反复冻融对血清HBV DNA定量检测结果的影响  

Effect of multiple freeze-thaw cycles on hepatitis B virus DNA quantification

作　　者：魏燕[1] 程远霞[1] 华泽钊[1] 

机构地区：[1]上海理工大学低温医学与食品冷冻研究所,200093

出　　处：《中国医药生物技术》2008年第1期45-48,共4页Chinese Medicinal Biotechnology

基　　金：国家自然科学基金(50676063)

摘　　要：目的研究反复冻融对血清HBV DNA定量检测结果的影响。方法采集15例慢性乙型肝炎患者静脉血，离心后获得血清标本。将各血清标本于-20℃保存24h，室温解冻lh，完成第1次冻融循环，此后按相同步骤依次进行第2-6次冻融循环。第1次冻融循环前（第0次冻融循环）和每次冻融循环后均取少量血清，采用荧光实时定量PCR（FQ-PCR）技术进行HBV DNA定量检测，计算各血清标本每次冻融循环相对于第0次冻融循环的HBV DNA拷贝数的相对变化量。采用一元线性回归分析方法分析处理实验数据。结果15份血清标本HBV DNA定量检测结果有效范围为5．21×10^2～3．43×10^7拷贝／ml。血清标本HBV DNA定量检测结果显示，在第6次冻融循环的15份血清标本中，有14份血清标本HBV DNA拷贝数与基准水平比较均略有降低；经6次冻融循环后，累计有18．89％（17／90）的血清标本HBV DNA拷贝数略高于基准水平，81．11％（73／90）的血清标本HBV DNA拷贝数略低于基准水平；经2、4、6次冻融循环后，HBV DNA拷贝数与基准水平比较升高〉20％的累计血清标本数（依次为1、2、2份）均分别低于降低〉20％的累计血清标本数（依次为2、4、5份）。一元线性回归分析显示，每次冻融循环后HBV DNA拷贝数与基准水平比较降低约1.4％。结论血清冷冻时间〈24h时，反复冻融后血清标本HBV DNA定量检测结果可基本保持稳定。Objective To study the effect of multiple freeze-thaw cycles on HBV DNA quantification in serum. Methods Blood samples were obtained from 15 patients with chronic hepatitis B, centrifugated, stored at -20℃ for 24 h, and then thawed at room temperature for 1 h as the first freeze-thaw cycle. The following freeze-thawing were done the same as the above. HBV DNA were tested by real-time FQ-PCR using Lightcycler. Quantification of HBV DNA of non-freeze-thawing were used as a standard. Average relative values of HBV DNA of every sample were calculated by comparing with the standards. Simple linear regression equation was used to analyze the data. Results The HBV DNA concentrations in the 15 specimens ranged from 5.21×10^2 to 3.43×10^7 copies/ml. Compared with the original serum samples HBV DNA concentration slightly deceased after 1 to 6 freeze-thaw cycles in 14 samples. During the 6 cycles, totally 18.89% of the samples （17/90） had a slightly higher concentration of HBV DNA, and 81.11% （73/90） slightly lower. The samples with an increased HBV DNA concentration by 20% was less than those with a decreased concentration by 20% after 2, 4, and 6 cycles （1, 2, 2 copies vs 2, 4, 5 copies, respectively）. Simple linear regression equation showed that the HBV DNA concentration decreased by a mean of 1.4% after each cycle. Conclusion When frozen time is shorter than 24 h, quantification results of HBV DNA is stable.

关 键 词：低温保存 肝炎病毒 乙型 DNA复制 聚合酶链反应 

分 类 号：R512.62[医药卫生—内科学] R512.610.3[医药卫生—临床医学]