Department of Laboratory Medicine, Chosun University Medical School, Gwangju, Korea.
Am J Clin Pathol. 2013 Jun;139(6):759-64. doi: 10.1309/AJCPN3ULH6YWBHPH.
We used pyrosequencing, peptide nucleic acid (PNA)-clamping polymerase chain reaction (PCR), and real-time PCR to detect the BRAF V600E mutation and to investigate the prognostic effect of the BRAF V600E mutation in paraffin block specimens from 100 patients diagnosed with papillary thyroid carcinoma. Positive rates of PNA-clamping PCR, real-time PCR, and pyrosequencing were 66%, 70%, and 68%, respectively. Pyrosequencing and PNA-clamping PCR detected mutant type in a 99:1 (wild-type: mutant) DNA concentration, and PNA-clamping PCR detected mutant type in a 99.5:0.5 DNA concentration. Clamping PCR showed higher κ value than real-time PCR (0.729 vs 0.626). The BRAF V600E mutation was associated with an advanced stage of cancer (P = .045) and was found to be associated with poor prognostic factors. This study suggests that pyrosequencing can be as sensitive as real-time PCR and that PNA-clamping PCR is a sensitive and reliable method to detect the BRAF V600E mutation.
我们使用焦磷酸测序、肽核酸（PNA）-夹式聚合酶链反应（PCR）和实时 PCR 来检测 100 例甲状腺乳头状癌石蜡块标本中的 BRAF V600E 突变，并探讨 BRAF V600E 突变的预后作用。PNA 夹式 PCR、实时 PCR 和焦磷酸测序的阳性率分别为 66%、70%和 68%。焦磷酸测序和 PNA 夹式 PCR 在 99:1（野生型:突变型）DNA 浓度下检测到突变型，而 PNA 夹式 PCR 在 99.5:0.5 DNA 浓度下检测到突变型。夹式 PCR 的 κ 值高于实时 PCR（0.729 比 0.626）。BRAF V600E 突变与癌症的晚期阶段相关（P =.045），并且与不良预后因素相关。本研究表明，焦磷酸测序可以像实时 PCR 一样敏感，而 PNA 夹式 PCR 是一种敏感且可靠的检测 BRAF V600E 突变的方法。
