Adapting a conventional PCR assay for Toxoplasma gondii detection to real-time quantitative PCR including a competitive internal control
Adapting a conventional PCR assay for Toxoplasma gondii detection to real-time quantitative PCR including a competitive internal control
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DOI：
10.1051/parasite/2007142149
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发表时间：
2007-06-01
期刊：
影响因子：
2.9
通讯作者：
Pelloux, H.
中科院分区：
文献类型：
--
作者：
Brenier-Pinchart, M. P.;Morand-Bui, V;Pelloux, H.
我们开发了一种定量 PCR 测定法 (LightCyder (R))，使用引物对 JW58 和 JW59 检测弓形虫的 35 倍重复 131 基因。这种实时 PCR 使用荧光共振能量转移 (FRET) 杂交探针，可以对弓形虫进行定量，并满足之前未描述的几个技术要求：i) 内部扩增对照（在单管中与一些引物），ii）尿嘧啶-N-糖基化酶和 iii）标准曲线，对应于一毫升羊水中 40 至 4.10(6) 寄生虫的校准弓形虫悬浮液的连续稀释（1 至 10(5) 弓形虫/PCR。在人工样品中，如果进行至少 3 个反应，则可以检测到一种寄生虫。
We have developed a quantitative PCR assay (LightCyder (R)) using the pair of primers JW58 and JW59 for the detection a( the 35-fold repeated 131 gene of Toxoplasma gondii. This real-time PCR, using fluorescence resonance energy transfert (FRET) hybridization probes, allows the quantification of T. gondii with several technical requirements not previously described: i) an internal amplification control (co-amplified in a single tube with the some primers), ii) Uracil-N-Glycosylase and iii) a standard curve corresponding to a serial dilution from a calibrated suspension of T. gondii ranging from 40 to 4.10(6) parasites in one ml of amniotic fluid (1 to 10(5) T. gondii/PCR. In artificial samples, one parasite could be detected if at least three reactions were performed.