Department of Biomedical Engineering, Vanderbilt University, Nashville, Tennessee 37235, United States.
Anal Chem. 2024 Jul 23;96(29):11897-11905. doi: 10.1021/acs.analchem.4c01611. Epub 2024 Jul 8.
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 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.
大多数实时聚合酶链反应（PCR）仪器中的熔解分析功能是一种简单的方法，用于确定是否存在预期或意外的产物。高分辨率熔解（HRM）分析旨在提高熔解温度测量的精度，以更好地对 PCR 产物序列进行特征分析。在结核病（TB）药物敏感性筛选领域，测序表明，单个碱基的改变足以使一线 TB 药物无效。在这项研究中，开发了一种基于试剂的校准策略，该策略基于合成左手（L）-DNA，称为 LHRM，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，将一定量的双链 L-DNA 熔解比较器添加到每个样品中，并用作样品内熔解标准。使用 9 种合成变体的测试床，每种变体包含已知对一线 TB 药物异烟肼（INH）具有抗性的单个或多个碱基突变，来测试 LHRM 和标准 HRM 的性能，将 PCR 产物分类为药物敏感或非药物敏感。LHRM 仅依靠 L-DNA 和未知 PCR 产物之间的样品内熔解差异，实现了与标准 HRM 相当的分类。使用最先进的校准仪器和多个样品分类分析，标准 HRM 的灵敏度为 66.7%，特异性为 98.8%。将用于试剂校准的 L-DNA 纳入每个样品的单个样品分析保持了高性能，灵敏度为 77.8%，特异性为 98.7%。LHRM 有望成为一种高分辨率的单个样品方法，用于验证已知预期序列的 PCR 产物。
