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首页> 外文期刊>Diagnostic microbiology and infectious disease >Pyrosequencing-based validation of a simple cell-suspension polymerase chain reaction assay for Campylobacter with application of high-processivity polymerase and novel internal amplification controls for rapid and specific detection
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Pyrosequencing-based validation of a simple cell-suspension polymerase chain reaction assay for Campylobacter with application of high-processivity polymerase and novel internal amplification controls for rapid and specific detection

机译：基于焦磷酸测序的弯曲杆菌简单细胞悬浮聚合酶链反应分析方法的验证，并应用高生产力聚合酶和新型内部扩增对照物进行快速，特异性检测

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摘要

Although Campylobacter is an important food-borne human pathogen, there remains a lack of molecular diagnostic assays that are simple to use, cost-effective, and provide rapid results in research, clinical, or regulatory laboratories. Of the numerous Campylobacter assays that do exist, to our knowledge none has been empirically tested for specificity using high-throughput sequencing. Here we demonstrate the power of next-generation sequencing to determine the specificity of a widely cited Campylobacter-specific polymerase chain reaction (PCR) assay and describe a rapid method for direct cell suspension PCR to quickly and easily screen samples for Campylobacter. We present a specific protocol which eliminates the need for time-consuming and expensive genomic DNA extractions and, using a high-processivity polymerase, demonstrate conclusive screening of samples in 1 h. Pyrosequencing results show the assay to be extremely (99%) sensitive, and spike-back experiments demonstrated a detection threshold of 10 2 CFU mL -1. Additionally, we present 2 newly designed broad-range bacterial primer sets targeting the 23S rRNA gene that have wide applicability as internal amplification controls. Empirical testing of putative taxon-specific assays using high-throughput sequencing is an important validation step that is now financially feasible for research, regulatory, or clinical applications.
机译：尽管弯曲杆菌是一种重要的食源性人类病原体，但仍缺乏易于使用，具有成本效益且在研究，临床或监管实验室中提供快速结果的分子诊断测定方法。在确实存在的众多弯曲杆菌测定中，据我们所知，尚未使用高通量测序对任何特异性进行经验性测试。在这里，我们展示了新一代测序的功能，可用来确定广为引用的弯曲杆菌特异性聚合酶链反应（PCR）分析的特异性，并描述了一种直接细胞悬液PCR的快速方法，可快速，轻松地筛选弯曲杆菌样品。我们提出了一种特定的方案，该方案消除了对耗时且昂贵的基因组DNA提取的需求，并使用高加工性聚合酶证明了在不到1小时的时间内对样品进行最终筛查。焦磷酸测序结果表明该测定法极其敏感（> 99％），加标定针实验表明检测阈值<10 2 CFU mL -1。此外，我们目前针对23S rRNA基因的2个新设计的大范围细菌引物集，作为内部扩增对照具有广泛的适用性。使用高通量测序对假定的分类单元特异性测定进行经验测试是重要的验证步骤，现在对于研究，监管或临床应用在财务上是可行的。

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