Ann Lab Med.  2015 Nov;35(6):624-629. 10.3343/alm.2015.35.6.624.

Evaluation of the Anyplex BRAF V600E Real-Time Detection Assay Using Dual-Priming Oligonucleotide Technology in Fine-Needle Aspirates of Thyroid Nodules

• Choi R
• Park KS
• Kim JW
• Ki CS

• KMID: 2363266
• DOI: http://doi.org/10.3343/alm.2015.35.6.624

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.

Keyword

BRAF V600E; Evaluation; Fine-needle aspiration; Real-time PCR; Seeplex; Anyplex

MeSH Terms

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
DNA
DNA Primers
Oligonucleotides
Proto-Oncogene Proteins B-raf

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