A simple and high sensitive method for detection of B-RAF 1799T>A (V600E) mutation in the thyroid fine needle aspirate

Abstract

OBJECTIVE: In our report, we prospectively estimated a cohort of patients with nodules considered suspicious sonographically, for PTC. We evaluated 24 samples from PTC patients. The sensitivity, specificity, and negative predictive values (NPV) of FNAB BRAF testing in this study were 0.1% (analytical sensitivity), 100%, and 78%, respectively. In terms of assay costs, our PNA PCR-based method could be like either Allele Specific-PCR (AS-PCR) and/or Restriction Fragment Length Polymorphism (RFLP). PATIENTS AND METHODS: PNA chemistry has been performed to amplify selectively minor mutant allele background variants in a large amount of wild type (wt) allele derived from DNA extracted by 24 thyroid biopsies; 14 of them were obtained from cytological slides and 10 from fresh Fine Needle Aspirate (FNA) of patients with papillary thyroid carcinoma (PTC). Finally, we compared the results with those gained by direct sequencing. RESULTS: The assay sensitivity of the method was 0.1% of mutant alleles, evaluated by serial dilution of DNA from ARO cell line (carrying heterozygous V600E), mixed to wild type DNA gained from healthy donors. Optimized concentration of the primer PNA clamping-wt DNA is 12 µM per reaction. Direct sequencing was able to detect mutation in only 33.3% (8/24), while PNA-based PCR assay 45.8% (11/24) of patients, carrying mutation at codon V600. The estimated reagents costs are about € 20.00 per sample, including controls and pre-analytical steps. This assay could be performed in a simple thermal cycler and results visualized by agarose ethidium bromide stained gels. CONCLUSIONS: PNA clamping wt-DNA could be performed in any laboratory with very cheap PCR equipment. It is very cost-effective and could easily be adapted to detect hot spot mutations in any other genes.

Keywords

• nb-raf mutations
• mutant minor allele detection
• pharmacogenomics
• pnaclamping pcr