摘要
利用微波加热原理,探索一种快速、简单、高效提取细菌DNA用于常规PCR快速检测的方法.在微波炉额定功率800 W条件下分别对大肠杆菌、金黄色葡萄球菌、单增李斯特氏菌、副溶血弧菌、沙门氏菌以及志贺氏菌6种常见食源性病原菌进行微波处理,离心取上清,获取细菌DNA,将样本DNA进行PCR扩增及琼脂糖凝胶电泳进行验证.并进一步采用微波加热方法针对6种病原菌最低检出浓度和混合提取6种病原菌DNA的PCR检测进行分析.6种病原菌在微波炉中加热40-130 s的上清DNA样本均能用于常规PCR扩增.针对检出浓度的分析表明,副溶血弧菌的最低检出浓度为103cfu/ml,金黄色葡萄球菌为104cfu/ml,肠出血性大肠杆菌为105cfu/ml,肠炎沙门氏菌和福氏志贺氏菌的最低检出浓度为106cfu/ml,针对单核细胞增生李斯特氏杆菌的的最低检出浓度为107cfu/ml.进行6种混合菌微波共提取时,其中5种病原菌的特异性基因都能扩出,并且条带很亮.该微波提取方法具有快速,简单,高效的特点,能满足大部分食源性病原菌的常规PCR检测需求,大大节约了时间和成本,具有广泛的适用性,为细菌快速分子检测提供了简便手段.
A fast,simple and efficient method with microwave heating for extracting pathogenic microorganisms genomic DNA was developed and applied for routine PCR amplification. Six foodbome pathogens （Escherichia coli, Staphylococcus aureus, Listeria monocytogenes, Vibrio parahaemolyticus ,Salmonella and Shigella） were used in this assay. The pellet was suspended in 100 μL TE buffer,treated in a microwave oven at rated power（800 W）for a certain time（40s - 130s） ,then centrifuged at 12,000 g for 2 min. The supernatant was transferred to a sterile tube and stored at - 20℃ for PCR testing and agarose gel electrophoresis. The same method was also used for extracting genomic DNA of different concentration of each pathogen and the mixture of six pathogens. All the supernatant after being treated in the microwave oven for 40 to 130s, could be used for PCR amplification. For concentration detection limit test, the limit concentration for V. parahaemolyticus was 10^3 cfu/ml, for S. aureus 10^4 cfu/ ml,for E. coli 10^5 cfu/ml,for Salmonella and Shigella 10^6 cfu/ml,and for L. monocytogenes 10^7 cfu/ml. For the mixture test of six bacteria DNA, five of them could be detected except for L. monocytogenes. The microwave heating method for extracting bacteria DNA could meet the requirments of PCR detection, which is simple ,fast and cost effective. This method will have broaden application, particularly for handling a large number of samples.
出处
《西南民族大学学报（自然科学版）》
CAS
2015年第2期150-155,共6页
Journal of Southwest Minzu University(Natural Science Edition)
基金
国家自然科学基金(31371781)
四川省应用基础项目(14JC0702)
教育部新世纪人才支持计划(NCET-11-0847)
