5种方法提取真菌基因组DNA作为PCR模板效果的比较

本研究旨在比较CTAB法、改良CTAB法、SDS法、氯化苄法和Chelex-100法提取真菌DNA作为PCR模板的效果,以生长速率、菌落形态差异较大的11个煤污病菌属真菌为研究对象,以核糖体RNA(r RNA)基因中的内转录间隔区(ITS)序列和28S r RNA基因部分序列的扩增率为指标,采用SPSS软件对结果进行方差分析。结果表明,CTAB法和改良CTAB法适用真菌范围最广,分别有7个和9个属真菌的扩增率都大于70%且无显著性差异;氯化苄法和Chelex-100法适用范围居中,分别有6个和8个属真菌的扩增率较高且无显著性差异,扩增率分别大于70%和50%;SDS法适用范围最窄,有6个属真菌的扩增率高于50%且无显著性差异。大多数煤污病菌至少有2种及以上的DNA提取方法能够取得较好的效果,但链丝孢属真菌只有用氯化苄法提取DNA效果最好。采用改良CTAB法结合氯化苄法,能够满足所有供试煤污病菌类群真菌提取基因组DNA用作PCR反应模板的需要。

Effect Comparison of Five Methods to Extract Fungal Genomic DNA as PCR Templates

The objective of this study was to compare the effect of fungal genomic DNA, which was extracted by five methods including CTAB method, modified CTAB method, SDS method, benzyl chloride method and Chelex-100 method, as PCR templates. Eleven sooty blotch and flyspeck (SBFS) fungal genera that exhibited different growth rates and colony characters were selected as tested fungi, and amplification percentages of PCR reactions of the internal transcribed spacers (ITS) and partial sequences of 28S rRNA gene of ribosomal RNA (rRNA) were used as indexes, and the results were analyzed with SPSS software. The results showed that the CTAB method and the modified CTAB method were suitable for most fungi, respectively with seven and nine tested fungal genera amplification percentage higher than 70% and had no significant differences. The benzyl chloride method and the Chelex-100 method were suitable for partial fungi, respectively with six and eight tested fungal genera amplification percentage higher than 70% and 50%, and had no significant differences. The SDS method was suitable for only a narrow range of fungi, with six tested fungal genera amplification percentage higher than 50% and had no significant differences. There were at least two methods that could perform well to extract DNA from most SBFS fungal genera, but only the benzyl chloride method gave the best DNA extraction result for the genus Scleroramularia. Using modified CTAB method combined with the benzyl chloride method could meet the need of extracting SBFS fungal genomic DNA as PCR templates.