五种DNA提取方法的比较及用于检测小鼠脑组织中弓形虫组织包囊的b1基因巢式PCR（nPCR）的优化

Comparison of five DNA extraction methods and optimization of a b1 gene nested PCR (nPCR) for detection of Toxoplasma gondii tissue cyst in mouse brain.

作者信息

Mahittikorn Aongart, Wickert Hannes, Sukthana Yaowalark

机构信息

Department of Protozoology, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand.

出版信息

Southeast Asian J Trop Med Public Health. 2005 Nov;36(6):1377-82.

PMID:16610637

Abstract

Toxoplasmosis, caused by Toxoplasma gondii, is an important parasitic disease worldwide. Different techniques have been developed for T. gondii detection. At present, polymerase chain reaction (PCR) has been widely used. However, PCR for identifying T. gondii remains unsatisfactory in many laboratories because of lack of standardization and variations in efficiency. In the present study, we optimized a nested PCR protocol (n-PCR) in order to compare the amplification of T. gondii DNA, after being extracted from mouse brain by five different DNA extraction methods including phenol chloroform, QIAamp DNA minikit, Genomic DNA purification kit and Chelex with or without proteinase K. All DNA extraction methods were able to extract DNA from a single tissue cyst from mouse brain. However, among the five DNA extraction methods, the Chelex without proteinase K appeared to be the most rapid and easiest.

摘要

由刚地弓形虫引起的弓形虫病是一种在全球范围内重要的寄生虫病。已经开发出了不同的技术用于检测刚地弓形虫。目前，聚合酶链反应（PCR）已被广泛应用。然而，由于缺乏标准化以及效率存在差异，许多实验室用于鉴定刚地弓形虫的PCR仍不尽人意。在本研究中，我们优化了一种巢式PCR方案（n-PCR），以便比较用五种不同的DNA提取方法（包括酚氯仿法、QIAamp DNA微量试剂盒法、基因组DNA纯化试剂盒法以及使用或不使用蛋白酶K的Chelex法）从小鼠脑中提取刚地弓形虫DNA后的扩增情况。所有DNA提取方法都能够从小鼠脑的单个组织包囊中提取DNA。然而，在这五种DNA提取方法中，不使用蛋白酶K的Chelex法似乎是最快速、最简单的。