| 摘要: |
| 为了研究苹果汁生产中棒曲霉素产生菌的快速检测,利用实时PCR方法快速检测扩展青霉的可能性,对扩增条件进行优化。在polygatacturonase基因引物设计和传统PCR扩增扩展青霉DNA的基础上,研究实时PCR扩增时退火温度、引物浓度以及底物质量浓度对扩增效果的影响,以获得最佳的参数。结果表明,用Lightercy-cler实时PCR扩增扩展青霉DNA的最佳退火温度为61 ℃,最佳引物浓度为0.20～0.40 μmol/L,底物质量浓度对实时PCR扩增的影响较小。在以上参数下,可以获得良好的PCR扩增曲线、产物溶解曲线以及清晰的产物条带。研究获得的快速检测扩展青霉的实时PCR参数,可用于浓缩苹果汁工业化生产中扩展青霉的快速检测,也可作为其他微生物实时PCR检测时的方法学参考。 |
| 关键词: 苹果汁 扩展青霉 PCR检测 |
| DOI： |
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| 基金项目:国家留学基金项目；国家科技支撑计划项目（2006BAK02A24）；陕西省科技攻关项目（2007K01-12） |
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| Condition optimization of real time PCR used in rapid detection of Penicillium expansum in apple juice concentrate. |
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| Abstract: |
| In order to to detect Penicillium expansum rapidly in apple juice concentration,the real time PCR method was used to detect Penicillium expansumand the conditions were also optimized.Based on the primers from polygatacturonase gene,the effect of annealing temperature,primer concentration and template concentration of real time PCR on the amplification was evaluated.The result showed that the amplification temperature of Penicillium expansum DNA was 61 ℃ when using Lightcycler,primer concentration was between 0.20-0.40 μmol/L,and the template concentration had little effect on amplification.Using above mentioned parameters,the best amplification,product melting curve and clear bands could be obtained.The parameters to detect Penicillium expansumobtained in this study can be used in the detection of Penicillium expansumin apple juice production,it can be also used as a reference for real time PCR detection for other microbes. |
| Key words: apple juice,Penicillium expansum,PCR detection |
