病原真菌DNA提取方法及简单重复序列聚合酶链反应体系的优化

DNA extraction method of pathogenic fungi and the optimization of simple sequence repeat PCR system

背景:真菌DNA的提取在真菌基因工程以及分子生物学研究中占有重要地位。DNA的提取效率,尤其是DNA的质量严重影响实验结果。目的:建立提取病原真菌基因组DNA的方法,探讨简单重复序列-PCR反应体系中各成分的最佳组合。设计、时间及地点:DNA提取方法对照分析及正交实验,于2004-06/2006-12在吉林大学白求恩医学院病原生物学教研室真菌学研究室进行。材料:将接种临床标本的马铃薯葡萄糖液体培养基、马铃薯葡萄糖琼脂培养基、酵母蛋白胨液体培养基28℃培养3～7d后,选取可疑菌落进行分离、纯化。方法:比较玻璃珠-盐析法、CTAB法、GeneTLETM抽提法3种不同DNA提取方法对菌体DNA质量的影响;利用正交设计,选择TaqDNA聚合酶、模板DNA、dNTP、引物4个因素,在3个水平上根据L9(34)正交表进行试验,对真菌简单重复序列-PCR(SSR-PCR)体系进行优化分析;并通过PCR选择适宜的退火温度及循环次数。主要观察指标:根据扩增获得带型的多态性和特异性,确定最佳反应体系。结果:GeneTLETM抽提法全部在相应的PCR体系中扩增出目的片段,且带型清楚度、明亮度优于其余两种方法。SSR-PCR反应体系正交试验结果显示,根据R值大小确定因素显著性顺序为模板DNA(2.67)>TaqDNA聚合酶(2.00)>dNTP(0.67)>引物(0.33);根据ki值大小确定各因素优化水平组合为:模板DNA30mg/L,TaqDNA聚合酶1U,dNTP150μmol/L,引物0.5μmol/L。但由于引物作为影响因素的R值小,是非显著因素,因此引物取A水平即0.25μmol/L。反应条件以53℃退火温度、35个循环次数为最佳。结论:GeneTLETM提取法提取DNA的效率更高、操作步骤快速简单。根据正交试验的优化结果,SSR-PCR最佳反应体系为模板DNA30mg/L,TaqDNA聚合酶1U,dNTP150μmol/L,引物0.25μmol/L。反应条件以53℃退火温度、35个循环次数为最佳。

BACKGROUND： Extraction of fungal DNA plays an important role in fungal genetic engineering and molecular biology research. The result of experiment is affected seriously by the efficiency of extracting DNA especially the quality of DNA. OBJECTIVE： To develop a method for extracting genomic DNA of pathogenic fungi and discuss the optimal combination of components in simple sequence repeat PCR （SSR-PCR） system. DESIGN, TIME AND SETTING： A comparative analysis of DNA extraction methods and an orthogonal experiment were conducted in the Mycology Research Lab of Department of Pathogenobiology in Norman Bethune College of Medicine, Jilin University from July 2004 to December 2006. MATERIALS： Clinical specimens were inoculated on Potato dextrose agar, Potato dextrose broth and Yeast extract peptone dextrose and cultivated under the temperature of 28 ℃ for 3-7 days, after which suspectable colonies were selected to be isolated and purified. METHODS： Three kinds of methods of extracting DNA（beading-salt fractionation method, CTAB method and Gene TLETM extraction method ） were compared in terms of their effects on DNA quality; Experiment was performed with orthogonal design to four factors （Taq DNA polymerase, template DNA, dNTP, primers） in three levels on the basis of L9（3^4） orthogonal table. The appropriate annealing temperature and cycleswere determined through PCR. MAIN OUTCOME MEASURES： The optimal reaction system determined according to the polymorphism and specificity of amplification of banding pattern. RESULTS： The objective fragments were all amplified by Gene TLETM extraction method, and the banding patterns obtained were clearer and brighter compared with the other two methods. The result of orthogonal experiment on SSR-PCR system showed that, according to the value of R, the significance of factors followed by ascending were template DNA （2.67）, Taq DNA polymerase （2.00）, dNTP （0.67） and primers （0.33）. According to the value of ki, the optimal level of each factor combination was 30 mg/L template DNA, 1 U Taq DNA polymerase, 150μmol/L dNTP, 0.5μmol/L primer. However, because primers were nonsignificant factors, which was presented by their small R value, we took A level of primer as 0.25μmol/L. The best reaction condition was 55℃ annealing temperature and 35 cycles. CONCLUSION： The Gene TLE^TM method shows higher efficiency of extracting DNA and its operation is fast and simple. According to the results of orthogonal experiment, the optimal SSR-PCR system was 30 mg/L template DNA, lU Taq DNA polymerase, 150μmol/L dNTP and 0.25μmol/L primer. The best reaction condition was 55℃ annealing temperature and 35 cycles.