Optimization and Validation of a Folate Ligand-Targeted qPCR Method for Detection of Folate Receptor-Positive...

Area of Science:

• Biomolecular Engineering
• Molecular Diagnostics
• Oncology

Background:

• Gastric cancer poses a significant global health challenge with limited early detection and poor prognosis.
• Folate receptor (FR)-positive circulating tumor cells (CTCs) are promising biomarkers due to FR-alpha's overexpression in cancer tissues.

Purpose of the Study:

• To develop and optimize a Folate-Ligand Targeted quantitative PCR (Folate-LT qPCR) method.
• To enable selective detection and quantification of FR-positive CTCs in gastric cancer patients.

Main Methods:

• Designed a folate-oligonucleotide conjugate and a specific stem-loop primer for complementary DNA synthesis.
• Optimized qPCR using candidate primers and validated the assay via spiking experiments and CTC enrichment.
• Assessed performance through amplification efficiency, melt curve analysis, and gel electrophoresis.

Main Results:

• Primer F1.1 showed superior performance with consistent amplification and no non-specific binding.
• Established optimal annealing temperatures (40-45°C) and demonstrated strong linearity (R2 = 0.9970) in the standard curve.
• Achieved 104% amplification efficiency, confirming reliable quantification across a wide dynamic range.

Conclusions:

• The optimized Folate-LT qPCR method is sensitive and specific for detecting FR-positive CTCs in gastric cancer.
• This technique overcomes limitations of existing methods, requiring minimal blood volume and avoiding marker-dependent issues.
• The assay holds promise for early cancer detection, disease monitoring, and treatment assessment, potentially improving patient outcomes.