Introduction: Cryptosporidium is an intestinal parasite responsible for gastroenteritis. Conventional diagnosis of Cryptosporidium is made by microscopy. The most frequent molecular detection method for this parasite is polymerase chain reaction (PCR). The objective of the present study was to identify the novel DNA targets and development of PCR-based assays for the specific detection of two major human infecting species Cryptosporidium parvum and Cryptosporidium hominis. Methodology: Sensitive and specific SYBR green quantitative PCR (qPCR) and TaqMan qPCR assays were developed and validated at both diagnostic and analytical level using the new identified targets TU502HP-1 and TU502HP-2. Results: Assay validation results showed that the newly developed real-time PCR assays are 100% specific with a reliable limit of detection. Overall repeatability and reproducibility of these assays showed good quality results over intra- and inter-laboratory analysis. Conclusion: Novel target-based qPCR assays can be rapid an efficient tool for simultaneous detection of a C. parvum and C. hominis. These genes could also be utilized for the development of innovative DNA-based Point-of-Care test development.


中文翻译：

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两种新型基因组 DNA 序列作为检测人型隐孢子虫和小隐孢子虫的常见诊断靶标：定量聚合酶链反应测定的开发和临床评估



简介：隐孢子虫是一种导致胃肠炎的肠道寄生虫。隐孢子虫的常规诊断是通过显微镜进行的。这种寄生虫最常见的分子检测方法是聚合酶链反应（PCR）。本研究的目的是确定新的 DNA 靶标并开发基于 PCR 的检测方法，用于特异性检测两种主要人类感染物种小隐孢子虫和人隐孢子虫。方法：使用新确定的靶点 TU502HP-1 和 TU502HP-2，开发了灵敏且特异的 SYBR green 定量 PCR (qPCR) 和 TaqMan qPCR 检测方法，并在诊断和分析水平上进行了验证。结果：检测验证结果表明，新开发的实时 PCR 检测具有 100% 特异性，具有可靠的检测限。与实验室内和实验室间分析相比，这些测定的总体重复性和再现性显示出良好的质量结果。结论：基于靶点的新型 qPCR 检测可以成为同时检测微小念珠菌和人念珠菌的有效工具。这些基因还可用于开发基于 DNA 的创新床旁测试开发。