Quick-multiplex-consensus (QMC)-PCR followed by high-resolution melting: a simple and robust method for mutation...

Area of Science:

• Oncology
• Molecular Diagnostics
• Pathology

Background:

• Predictive mutation identification is crucial for pathological diagnosis in oncology.
• There is a need for cost-effective and reliable mutation detection methods using formalin-fixed paraffin-embedded (FFPE) tissues.

Purpose of the Study:

• To develop and evaluate the Quick-Multiplex-Consensus PCR (QMC-PCR) protocol combined with High-Resolution Melting (HRM) analysis.
• To assess the sensitivity, specificity, and robustness of QMC-PCR with HRM for mutation detection in FFPE tissues compared to Sanger sequencing.

Main Methods:

• Developed the QMC-PCR protocol for HRM analysis.
• Compared QMC-PCR with Sanger sequencing for mutation detection.
• Validated the assay using DNA from FFPE tissues and cell lines, screening multiple mutation hotspots.

Main Results:

• QMC-PCR with HRM detected as little as 2.5% mutant alleles, significantly more sensitive than Sanger sequencing (20%).
• Screened ten mutation hotspots in KRAS, BRAF, PIK3CA, and CDC4 in cell lines with 100% sensitivity and specificity.
• In FFPE colorectal tumors, QMC-PCR demonstrated 100% sensitivity for KRAS and PIK3CA mutations, with high positive predictive values (up to 84%) across all hotspots tested.

Conclusions:

• QMC-PCR with HRM is a simple, robust, and inexpensive technique for mutation detection.
• The assay offers superior sensitivity compared to Sanger sequencing.
• Highly suitable for rapid screening of multiple mutation hotspots in DNA from FFPE tissues for pathological diagnosis.