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[用于通过聚合酶链反应鉴定鼻疽和类鼻疽病原体的DNA提取方法的比较评估]

[Comparative assessment of DNA extraction methods for identification of glanders and melioidosis etiological agents by PCR].

作者信息

Zinchenko O V, Antonov V A, Tkachenko G A, Altukhova V V, Zamaraev V S, Piven' N N, Goloseev Iu A, Vasil'ev V P, Lomova L V, Alekseev V V

出版信息

Zh Mikrobiol Epidemiol Immunobiol. 2008 Jan-Feb(1):55-60.

Abstract

Pathogenic Burkholderia are considered as a cause of dangerous infections and potential agents of bioterrorism. Comparative assessment of different methods of extraction and purification of DNA for PCR analysis of pure cultures and samples contaminated by etiological agents of glanders and melioidosis was performed. Samples of soil and food artificially contaminated by pathogenic Burkholderia as well as organs of infected animals were tested. DNA was extracted by methods of boiling, nucleosorption with presence of guanidine thiocyanate, guanidine thiocyanatephenol extraction, guanidine thiocyanate-phenol extraction with additional purification of DNA by nucleosorption. Amplification was performed by "Flash" technique and detector of fluorescence was used for analysis of PCR products. Utilization of the recommended methods of preparation depending on the nature of sample let to detect by the "Flash" technique the etiological agents of glanders and melioidosis in concentration =10(3) microbial cells per ml. Choice of DNA extraction and purification methods is determined by type of a sample and presence in it of admixtures inhibiting PCR.

摘要

致病性伯克霍尔德菌被认为是危险感染的病因和生物恐怖主义的潜在媒介。对用于鼻疽和类鼻疽病原体纯培养物及受污染样本PCR分析的不同DNA提取和纯化方法进行了比较评估。对人工被致病性伯克霍尔德菌污染的土壤和食物样本以及感染动物的器官进行了检测。采用煮沸法、存在硫氰酸胍时的核吸附法、硫氰酸胍-苯酚提取法、硫氰酸胍-苯酚提取并通过核吸附对DNA进行额外纯化的方法提取DNA。通过“Flash”技术进行扩增,并使用荧光检测器分析PCR产物。根据样本性质采用推荐的制备方法,能够通过“Flash”技术检测到浓度为每毫升10(3)个微生物细胞的鼻疽和类鼻疽病原体。DNA提取和纯化方法的选择取决于样本类型及其是否存在抑制PCR的混合物。

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