| 韩文超1, 许景升1, 黄宏坤2, 李志明1, 杨晓欣1, 徐 进1*, 冯 洁1*.PMAxx-qPCR定量检测梨火疫活菌方法的建立[J].植物保护,2025,51(4):290-297. |
| PMAxx-qPCR定量检测梨火疫活菌方法的建立 |
| Development of a PMAxx-qPCR method for quantitative detection of viable Erwinia amylovora |
| 投稿时间：2024-08-28 修订日期：2024-10-11 |
| DOI：10.16688/j.zwbh.2024448 |
| 中文关键词: 梨火疫病 PMAxx-qPCR检测技术 活菌检测 |
| 英文关键词:fire blight PMAxx-qPCR viable cell detection |
| 基金项目:国家重点研发计划（2022YFC2602204） |
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| 摘要点击次数: 65 |
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| 中文摘要: |
| 本研究将改良的叠氮溴化丙锭(PMAxx)与荧光实时定量PCR技术(qPCR)相结合, 开发出一种精准高效、灵敏特异的PMAxx-qPCR检测方法, 用于梨火疫病菌Erwinia amylovora活细胞的定量检测。通过控制变量的单因素变化试验对PMAxx预处理体系中的各项参数进行优化, 确立了PMAxx终浓度为15 μmol/L, 黑暗孵育时间为15 min, 曝光时间为8 min的PMAxx预处理体系, 可有效抑制1. 0×108 cfu/mL梨火疫死菌的DNA扩增, 对梨火疫活菌DNA的扩增无影响。本文开发的PMAxx-qPCR检测技术可于特定范围内有效地排除死菌干扰, 适用于菌液浓度在103~108 cfu/mL范围内的活菌数精准定量。为进一步深入探究梨火疫病的流行规律提供新的技术支持, 并有效防止在实际样本的PCR检测中出现假阳性的现象。 |
| 英文摘要: |
| In this study, an improved propidium monoazide dye (PMAxx) was combined with quantitative real-time PCR (qPCR) to establish a rapid, sensitive, and highly specific method (PMAxx-qPCR) for the quantitative detection of viable Erwinia amylovora cells, the causative agent of fire blight. Key parameters of the PMAxx treatment system were optimized through single-variable tests. The optimal conditions were determined as a final PMAxx concentration of 15 μmol/L, dark incubation for 15 min, and light exposure for 8 min. Under these conditions, amplification of inactivated cells at a concentration of 1.0×108 cfu/mL was effectively suppressed, while amplification of viable cells was not affected. The PMAxx-qPCR assay demonstrated reliable quantification of viable E. amylovora cells within the range of 103 to 108 cfu/mL, effectively eliminating interference from dead cells. This method provides robust technical support for epidemiological studies of fire blight and offers a solution to the issue of false positives in routine PCR diagnostics due to the presence of dead bacterial cells. |
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