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

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Real-time polymerase chain reaction (PCR) with melt analysis is used to detect PCR products.
• High-resolution melt (HRM) analysis enhances PCR product characterization by improving melt temperature precision.
• Single base changes in tuberculosis (TB) genes like *katG* can confer resistance to first-line drugs such as isoniazid (INH).

Purpose of the Study:

• To develop and validate a reagent-based calibration strategy, LHRM (left-handed HRM), for single-base resolution PCR product characterization.
• To assess the performance of LHRM in classifying *katG* variants associated with isoniazid resistance in TB.

Main Methods:

• A synthetic double-stranded left-handed DNA (L-DNA) melt comparator was added to each sample as an internal standard.
• LHRM and standard HRM were employed to classify nine synthetic *katG* variants with known mutations conferring INH resistance.
• Performance was evaluated using sensitivity and specificity metrics for both single-sample and multiple-sample analyses.

Main Results:

• LHRM achieved classification performance comparable to standard HRM by utilizing within-sample melt differences between L-DNA and unknown PCR products.
• Standard HRM in multiple sample analysis showed 66.7% sensitivity and 98.8% specificity.
• Single-sample LHRM analysis, incorporating L-DNA calibration, demonstrated improved performance with 77.8% sensitivity and 98.7% specificity.

Conclusions:

• LHRM provides a robust, single-sample method for validating PCR products with single-base resolution.
• This approach shows significant promise for applications requiring precise sequence characterization, such as TB drug susceptibility screening.
• LHRM offers a reliable alternative for identifying genetic variations impacting drug efficacy.