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首页> 外文期刊>Journal of Clinical Microbiology >Detection and Differentiation of Cryptosporidium spp. in Human Clinical Samples by Use of Real-Time PCR
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Detection and Differentiation of Cryptosporidium spp. in Human Clinical Samples by Use of Real-Time PCR

机译：隐孢子虫的检测和鉴别。实时荧光定量PCR检测人体临床样品

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摘要

Real-time PCR has the potential to streamline detection and identification of Cryptosporidium spp. in human clinical samples. In the present article, we report the first such assay to allow not only detection and differentiation of the most common human pathogens, Cryptosporidium hominis and Cryptosporidium parvum, but also simultaneous amplification of a region of the small subunit (SSU) rRNA gene, permitting direct sequence analysis to identify any Cryptosporidium species. An internal control is incorporated to identify the presence of PCR inhibitors. Analytical sensitivity was determined to be as low as 200 oocysts per gram of feces processed, equivalent to 2 oocysts per PCR. The C. hominis and C. parvum PCRs specifically detected only species/genotypes in their respective target clades. Diagnostic sensitivity and specificity, evaluated against a widely used conventional nested SSU rRNA gene PCR as a nominated gold standard using a panel of 258 (151 positive and 107 negative) samples, were 100% and 99.1%, respectively. The assay agreed with PCR-restriction fragment length polymorphism analysis of the Cryptosporidium oocyst wall protein gene for 134 of 136 (98.5%) samples tested prospectively and typed two additional isolates. The real-time PCR assay was sensitive, specific, and reproducible and significantly improved laboratory work flow and turnaround times.
机译：实时PCR可以简化隐孢子虫的检测和鉴定。在人类临床样品中。在本文中，我们报告了第一个这样的分析方法，它不仅可以检测和区分最常见的人类病原体，人隐隐孢子虫和小隐孢子虫，而且还可以同时扩增小亚基（SSU）rRNA基因区域，从而可以直接序列分析以鉴定任何隐孢子虫种类。掺入内部对照以鉴定PCR抑制剂的存在。确定的分析灵敏度低至每克加工的粪便200个卵囊，相当于每个PCR 2个卵囊。 C. hominis和C. parvum PCRs仅在其各自的目标进化枝中仅检测到物种/基因型。使用一组258个样本（151个阳性和107个阴性）作为标准金标准，针对广泛使用的常规嵌套SSU rRNA基因PCR评估的诊断敏感性和特异性分别为100％和99.1％。该测定方法与对预期进行测试的136个样本（98.5％）中的134个隐孢子虫卵囊壁蛋白基因的PCR限制性片段长度多态性分析相符，并确定了另外两个分离株。实时PCR检测灵敏，特异，可重复，可显着改善实验室工作流程和周转时间。

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