L-DNA-Based Melt Analysis Enables Within-Sample Validation of PCR Products.

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.