Abstract：
UNASSIGNED: Droplet digital (dd)PCR is a new-generation PCR technique with high precision and sensitivity; however, the positive and negative droplets are not always effectively separated because of the \"rain\" phenomenon. We aimed to develop a practical optimization and evaluation process for the ddPCR assay and to apply it to the detection of BRAF V600E in fine-needle aspiration (FNA) specimens of thyroid nodules, as an example.
UNASSIGNED: We optimized seven ddPCR parameters that can affect \"rain.\" Analytical and clinical performance were analyzed based on histological diagnosis after thyroidectomy using a consecutive prospective series of 242 FNA specimens.
UNASSIGNED: The annealing time and temperature, number of PCR cycles, and primer and probe concentrations were found to be more important considerations for assay optimization than the denaturation time and ramp rate. The limit of blank and 95% limit of detection were 0% and 0.027%, respectively. The sensitivity of ddPCR for histological papillary thyroid carcinoma (PTC) was 82.4% (95% confidence interval [CI], 73.6%-89.2%). The pooled sensitivity of BRAF V600E in FNA specimens for histological PTC was 78.6% (95% CI, 75.9%-81.2%, I2=60.6%).
UNASSIGNED: We present a practical approach for optimizing ddPCR parameters that affect the separation of positive and negative droplets to reduce rain. Our approach to optimizing ddPCR parameters can be expanded to general ddPCR assays for specific mutations in clinical laboratories. The highly sensitive ddPCR can compensate for uncertainty in cytological diagnosis by detecting low levels of BRAF V600E.
UNASSIGNED: We optimized seven ddPCR parameters that can affect \"rain.\" Analytical and clinical performance were analyzed based on histological diagnosis after thyroidectomy using a consecutive prospective series of 242 FNA specimens.
UNASSIGNED: The annealing time and temperature, number of PCR cycles, and primer and probe concentrations were found to be more important considerations for assay optimization than the denaturation time and ramp rate. The limit of blank and 95% limit of detection were 0% and 0.027%, respectively. The sensitivity of ddPCR for histological papillary thyroid carcinoma (PTC) was 82.4% (95% confidence interval [CI], 73.6%-89.2%). The pooled sensitivity of BRAF V600E in FNA specimens for histological PTC was 78.6% (95% CI, 75.9%-81.2%, I2=60.6%).
UNASSIGNED: We present a practical approach for optimizing ddPCR parameters that affect the separation of positive and negative droplets to reduce rain. Our approach to optimizing ddPCR parameters can be expanded to general ddPCR assays for specific mutations in clinical laboratories. The highly sensitive ddPCR can compensate for uncertainty in cytological diagnosis by detecting low levels of BRAF V600E.
摘要：
■液滴数字（dd）PCR是一种高精度和高灵敏度的新一代PCR技术；但是，由于“雨”现象，正负液滴并不总是有效地分离。我们旨在为ddPCR测定开发实用的优化和评估过程，并将其应用于甲状腺结节细针穿刺（FNA）标本中BRAFV600E的检测，作为一个例子。
■我们优化了七个可以影响“雨”的ddPCR参数。“根据甲状腺切除术后的组织学诊断，使用242个FNA标本的连续前瞻性系列进行分析和临床表现。
■退火时间和温度，PCR循环数,发现引物和探针浓度是比变性时间和升温速率更重要的测定优化考虑因素。空白和95%检出限分别为0%和0.027%,分别。ddPCR对甲状腺乳头状癌(PTC)的敏感性为82.4%(95%可信区间[CI],73.6%-89.2%）。FNA标本中BRAFV600E对组织学PTC的合并敏感性为78.6％（95％CI，75.9％-81.2％，I2=60.6%）。
■我们提出了一种实用的方法，用于优化ddPCR参数，该参数影响正负液滴的分离以减少雨水。我们优化ddPCR参数的方法可以扩展到临床实验室中针对特定突变的一般ddPCR测定。高度敏感的ddPCR可以通过检测低水平的BRAFV600E来补偿细胞学诊断中的不确定性。
■我们优化了七个可以影响“雨”的ddPCR参数。“根据甲状腺切除术后的组织学诊断，使用242个FNA标本的连续前瞻性系列进行分析和临床表现。
■退火时间和温度，PCR循环数,发现引物和探针浓度是比变性时间和升温速率更重要的测定优化考虑因素。空白和95%检出限分别为0%和0.027%,分别。ddPCR对甲状腺乳头状癌(PTC)的敏感性为82.4%(95%可信区间[CI],73.6%-89.2%）。FNA标本中BRAFV600E对组织学PTC的合并敏感性为78.6％（95％CI，75.9％-81.2％，I2=60.6%）。
■我们提出了一种实用的方法，用于优化ddPCR参数，该参数影响正负液滴的分离以减少雨水。我们优化ddPCR参数的方法可以扩展到临床实验室中针对特定突变的一般ddPCR测定。高度敏感的ddPCR可以通过检测低水平的BRAFV600E来补偿细胞学诊断中的不确定性。
