扩展青霉DNA提取及PCR检测条件的优化

Optimization of DNA Extraction and PCR Detection from Penicillium expansum

研究扩展青霉DNA的提取及其聚合酶链式反应(polymerase chain reaction,PCR)检测条件的优化。分别采用玻璃珠法、氯化苄法、玻璃珠+氯化苄法提取扩展青霉菌及对照菌株的基因组DNA,经紫外测定和电泳检测实验,分析提取DNA的质量浓度和纯度;同时,根据扩展青霉多聚半乳糖醛酸酶基因内一段保守序列设计合成一对288bp的扩增引物,并对PCR检测条件进行优化。结果表明:玻璃珠+氯化苄法提取到的DNA质量浓度和纯度较理想,扩展青霉DNA可获得良好的特异性扩增,PCR扩增的最适退火温度为53～59℃,最适引物浓度为0.04～0.16μmol/L,最适模板质量浓度为2.40～5.28μg/mL,dNTPs浓度对PCR扩增影响不大。运用实验所得优化参数检测扩展青霉菌,整个过程仅需3～4h,和传统的培养检测法相比,该方法可有效提高检测效率,可进一步将该方法应用于实践。

The genome DNAs of Penicillium expansum and 6 control fungi were extracted by glass bead method,benzyl chloride method and their combination,respectively.The concentration and purity of DNAs were analyzed by UV spectroscopy and gel electrophoresis.Meanwhile,a pair of primers with a size of 288 bp were designed and synthesized based on a conservative sequence of Penicillium expansum＇s polygalacturonase gene to conduct the PCR detection.The results showed that combined use of glass bead and benzyl chloride was more effective than either alone.Good specific amplification of DNA was obtained,and the optimal range of annealing temperature was between 53 ℃ and 59 ℃,and the optimal range of primer concentration between 0.04 μmol/L and 0.16 μmol/L,and the optimal range of template concentration between 2.40 μg/mL and 5.28 μg/mL.Meanwhile,dNTPs concentration had little influence on PCR amplification.The method requiring only 3 to 4 hours could evidently enhance detection efficiency when compared to traditional methods.As a result,it has promising potential to be further applied in practice.