修订日期：2015-02-09 |
| DOI： |
| 中文关键词: 青枯菌 叠氮溴化丙锭 活的非可培养状态 |
| 英文关键词:Ralstonia solanacerum propidum monoazide viable but non-culturable |
| 基金项目:公益性行业(农业)科研专项(201303015); 国家自然科学基金(31272008, 31371908); 天津市农业科技成果转化与推广项目(201002250) |
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| 中文摘要: |
| 青枯菌为应对逆境胁迫, 可进入活的但非可培养状态(viable but non-culturable, VBNC)。本文利用叠氮溴化丙锭(PMA)与PCR技术相结合, 建立了一种快速有效区分青枯菌死活细胞的分子检测方法。基于hrcS基因序列, 设计了一对青枯菌种特异性检测引物hrcSf/hrcSr; 利用PMA对青枯菌Po82菌株的细胞悬浮液样品进行预处理, 随后进行常规 PCR扩增。结果表明, 当样品中PMA质量浓度为3μg/mL、曝光时间大于5 min时, PMA可有效抑制死亡菌体细胞中的DNA扩增; 且对可培养和VBNC状态细胞中的DNA扩增没有影响; 本试验建立的PMA-PCR方法能有效对包括VBNC状态在内的青枯菌活菌进行检测, 避免了假阳性与假阴性结果的产生。 |
| 英文摘要: |
| Ralstonia solanacearum can enter into the VBNC state (viable but non-culturable) in response to adverse environmental conditions. A novel method to differentiate viable/dead cells of R. solanacearum was established by using a DNA dye of propidium monoazide (PMA) in combination with the polymerase chain reaction (PMA-PCR). On the basis of hrcS gene sequence, a species-specific primer set hrcSf/hrcSr was designed for detection of R. solanacearum. Samples were pre-treated with PMA which bound DNA from dead cells so that only viable cells can be amplified by PCR. A final concentration of 3 μg/mL PMA and 5-min exposure time was found to completely inhibit PCR amplification from DNA of dead cells, and had no inhibition effect on viable and viable but non-culturable (VBNC) cells. The PMA-PCR method established in this work can detect viable and viable but non-culturable (VBNC) cells and avoid false positive and false negation result of R. solanacerum. |
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