基于B1序列的实时定量PCR：一种直接测量γ射线照射后小鼠血浆DNA水平的灵敏方法。

B1 sequence-based real-time quantitative PCR: a sensitive method for direct measurement of mouse plasma DNA levels after gamma irradiation.

作者信息

Zhang Hengshan, Zhang Steven B, Sun Weimin, Yang Shanmin, Zhang Mei, Wang Wei, Liu Chaomei, Zhang Kunzhong, Swarts Steven, Fenton Bruce M, Keng Peter, Maguire David, Okunieff Paul, Zhang Lurong

机构信息

Department of Radiation Oncology, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642-8647, USA.

出版信息

Int J Radiat Oncol Biol Phys. 2009 Aug 1;74(5):1592-9. doi: 10.1016/j.ijrobp.2009.03.009.

DOI:10.1016/j.ijrobp.2009.03.009

PMID:19616745

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC2883165/

Abstract

PURPOSE

Current biodosimetric techniques for determining radiation exposure have inherent delays, as well as quantitation and interpretation limitations. We have identified a new technique with the advantage of directly measuring circulating DNA by amplifying inter-B1 regions in the mouse genome, providing a sensitive method for quantitating plasma DNA.

METHODS AND MATERIALS

Real-time quantitative polymerase chain reaction (PCR) was used to detect levels of DNA by amplifying inter-B1 genomic DNA in plasma samples collected at 0-48 h from mice receiving 0-10 Gy total- or partial-body irradiation ((137)Cs gamma-ray source at approximately 1.86 Gy/min; homogeneity: +/- 6.5%).

RESULTS

The correlation coefficient between DNA levels and the threshold cycle value (C(T)) was 0.996, and the average recoveries of DNA in the assay were 87%. This assay revealed that when BALB/c mice were exposed to 10 Gy total-body irradiation (TBI), plasma DNA levels gradually increased beginning at 3 h after irradiation, peaked at 9 h, and returned to baseline within 48 h. Increased plasma DNA levels were also detected following upper-torso or lower-torso partial-body irradiation; however, TBI approximately doubled those plasma DNA levels at the same radiation dose. This technique therefore reflects total body cell damage. The advantages of this assay are that DNA extraction is not required, the assay is highly sensitive (0.002 ng), and results can be obtained within 2.5 h after collection of plasma samples.

CONCLUSIONS

A radiation dose-dependent increase of plasma DNA was observed in the dose range from 2 to 10 Gy, suggesting that plasma DNA may be a useful radiation biomarker and adjunct to existing cell-based assays.

摘要

目的

当前用于确定辐射暴露的生物剂量测定技术存在固有的延迟，以及定量和解释方面的局限性。我们已经确定了一种新技术，其优点是通过扩增小鼠基因组中的B1区间直接测量循环DNA，为定量血浆DNA提供了一种灵敏的方法。

方法和材料

使用实时定量聚合酶链反应（PCR）通过扩增在0至48小时从接受0至10 Gy全身或局部照射的小鼠（以约1.86 Gy/min的(137)Csγ射线源；均匀性：±6.5%）采集的血浆样本中的B1基因组DNA来检测DNA水平。

结果

DNA水平与阈值循环值（C(T)）之间的相关系数为0.996，该测定中DNA的平均回收率为87%。该测定显示，当BALB/c小鼠接受10 Gy全身照射（TBI）时，血浆DNA水平在照射后3小时开始逐渐升高，在9小时达到峰值，并在48小时内恢复到基线。在上半身或下半身局部照射后也检测到血浆DNA水平升高；然而，在相同辐射剂量下，TBI使那些血浆DNA水平大约增加了一倍。因此，该技术反映了全身细胞损伤。该测定的优点是不需要提取DNA，测定高度灵敏（0.002 ng），并且在采集血浆样本后2.5小时内即可获得结果。