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首页> 外文期刊>Parasitology >Developing a real-time PCR assay based on multiplex high-resolution melt-curve analysis: a pilot study in detection and discrimination of soil-transmitted helminth and schistosome species
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Developing a real-time PCR assay based on multiplex high-resolution melt-curve analysis: a pilot study in detection and discrimination of soil-transmitted helminth and schistosome species

机译：基于多重高分辨率熔融曲线分析开发实时PCR测定：土壤传播蠕虫和血吸虫物种检测和辨别中的试验研究

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With the push towards control and elimination of soil-transmitted helminthiasis and schistosomiasis in low- and middle-income countries, there is a need to develop alternative diagnostic assays that complement the current in-country resources, preferably at a lower cost. Here, we describe a novel high-resolution melt (HRM) curve assay with six PCR primer pairs, designed to sub-regions of the nuclear ribosomal locus. Used within a single reaction and dye detection channel, they are able to discriminate Ancylostoma duodenale, Necator americanus, Strongyloides stercoralis, Ascaris lumbricoides, Trichuris trichiuria and Schistosoma spp. by HRM curve analysis. Here we describe the primers and the results of a pilot assessment whereby the HRM assay was tested against a selection of archived fecal samples from Ghanaian children as characterized by Kato–Katz and real-time PCR analysis with species-specific TaqMan hydrolysis probes. The resulting sensitivity and specificity of the HRM was 80 and 98.6% respectively. We judge the assay to be appropriate in modestly equipped and resourced laboratories. This method provides a potentially cheaper alternative to the TaqMan method for laboratories in lower resource settings. However, the assay requires a more extensive assessment as the samples used were not representative of all target organisms.
机译：随着在低收入和中等收入国家的控制和消除土壤传播的蠕虫症和血吸虫病的推动，需要开发替代诊断测定，这些诊断测定可以补充当前的国内资源，最好是较低的成本。这里，我们描述了一种具有六个PCR引物对的新型高分辨率熔体（HRM）曲线测定，所述PCR引物对设计为核核糖体基因座的子区域。它们在单一反应和染料检测通道中使用，它们能够区分猴头生瘤Duodenale，Necator Americanus，Strickyloides Stercoralis，Ascanis Lumbroides，Trichuris Trichiuria和Schistosoma SPP。通过HRM曲线分析。在这里，我们描述了引物和试验评估的结果，其中HRM测定对来自加纳儿童的一系列存档的粪便样本进行了测试，如Kato-Katz的特征在于用特定于特异性Taqman水解探针的实时PCR分析。得到的HRM的敏感性和特异性分别为80％和98.6％。我们在适用于谦虚和资源的实验室审理该检验。该方法提供了在较低资源设置中的实验室的Taqman方法潜在更便宜的替代方法。然而，该测定需要更广泛的评估，因为所用的样品没有代表所有目标生物。

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