真菌基因组DNA的提取和通用PCR检测方法的建立

The extraction of fungal genome DNA and the establishment of a broad-range fungal PCR

目的改良真菌基因组DNA的提取方法,建立真菌通用聚合酶链反应(PCR),为目前临床真菌感染的早期诊断、预防和治疗提供有效工具。方法参考前人报道的多种真菌基因组DNA的提取方法,作改良以确立本研究中所采纳的手段,并应用真菌核糖体DNA(rDNA)通用引物,以实验室保存的标准菌株及临床分离菌株来建立临床真菌感染检测用通用PCR。结果白念珠菌和烟曲霉在75℃温度下分别作用60和80 min可以完全被灭活。经目前多种破壁方法的探讨,发现对于白念珠菌(单细胞真菌)选用酶消化法,破壁效率高达98.29%,而烟曲霉(多细胞真菌)则需要酶消化法与打击器振荡法联合应用,其破壁率也可达66.68%,进一步用酚氯仿法抽提其基因组DNA,能够获得相对纯度高且有一定得量。当选择真菌rDNA ITS2区间的一对通用引物,通过PCR反应体系的优化,使得本研究中建立的通用PCR,对于白念珠菌和烟曲霉的检测下限分别为5个和9.7个,其PCR产物测序结果与数据库比对完全一致,同时选择临床分离的或实验室保存的其他真菌、细菌和病毒株进行验证,该方法仅针对真菌群,结合测序分析可以实现种属水平的鉴定。结论改良真菌基因组DNA提取后所建立的针对真菌rDNA的通用PCR敏感且特异,适宜实验室操作。

Objective To improve a method for the extraction of fungal genome DNA, and establish a broad-range fungal polymerase chain reaction （PCR） ,in order to provide the reference for early diagnosis, prevention and treatment of fungal infection disease. Methods According to the former fungal genome DNA extraction methods, a method was formed and improved in this experiment. The fungal ribosome deoxyribonucleic acid （rDNA） was applied as universal primer. A broad-range fungal PCR was established by testing the standard strains and the isolaled clinical strains stored. Results Candida albicans and Aspergillusfumigatus could be inactivated at 75℃ for processed 60 min and absolutely 80 rain respectively. By a series of tries, it suggested that the cell walls of Candida albicans could be fully degraded by enzymes, especially the lysozyme. The disruption rate of the Candida cell walls could be up to 98.29%. Referring to Aspergillus fumigatus, in order to achieve good result, it should be processed by Minibeadbeater-l further, with 66.68% cell disruption rate. The genome DNA was extracted by the phenol-chloroform-isoamyl isovalerate method. Enough quantity and good quality of DNA were obtained. The broad-range fungal PCR was established with its target region in internal transcribed spacer 2 （ ITS2 ） of the rDNA gene. The detection low limits of this method were 5 and 9.7 fungal cells on average, respectively. The sequence of the PCR products was highly consistent with the gene bank. The species identified by sequence analysis of the PCR products were the same as that identified by other methods. The method was used to amplify a series of samples including fungal genome DNA, bacterium genome DNA , virus genome DNA and so on, proving that the method established was only used to amplify the fungal genome DNA specially. Conclusions The improved extraction method could fulfill the research use, and the broad-range fungal PCR has good sensitivity, specificity and stability. It is suitable for the clinical application.