不同破壁方法提取真菌DNA的比较

Comparison of lysis methods for fungal DNA extraction

目的探索和评价提取致病性真菌DNA的简便方法。方法分别用6种方法(液氮冻融、-70℃冻融、溶胞酶消解、超声破碎、高盐溶液、尿素溶液)裂解真菌细胞壁,再用市售DNeasy B lood&Tissue K it试剂盒提取DNA,最后通过对提取物进行比色定量和PCR检测评价各种提取方法的优弊。结果酶消解破壁法提取真菌DNA效率最高,重复性良好。模拟全血标本中白色念珠菌检测限为102个孢子,烟曲霉及新型隐球菌检测限为103个孢子。结论酶消解破壁法提取真菌DNA在6种方法中效率最高,可用于临床常见真菌感染的PCR快速诊断。

Objective To investigate and evaluate simple DNA extraction method for fungal diagnosis. Methods Six cell lysis methods ineluding freeze thawing in liquid nitrogen or at -70℃, enzyme digestion, sonication, chaotropic salts, and urea buffer were investigated. Each lysis procedure was followed by DNA puiification using the Qiagen kit. The efficiency was estimated by quantitation and PCR. Resuits Of all six extraction methods, enzyme digestion combined with the kit resulted in the highest yield. The detection limit for Candida aIbieans, As.pergillus fumigatus, and Cryptococcus neoformans in simulated blood samples with universal primer PCR was 102 , 103 , and 103 cells per reaction respectively. Conclusions Enzyme digestion combined with the kit for extracting DNA from fungi is useful for diagnosing fungal infection.