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首页> 外文期刊>Journal of Clinical Microbiology >Real-Time PCR for Quantitative Detection ofToxoplasma gondii
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Real-Time PCR for Quantitative Detection ofToxoplasma gondii

机译：实时PCR定量检测弓形虫

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The protozoan Toxoplasma gondii is one of the most common infectious pathogenic parasites and can cause severe medical complications in infants and immunocompromised individuals. We report here the development of a real-time PCR-based assay for the detection of T. gondii. Oligonucleotide primers and a fluorescence-labeled TaqMan probe were designed to amplify the T. gondii B1 gene. After 40 PCR cycles, the cycle threshold values (CT) indicative of the quantity of the target gene were determined. Typically, a CT of 25.09 was obtained with DNA from 500 tachyzoites of the T. gondii RH strain. The intra-assay coefficients of variation (CV) were 0.4, 0.16, 0.24, and 0.79% for the four sets of quadruplicate assays, with a mean interassay CV of 0.4%. These values indicate the reproducibility of this assay. Upon optimization of assay conditions, we were able to obtain a standard curve with a linear range (correlation coefficient = 0.9988) across at least 6 logs of DNA concentration. Hence, we were able to quantitatively detect as little as 0.05 T. gondii tachyzoite in an assay. When tested with 30 paraffin-embedded fetal tissue sections, 10 sections (33%) showed a CT of <40 and were scored as positive for this test. These results were consistent with those obtained through our nested-PCR control experiments. We have developed a rapid, sensitive, and quantitative real-time PCR for detection of T. gondii. The advantages of this technique for the diagnosis of toxoplasmosis in a clinical laboratory are discussed.
机译：刚体弓形虫是最常见的传染性病原性寄生虫之一，可对婴儿和免疫功能低下的人造成严重的医学并发症。我们在这里报告了基于实时PCR的检测法检测 T的进展。刚地。设计寡核苷酸引物和荧光标记的TaqMan探针以扩增 T。刚地 B1基因。经过40个PCR循环后，确定了指示目标基因数量的循环阈值（C T ）。通常，用500个 T速殖子的DNA测得的C T 为25.09。刚地菌RH菌株。对于四组一式四份测定，测定内变异系数（CV）为0.4、0.16、0.24和0.79％，平均测定间变异系数为0.4％。这些值表明该测定法的可重复性。通过优化分析条件，我们能够获得至少6对数的DNA浓度具有线性范围（相关系数= 0.9988）的标准曲线。因此，我们能够定量检测低至0.05 Tem。测定中的弓形虫速殖子。用30个石蜡包埋的胎儿组织切片进行测试时，有10个切片（33％）显示C T <40，在该测试中被评为阳性。这些结果与通过我们的巢式PCR对照实验获得的结果一致。我们已经开发了一种快速，灵敏和定量的实时PCR，用于检测 T。刚地。讨论了该技术在临床实验室中诊断弓形虫病的优势。

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