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Acta Cytol. 2011;55(6):576-83. doi: 10.1159/000333453. Epub 2011 Dec 9.

Allele-specific PCR with competitive probe blocking for sensitive and specific detection of BRAF V600E in thyroid fine-needle aspiration specimens.

Author information

1
ARUP Institute for Clinical and Experimental Pathology®, ARUP Laboratories, Inc., and Department of Pathology, University of Utah, Salt Lake City, Utah, USA.

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%.

PMID:
22156469
DOI:
10.1159/000333453
[Indexed for MEDLINE]

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