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Allele-Specific PCR with Competitive Probe Blocking for Sensitive and Specific Detection of BRAF V600E in Thyroid Fine-Needle Aspiration Specimens

Allele-Specific PCR with Competitive Probe Blocking for Sensitive and Specific Detection of BRAF... Objective: To detect BRAF V600E mutation in thyroid fine-needle aspiration (FNA) slides and needle rinses (NR). Study Design: Tumor-enriched DNA was extracted from FNA smears, formalin-fixed paraffin-embedded (FFPE) sections, or NR specimens from 37 patients with confirmed papillary thyroid carcinoma or benign findings. An allele-specific primer selectively amplified the 1799 T>A BRAF mutation while simultaneously blocking amplification of wild-type (WT) BRAF with an unlabeled probe during PCR. Mutation detection was accomplished by melting analysis of the probe. Results: Allele-specific/blocking probe PCR confirmed the BRAF mutation status for 20 of 24 paired FNA/FFPE samples previously tested by fluorescent probe real-time PCR. For the other 4 cases, the sensitive PCR method detected the BRAF mutation in all paired FNA/FFPE samples. Previously, the mutation had been detected in only the FFPE samples. The BRAF mutation was also detected in some NR specimens. Conclusion: Treatment of patients with thyroid nodules is guided by FNA biopsy, which can be scantly cellular, necessitating a sensitive test that can detect low levels of BRAF V600E mutation in a WT background. We report increased detection of BRAF V600E in FNA specimens using allele-specific/blocking probe PCR, which has an analytical sensitivity of 0.01%. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Acta Cytologica Karger

Allele-Specific PCR with Competitive Probe Blocking for Sensitive and Specific Detection of BRAF V600E in Thyroid Fine-Needle Aspiration Specimens

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References (23)

Publisher
Karger
Copyright
© 2011 S. Karger AG, Basel
ISSN
0001-5547
eISSN
1938-2650
DOI
10.1159/000333453
Publisher site
See Article on Publisher Site

Abstract

Objective: To detect BRAF V600E mutation in thyroid fine-needle aspiration (FNA) slides and needle rinses (NR). Study Design: Tumor-enriched DNA was extracted from FNA smears, formalin-fixed paraffin-embedded (FFPE) sections, or NR specimens from 37 patients with confirmed papillary thyroid carcinoma or benign findings. An allele-specific primer selectively amplified the 1799 T>A BRAF mutation while simultaneously blocking amplification of wild-type (WT) BRAF with an unlabeled probe during PCR. Mutation detection was accomplished by melting analysis of the probe. Results: Allele-specific/blocking probe PCR confirmed the BRAF mutation status for 20 of 24 paired FNA/FFPE samples previously tested by fluorescent probe real-time PCR. For the other 4 cases, the sensitive PCR method detected the BRAF mutation in all paired FNA/FFPE samples. Previously, the mutation had been detected in only the FFPE samples. The BRAF mutation was also detected in some NR specimens. Conclusion: Treatment of patients with thyroid nodules is guided by FNA biopsy, which can be scantly cellular, necessitating a sensitive test that can detect low levels of BRAF V600E mutation in a WT background. We report increased detection of BRAF V600E in FNA specimens using allele-specific/blocking probe PCR, which has an analytical sensitivity of 0.01%.

Journal

Acta CytologicaKarger

Published: Dec 1, 2011

Keywords: BRAF V600E mutation; Papillary thyroid carcinoma; Aspiration biopsy; Fine-needle aspiration; Thyroid cancer; Allele-specific PCR; Competitive probe blocking

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