分支DNA技术检测血浆游离DNA方法的建立及在急性心肌梗死患者中的初步应用

Establishment and application of a ALU-based bDNA for the detection of plasma circulating free DNA in acute myocardial infarction

目的采用分支DNA(bDNA)信号放大定量技术建立检测血中游离DNA(cf-DNA)-ALU基因表达的方法并分析其在急性心肌梗死(AMI)患者血浆中的含量。方法根据ALU基因序列特点设计探针,建立检测血ALU的bDNA信号放大定量方法,并对其线性、灵敏度及精密度进行评价;根据标准曲线计算出AMI患者血浆中ALU的含量,并与肌钙蛋白I(cTnI)、肌酸激酶同功酶MB(CK-MB)和肌红蛋白(MYO)进行相关性分析。结果线性范围为0～400 ng/mL,相关系数为0.99,批内变异系数(CV)为7.85%～11.75%,批间CV为10.05%～14.32%。AMI患者和健康对照组血浆ALU的含量分别是中位数4 223 ng/mL和118 ng/mL,四分位数区间(2 285～7 864)ng/mL和(81～218)ng/mL(P<0.001),提示AMI患者血浆ALU含量上调,但与cTnI、CK-MB和MYO浓度无相关性。ALU的ROC曲线下面积为0.99,敏感度98.8%,特异性100.0%。结论本研究建立的检测方法具有灵敏、重复性好等优点,升高的ALU含量与AMI存在一定相关性。

Objective To establish a ALU-based branched DNA （bDNA） method for detection of human plasma circulating free DNA （cf-DNA） and to investigate its concentration in acute myocardial infarction （AMI）. Methods Based on the sequence of ALU, a bDNA method was established, Liner, sensitivity and reproducibility were evalu- ated according to the standard curve created, the concentration of ALU in AMI was determined and the relationship of cf-DNA concentrations with cardiac enzymes were presented. Results The linear rang was 0 ~ 400 ng/mL, thecoefficient correlation was 0. 99, the intra-assay coefficient variation was 7. 85 % ＋ 11.75%, the inter-assay coeffi- cient variation wasl0. 05% ~ 14. 32%. The concentration of ALU in AMI and healthy controls were 4 223 ng/mL and 118 ng/mL （P 〈0. 001 ） respectively indicating that ALU concentration was increased in AMI. No correlation was found between ALU and cardiac enzymes. The area under ROC was 0. 99, the sensitivity was 98.8% and the specificity was 100.0%. Conclusions The ALU-based bDNA method is sensitive and reproductive ; cf-DNA con- centration is associated with AMI.