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首页> 外文期刊>Journal of Clinical Microbiology >Serial Quantitative PCR Assay for Detection, Species Discrimination, and Quantification of Leishmania spp. in Human Samples
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Serial Quantitative PCR Assay for Detection, Species Discrimination, and Quantification of Leishmania spp. in Human Samples

机译：用于检测，物种鉴别和定量利什曼原虫的串行定量PCR分析。在人类样本中

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The Leishmania species cause a variety of human disease syndromes. Methods for diagnosis and species differentiation are insensitive and many require invasive sampling. Although quantitative PCR (qPCR) methods are reported for leishmania detection, no systematic method to quantify parasites and determine the species in clinical specimens is established. We developed a serial qPCR strategy to identify and rapidly differentiate Leishmania species and quantify parasites in clinical or environmental specimens. SYBR green qPCR is mainly employed, with corresponding TaqMan assays for validation. The screening primers recognize kinetoplast minicircle DNA of all Leishmania species. Species identification employs further qPCR set(s) individualized for geographic regions, combining species-discriminating probes with melt curve analysis. The assay was sufficient to detect Leishmania parasites, make species determinations, and quantify Leishmania spp. in sera, cutaneous biopsy specimens, or cultured isolates from subjects from Bangladesh or Brazil with different forms of leishmaniasis. The multicopy kinetoplast DNA (kDNA) probes were the most sensitive and useful for quantification based on promastigote standard curves. To test their validity for quantification, kDNA copy numbers were compared between Leishmania species, isolates, and life stages using qPCR. Maxicircle and minicircle copy numbers differed up to 6-fold between Leishmania species, but the differences were smaller between strains of the same species. Amastigote and promastigote leishmania life stages retained similar numbers of kDNA maxi- or minicircles. Thus, serial qPCR is useful for leishmania detection and species determination and for absolute quantification when compared to a standard curve from the same Leishmania species.
机译：利什曼原虫物种引起多种人类疾病综合征。诊断和物种区分的方法不敏感，许多方法需要侵入性采样。尽管报告了定量PCR（qPCR）方法用于利什曼原虫的检测，但尚无建立系统的方法来定量寄生虫并确定临床标本中的物种。我们开发了一种串行qPCR策略，以识别和快速区分利什曼原虫种类并量化临床或环境标本中的寄生虫。主要使用SYBR green qPCR，以及相应的TaqMan分析进行验证。筛选引物识别所有利什曼原虫物种的运动塑料微环DNA。物种鉴定采用了针对地理区域的进一步qPCR集，将区分物种的探针与熔解曲线分析相结合。该测定足以检测利什曼原虫的寄生虫，进行物种测定并定量利什曼原虫的种类。血清，皮肤活检标本或孟加拉国或巴西患有不同形式利什曼病的受试者的培养分离株。基于前鞭毛体标准曲线，多拷贝动植物体DNA（kDNA）探针对定量最为敏感和有用。为了测试其定量的有效性，使用qPCR比较了利什曼原虫物种，分离株和生命阶段的kDNA拷贝数。在利什曼原虫物种之间，最大圆和最小圆的拷贝数相差最多6倍，但是同一物种的品系之间的差异较小。鞭毛体和鞭毛体利什曼原虫的生命阶段保留了相似数量的kDNA上或下圆。因此，与来自相同利什曼原虫物种的标准曲线相比，串行qPCR可用于利什曼原虫的检测和物种确定以及绝对定量。

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