Lyons R E, Lyons K, McLeod R, Roberts C W
Department of Immunology, Strathclyde Institute for Biomedical Sciences, University of Strathclyde, Glasgow, Scotland, UK.
Parasitology. 2001 Nov;123(Pt 5):433-9. doi: 10.1017/s003118200100868x.
The obligate intracellular protozoan parasite, Toxoplasma gondii exists as 2 life-cycle forms in intermediate hosts. The rapidly dividing tachyzoites responsible for acute disease, present in the first 14 days of infection, give rise to slowly dividing bradyzoites that reside in tissue cysts. Reactivation of disease is associated with conversion of bradyzoites to tachyzoites. A sensitive method for detection and assessment of the number of each life-cycle stage would be useful for following these events. Herein we describe the construction and validation of a plasmid (pSWITCH) containing a polycompetitor construct (SWITCH) for use in competitive reverse transcriptase-PCR (cRT-PCR). pSWITCH contains competitors for SAG2A and LDH2 genes, which are exclusively expressed by tachyzoite and bradyzoite stages respectively, and for beta-tubulin, a gene expressed by both stages. Using cRT-PCR, samples can first be accurately normalized for expression of the housekeeping gene, beta-tubulin and then the relative levels of SAG2A and LDH2 expression compared to follow stage conversion. The abundance of transcripts for other genes of interest can then be followed during this process as demonstrated here for the SAG2-related family of genes. This technique offers a powerful tool for studying the processes involved in tachyzoite and bradyzoite interconversion.
专性细胞内原生动物寄生虫刚地弓形虫在中间宿主体内以两种生命周期形式存在。负责急性疾病的快速分裂速殖子出现在感染的前14天，之后会产生驻留在组织包囊中的缓慢分裂缓殖子。疾病的复发与缓殖子向速殖子的转化有关。一种用于检测和评估每个生命周期阶段数量的灵敏方法对于追踪这些过程将是有用的。在此，我们描述了一种质粒（pSWITCH）的构建和验证，该质粒包含一个用于竞争性逆转录聚合酶链反应（cRT-PCR）的多竞争构建体（SWITCH）。pSWITCH包含分别由速殖子和缓殖子阶段特异性表达的SAG2A和LDH2基因的竞争物，以及由两个阶段都表达的β-微管蛋白基因的竞争物。使用cRT-PCR，首先可以针对管家基因β-微管蛋白的表达对样品进行准确归一化，然后比较SAG2A和LDH2的相对表达水平以追踪阶段转化。在此过程中，可以追踪其他感兴趣基因的转录本丰度，如这里针对SAG2相关基因家族所展示的那样。该技术为研究速殖子和缓殖子相互转化所涉及的过程提供了一个强大的工具。
