A novel Alu-based real-time PCR method for the quantitative detection of plasma circulating cell-free DNA: sensitivity and specificity for the diagnosis of myocardial infarction.
In the present study, we aimed to develop and validate a rapid and sensitive, Alu-based real-time PCR method for the detection of circulating cell-free DNA (cfDNA). This method targeted repetitive elements of the Alu reduplicative elements in the human genome, followed by signal amplification using fluorescence quantification. Standard Alu-puc57 vectors were constructed and 5 pairs of specific primers were designed. Valuation was conducted concerning linearity, variation and recovery. We found 5 linear responses (R1-5=0.998-0.999). The average intra- and inter-assay coefficients of variance were 12.98 and 10.75%, respectively. The recovery was 82.33-114.01%, with a mean recovery index of 101.26%. This Alu-based assay was reliable, accurate and sensitive for the quantitative detection of cfDNA. Plasma from normal controls and patients with myocardial infarction (MI) were analyzed, and the baseline levels of cfDNA were higher in the MI group. The area under the receiver operating characteristic (ROC) curve for Alu1, Alu2, Alu3, Alu4, Alu5 and Alu (Alu1 + Alu2 + Alu3 + Alu4 + Alu5) was 0.887, 0.758, 0.857, 0.940, 0.968 and 0.933, respectively. The optimal cut-off value for Alu1, Alu2, Alu3, Alu4, Alu5 and Alu to predict MI was 3.71, 1.93, 0.22, 3.73, 6.13 and 6.40 log copies/ml. We demonstrate that this new method is a reliable, accurate and sensitive method for the quantitative detection of cfDNA and that it is useful for studying the regulation of cfDNA in certain pathological conditions. Alu4, Alu5 and Alu showed better sensitivity and specificity for the diagnosis of MI compared with cardiac troponin I (cTnI), creatine kinase MB (CK-MB) isoenzyme and lactate dehydrogenase (LDH). Alu5 had the best prognostic ability.
Department of Central Laboratory, Songjiang Hospital Affiliated First People's Hospital, Shanghai Jiao Tong University, Shanghai 201600, P.R. China.
Xiaoli
X
Lou
Department of Central Laboratory, Songjiang Hospital Affiliated First People's Hospital, Shanghai Jiao Tong University, Shanghai 201600, P.R. China.
Yanqiang
Y
Hou
Department of Central Laboratory, Songjiang Hospital Affiliated First People's Hospital, Shanghai Jiao Tong University, Shanghai 201600, P.R. China.
Dongyu
D
Liang
Department of Central Laboratory, Songjiang Hospital Affiliated First People's Hospital, Shanghai Jiao Tong University, Shanghai 201600, P.R. China.
Liang
L
Peng
Department of Central Laboratory, Songjiang Hospital Affiliated First People's Hospital, Shanghai Jiao Tong University, Shanghai 201600, P.R. China.
Hongwei
H
Chen
Department of Central Laboratory, Songjiang Hospital Affiliated First People's Hospital, Shanghai Jiao Tong University, Shanghai 201600, P.R. China.
Shanyuan
S
Ma
Department of Radiation Oncology, Shands Cancer Center, University of Florida, Gainesville, FL 32610, USA.
Lurong
L
Zhang
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D020087:Alu Elements / Q000235:genetics*; D016022:Case-Control Studies; D004247:DNA / Q000097:blood*; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D009203:Myocardial Infarction / Q000097:blood* / Q000175:diagnosis / Q000235:genetics*; D012372:ROC Curve; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D004247:DNA