title: Allele-specific PCR with competitive probe blocking for sensitive and specific detection of BRAF V600E in thyroid fine-needle aspiration specimens.
abstract: OBJECTIVE
  To detect BRAF V600E mutation in thyroid fine-needle aspiration (FNA) slides and needle rinses (NR).
  
  
  METHODS
  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.
  
  
  CONCLUSIONS
  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%.
authors.affiliation: ARUP Institute for Clinical and Experimental Pathology®, ARUP Laboratories, Inc., and Department of Pathology, University of Utah, Salt Lake City, Utah, USA.
authors.forename: G Denice
authors.initials: GD
authors.lastname: Smith
authors.forename: Luming
authors.initials: L
authors.lastname: Zhou
authors.forename: Leslie R
authors.initials: LR
authors.lastname: Rowe
authors.forename: Elke A
authors.initials: EA
authors.lastname: Jarboe
authors.forename: Brian T
authors.initials: BT
authors.lastname: Collins
authors.forename: Joel S
authors.initials: JS
authors.lastname: Bentz
authors.forename: Carl T
authors.initials: CT
authors.lastname: Wittwer
authors.forename: Barbara E
authors.initials: BE
authors.lastname: Chadwick
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D000483:Alleles; D044963:Biopsy, Fine-Needle; D002277:Carcinoma / Q000175:diagnosis / Q000235:genetics* / Q000473:pathology; D002291:Carcinoma, Papillary; D004252:DNA Mutational Analysis / Q000379:methods*; D017931:DNA Primers / Q000235:genetics; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D016133:Polymerase Chain Reaction / Q000379:methods*; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics*; D012189:Retrospective Studies; D012306:Risk; D012680:Sensitivity and Specificity; D000077273:Thyroid Cancer, Papillary; D013961:Thyroid Gland / Q000378:metabolism / Q000473:pathology; D013964:Thyroid Neoplasms / Q000175:diagnosis / Q000235:genetics* / Q000473:pathology; D016606:Thyroid Nodule / Q000175:diagnosis / Q000235:genetics* / Q000473:pathology
chemical_list: D017931:DNA Primers; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf