血红蛋白对临床FQ PCR检测结果的影响

Influence of hematoglobin to clinical serum FQ PCR detection

目的分析血红蛋白对血清FQ-PCR检测的影响。方法通过Grubbs离散值分析法和置信区间估计法对扩增效率进行分析;制备不同血红蛋白含量血清标本进行FQ PCR法HBV-DNA定量检测,分析测定值。结果扩增曲线分析中插入2批次中溶血标本的扩增效率与同批质控样本、标准参考品E0.05-0.1数值组差异无统计学意义;含血红蛋白0.6、3.0、5.0、10.0、20.0G/L的高DNA浓度(1.35E+7 copies/ml)样本浓度测定值分别为对照的83%、51%、22%、15%、8%,呈明显趋势性变化;低浓度(3.43E+3 copies/ml)样本浓度测定值则未见明显趋势性变化。结论血红蛋白对FQ-PCR检测的干扰可能主要产生于标本制备过程中有效模板的损失。

Objective To evaluate the influence of Hematoglobin to clinical HBV-DNA quantitative detection.Methods Grubbs outlier analysis and confidence range estimation were applied to analyze the amplification efficiencies.In order to investigate the influence,the samples with different designed levels of Hematoglobin were made up for HBV-DNA quantitative detection using real time FQ-PCR.Results Amplification efficiencies E0.05-0.1 of hemolytic samples illustrated no significant variance with E0.05-0.1 with normal serum samples and standard reference samples in same batch.However,the samples of high DNA level（1.35E＋7 copies/ml） with different Hematoglobin concentrations of 0.6、3.0、5.0、10.0、20.0G/L measured 83%,51%,22%,15% and 8% HBV DNA copies of samples without Hematoglobin,while samples of low level（3.43E＋3 copies/ml）,the samples with different Hematoglobin concentrations showed no distinct trend of HBV DNA level.Conclusion Hematoglobin may influence HBV DNA detection using FQ-PCR through template loss during sample preparation other than inhibits of amplification efficiency.