耐热菌的竞争定量PCR检测方法优化与建立

 【目的】研究苹果浓缩汁中耐热菌（Alicyclobacillus acidoterrestris）的定量PCR快速检测法。【方法】通过引物设计、PCR扩增及凝胶纯化试剂盒回收，构建并获得耐热菌的PCR竞争模板；用获得的竞争模板作为定量内标物建立耐热菌的竞争定量PCR（QC-PCR）检测体系。【结果】经对建立的QC-PCR检测体系优化，目标模板检测灵敏度由5×104个分子/PCR 体系，提高到50个分子/PCR 体系，竞争模板和目标模板分子共扩增可检测到5×102个目标模板分子/PCR 体系，并能从人工回添苹果浓缩汁样品中定量检测到5×103 cfu/ PCR体系的耐热菌，整个检测时间为4～5 h，比传统的细菌培养皿培养记数法时间（4～5 d）大大缩短。【结论】本研究建立的耐热菌竞争定量PCR检测法特异、快速，可作为微生物PCR竞争模板构建和建立竞争定量PCR的方法学参考，也可用于商品果汁和苹果浓缩汁工业化生产中耐热菌的快速检测和质量安全控制。

Optimization and Establishment of Quantitatively Competitive PCR System for the Detection of Alicyclobacillus acidoterrestris

【Objective】To study the detective method of Alicyclobacillus acidoterrestris in apple juice concentrate (AJC) by quantitatively competitive polymerase chain reaction (QC-PCR) system. 【Method】 The QC-PCR system for detection of Alicyclobacillus acidoterrestris was established through the primer design, PCR amplification, and reclaim of gel purification reagent box to construct and obtain the competitive template which was used as quantitative internal standards. 【Result】 The QC-PCR system was optimized and established in this study, and the detective sensitivity of the target template had gotten better from 5×104 to 50 molecules /PCR system. As a result of this, 5×102 target template molecules /PCR system was detected when the two templates co-amplified, and 5×103 cfu / PCR system of Alicyclobacillus acidoterrestris in AJC was detected. The detective time (4 h to 5 h) is remarkably shortened from traditional method（4 d or 5 d）using plate culture counting. 【Conclusion】 The method in this study is better than others in efficiency and specificity, which can be a reference for competitive template construction of microbe PCR and constructive methodology of QC-PCR. It has potential to be applied in AJC commercial production process for the rapid detection of Alicyclobacillus acidoterrestris.