Institute for Research in Molecular Medicine, Universiti Sains Malaysia, Penang, Malaysia.
Exp Parasitol. 2012 Jun;131(2):231-8. doi: 10.1016/j.exppara.2012.04.009. Epub 2012 Apr 25.
Molecular methods are used increasingly for the detection of Toxoplasma gondii infection. This study developed a rapid, sensitive, and specific conventional triplex PCR for the detection of the B1 gene and ITS1 region of T. gondii using newly designed primers and an internal control based on the Vibrio cholerae HemM gene. The annealing temperature and concentrations of the primers, MgCl(2), and dNTPs were optimized. Two sets of primers (set 1 and 2) were tested, which contained different segments of the T. gondii B1 gene, 529 repeat region and ITS1 region. A series of sensitivity tests were performed using parasite DNA, whole parasites, and spiked human body fluids. Specificity tests were performed using DNA from common protozoa and bacteria. The newly developed assay based on set 2 primers was found to be specific and sensitive. The test was capable of detecting as little as 10 pg T. gondii DNA, 10(4) tachyzoites in spiked body fluids, and T. gondii DNA in the organ tissues of experimentally infected mice. The assay developed in this study will be useful for the laboratory detection of T. gondii infection.
分子方法越来越多地用于检测弓形虫感染。本研究使用新设计的引物和基于霍乱弧菌 HemM 基因的内部对照，开发了一种快速、敏感、特异的常规三重 PCR 方法，用于检测弓形虫的 B1 基因和 ITS1 区。优化了引物、MgCl2 和 dNTPs 的退火温度和浓度。测试了两套引物（set1 和 set2），它们包含了弓形虫 B1 基因的不同片段，529 重复区和 ITS1 区。使用寄生虫 DNA、全寄生虫和加标人体体液进行了一系列灵敏度测试。使用常见原生动物和细菌的 DNA 进行了特异性测试。基于 set2 引物的新开发的检测方法被发现是特异和敏感的。该检测法能够检测到低至 10pg 弓形虫 DNA、加标体液中的 10^4^速殖子以及实验感染小鼠的器官组织中的弓形虫 DNA。本研究中开发的检测方法将有助于实验室检测弓形虫感染。
