PDF(Pubmed)
Abstract：
The melt analysis feature in most real-time polymerase chain reaction (PCR) instruments is a simple method for determining if expected or unexpected products are present. High-resolution melt (HRM) analysis seeks to improve the precision of melt temperature measurements for better PCR product sequence characterization. In the area of tuberculosis (TB) drug susceptibility screening, sequencing has shown that a single base change can be sufficient to make a first-line TB drug ineffective. In this study, a reagent-based calibration strategy based on synthetic left-handed (L)-DNA, designated LHRM, was developed to confirm validation of a PCR product with single base resolution. To test this approach, a constant amount of a double-stranded L-DNA melt comparator was added to each sample and used as a within-sample melt standard. The performance of LHRM and standard HRM was used to classify PCR products as drug-susceptible or not drug-susceptible with a test bed of nine synthetic katG variants, each containing single or multiple base mutations that are known to confer resistance to the first-line TB drug isoniazid (INH). LHRM achieved comparable classification to standard HRM relying only on within-sample melt differences between L-DNA and the unknown PCR product. Using a state-of-the-art calibrated instrument and multiple sample classification analysis, standard HRM was performed at 66.7% sensitivity and 98.8% specificity. Single sample analysis incorporating L-DNA for reagent-based calibration into every sample maintained high performance at 77.8% sensitivity and 98.7% specificity. LHRM shows promise as a high-resolution single sample method for validating PCR products in applications where the expected sequence is known.
摘要：
大多数实时聚合酶链反应（PCR）仪器中的解链分析功能是确定是否存在预期或意外产物的简单方法。高分辨率熔解（HRM）分析旨在提高熔解温度测量的精度，以实现更好的PCR产物序列表征。在结核病（TB）药物敏感性筛查领域，测序表明，单个碱基的变化足以使一线TB药物无效。在这项研究中,基于合成左手（L）-DNA的基于试剂的校准策略，指定的LHRM,开发用于确认具有单碱基分辨率的PCR产物的验证。为了测试这种方法，向每个样品中加入恒定量的双链L-DNA解链比较剂,并用作样品内解链标准品。LHRM和标准HRM的性能用于将PCR产物分类为药物敏感或非药物敏感的九种合成katG变体的测试床，每个包含已知赋予一线TB药物异烟肼(INH)耐药性的单个或多个碱基突变.仅依赖于L-DNA和未知PCR产物之间的样品内解链差异,LHRM实现了与标准HRM相当的分类。使用先进的校准仪器和多样本分类分析，标准HRM的敏感性为66.7%,特异性为98.8%.将用于基于试剂的校准的L-DNA并入每个样品中的单样品分析保持77.8%灵敏度和98.7%特异性的高性能。在已知预期序列的应用中，LHRM有望作为验证PCR产物的高分辨率单样品方法。
