用于PCR检测的枣中霉菌DNA提取条件优化  

Optimization of DNA extracting conditions for detection of mould in jujube

作　　者：郝莉花 蒋祎丽 纵伟[2] 吴晓宗[2] 

机构地区：[1]河南省产品质量监督检验院,河南郑州450000 [2]郑州轻工业学院食品与生物工程学院,河南郑州450002

出　　处：《中国卫生检验杂志》2014年第21期3050-3052,共3页Chinese Journal of Health Laboratory Technology

基　　金：国家质检总局科技计划项目(2013QK232)

摘　　要：目的提取霉变红枣中霉菌DNA,研究其适宜的提取条件。方法采用氯化苄提取方法,以DNA浓度为指标,从提取溶剂p H、提取时间和氯化苄加入量3个方面对DNA的提取条件进行研究,在单因素实验的基础上,采用正交实验进行优化。结果氯化苄提取红枣中霉菌DNA的最佳条件为:提取溶剂p H=8.8,提取时间为55 min,氯化苄量为320μl。各因素对CDNA影响的次序为:提取溶剂p H>提取时间>氯化苄量。方差分析表明,提取溶剂p H在所考察的范围内对CDNA影响显著,而提取时间和氯化苄量对CDNA在所考察的范围内影响不显著,最佳提取条件下,CDNA平均值为20.11μg/ml。结论应用氯化苄法提取枣中真菌DNA,不需要添加蛋白酶消解蛋白质,也不需要氯仿、苯酚等抽提蛋白质,且破壁与DNA提取可同时进行,方法简单、效率高,是检测霉变红枣中真菌的适宜方法。Objective To extract mould DNA in jujube and study its suitable parameter. Methods The mould DNA was ex- tract with benzyl chloride by DNA concentration as index, the effect of extraction conditions （ pH, time and benzyl chloride ad- dition） on extract parameters were studied. Based on signal factor test, orthogonal test was used to optimum extract conditions. Results The optimum extraction conditions were as.follows ： pH 8.8, time 55 min and benzyl chloride addition 320 μl. The order of the factors influence on CDNA were pH 〉 time 〉 benzyl chloride addition. Variance analysis showed that pH has significant effects on CDNA in the investigation scope, time and benzyl chloride addition has not significant effects on CDNA in the investigation scope. CDNA was 20. 11 μg/ml in the optimum extract conditions. Conclusion Benzyl chloride method don＇t need to add protease to digestion of protein, don＇t need to add chloroform and phenol to extract protein. Moreover, wall -breaking and DNA extraction can be made at the same time. This method was a simple and effective method for extracting DNA from mould in jujube.

关 键 词：聚合酶链式反应 霉菌 脱氧核糖核酸 提取 

分 类 号：Q939.5[生物学—微生物学]