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首页> 外文期刊>Applied Microbiology >Detection and Resolution of Cryptosporidium Species and Species Mixtures by Genus-Specific Nested PCR-Restriction Fragment Length Polymorphism Analysis, Direct Sequencing, and Cloning
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Detection and Resolution of Cryptosporidium Species and Species Mixtures by Genus-Specific Nested PCR-Restriction Fragment Length Polymorphism Analysis, Direct Sequencing, and Cloning

机译：通过属特异性嵌套式PCR-限制性片段长度多态性分析，直接测序和克隆检测和鉴定隐孢子虫物种和物种混合物

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Molecular methods incorporating nested PCR-restriction fragment length polymorphism (RFLP) analysis of the 18S rRNA gene of Cryptosporidium species were validated to assess performance based on limit of detection (LoD) and for detecting and resolving mixtures of species and genotypes within a single sample. The 95% LoD was determined for seven species ( Cryptosporidium hominis , C. parvum , C. felis , C. meleagridis , C. ubiquitum , C. muris , and C. andersoni ) and ranged from 7 to 11 plasmid template copies with overlapping 95% confidence limits. The LoD values for genomic DNA from oocysts on microscope slides were 7 and 10 template copies for C. andersoni and C. parvum , respectively. The repetitive nested PCR-RFLP slide protocol had an LoD of 4 oocysts per slide. When templates of two species were mixed in equal ratios in the nested PCR-RFLP reaction mixture, there was no amplification bias toward one species over another. At high ratios of template mixtures (>1:10), there was a reduction or loss of detection of the less abundant species by RFLP analysis, most likely due to heteroduplex formation in the later cycles of the PCR. Replicate nested PCR was successful at resolving many mixtures of Cryptosporidium at template concentrations near or below the LoD. The cloning of nested PCR products resulted in 17% of the cloned sequences being recombinants of the two original templates. Limiting-dilution nested PCR followed by the sequencing of PCR products resulted in no sequence anomalies, suggesting that this method is an effective and accurate way to study the species diversity of Cryptosporidium , particularly for environmental water samples, in which mixtures of parasites are common.
机译：验证了结合隐孢子虫属物种18S rRNA基因的嵌套式PCR限制性片段长度多态性（RFLP）分析的分子方法，以基于检测限（LoD）评估性能并检测和解析单个样品中物种和基因型的混合物。确定了七个物种（隐孢子虫，小隐孢子虫，猫形隐孢子虫，米氏隐孢子虫，泛酸隐孢子虫，毛孢隐孢子虫和安德森隐孢子虫）的95％LoD，范围为7至11个质粒模板，其中95个重叠％置信度限制。显微镜载玻片上来自卵囊的基因组DNA的LoD值分别为C. andersoni和C. parvum的7和10个模板副本。重复的嵌套式PCR-RFLP玻片方案的LoD为每个玻片4个卵囊。在巢式PCR-RFLP反应混合物中将两种物种的模板以相等的比例混合时，对一种物种的扩增偏向于另一种。在高比例的模板混合物（> 1:10）下，通过RFLP分析检测到的较不丰富的物种有所减少或丢失，这很可能归因于PCR后期循环中异源双链体的形成。复制嵌套式PCR成功地解决了LoD附近或以下的模板浓度的隐孢子虫的许多混合物。巢式PCR产物的克隆导致17％的克隆序列是两个原始模板的重组体。有限稀释巢式PCR和随后的PCR产物测序不会导致序列异常，这表明该方法是研究隐孢子虫物种多样性的一种有效且准确的方法，特别是对于寄生虫混合物普遍存在的环境水样品而言。

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