Evaluation of the Anyplex BRAF V600E Real-Time Detection Assay Using Dual-Priming Oligonucleotide Technology in Fine-Needle Aspirates of Thyroid Nodules.
10.3343/alm.2015.35.6.624
- Author:
Rihwa CHOI
1
;
Kyung Sun PARK
;
Jong Won KIM
;
Chang Seok KI
Author Information
1. Department of Laboratory Medicine and Genetics, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea. changski@skku.edu
- Publication Type:Evaluation Studies ; Original Article ; Research Support, Non-U.S. Gov't
- Keywords:
BRAF V600E;
Evaluation;
Fine-needle aspiration;
Real-time PCR;
Seeplex;
Anyplex
- MeSH:
Adult;
Aged;
Asian Continental Ancestry Group/genetics;
Biopsy, Fine-Needle;
DNA/chemistry/metabolism;
DNA Mutational Analysis/*methods;
DNA Primers/*metabolism;
Female;
Humans;
Male;
Middle Aged;
Multiplex Polymerase Chain Reaction;
Oligonucleotides/metabolism;
Polymorphism, Single Nucleotide;
Proto-Oncogene Proteins B-raf/*genetics;
Republic of Korea;
Thyroid Nodule/*metabolism/pathology
- From:Annals of Laboratory Medicine
2015;35(6):624-629
- CountryRepublic of Korea
- Language:English
-
Abstract:
BACKGROUND: Several molecular assays have been developed to detect the BRAF V600E mutation in fine needle aspirates (FNAs) for the diagnosis of papillary thyroid cancer. Using a multiplex PCR technique, we evaluated the Anyplex BRAF V600E Real-time Detection (Anyplex) assay and compared its efficacy with that of the Seeplex BRAF V600E ACE Detection (Seeplex) method. METHODS: We tested 258 consecutive FNA specimens using the Seeplex and Anyplex assays. Any conflicting results between the two assays were confirmed by using mutant enrichment with 3'-modified oligonucleotide (MEMO) sequencing. The limits of detection (LODs) and reproducibility for each assay were evaluated with serially diluted DNA from a BRAF V600E-positive cell line. RESULTS: The BRAF V600E mutation was detected in 36.4% (94/258) FNA specimens by either the Seeplex or Anyplex assay. Results for the two assays showed 93.4% (241/258) agreement, with a kappa value of 0.861 (95% confidence interval, 0.798-0.923). Of the eight specimens that were BRAF V600E-positive by the Anyplex assay but not by the Seeplex assay, five were found to be BRAF V600E-positive by MEMO sequencing. The mutation detection rate of the Seeplex and Anyplex assays was 79.0% and 84.0%, respectively, in the FNA specimens diagnosed as malignant (n=81). The LOD as determined by probit analysis was 0.046% (95% confidence interval, 0.019-0.532%). CONCLUSIONS: The Anyplex assay performed better than the Seeplex assay with respect to the detection of the BRAF V600E mutation.
