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

Abstract

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.

Circulating DNA in rheumatoid arthritis: pathological changes and association with clinically used serological markers

“…Mitochondrial circulating DNA (m-cirDNA) was also elevated in patients with cancer and disorders associated with massive cell damage, such as acute ischemic stroke [11], myocardial infarction [12], trauma [13], and severe sepsis [14]. The biological role of n/m-cirDNA remains enigmatic in both health and pathology.…”

Droplet digital PCR combined with minisequencing, a new approach to analyze fetal DNA from maternal blood: application to the non-invasive prenatal diagnosis of achondroplasia

“…The study of cfDNA is becoming a crucial tool for diagnosis and management in various clinical disorders. Indeed, increased levels of cfDNA have been reported in a number of clinicopathological conditions such as cancer, stroke, trauma, myocardial infarction, autoimmune disorders and pre‐eclampsia . Additionally, cfDNA from fetal origin can also be detected in the plasma of the mother as early as from the 5th week of gestation, opening the field for many applications of non‐invasive prenatal testing such as fetal gender determination or RHD genotyping, chromosomal aneuploidies, and an increasing number of single gene disorders …”

“…Indeed, increased levels of cfDNA have been reported in a number of clinicopathological conditions such as cancer, stroke, trauma, myocardial infarction, autoimmune disorders and preeclampsia. [8][9][10][11][12][13][14][15] Additionally, cfDNA from fetal origin can also be detected in the plasma of the mother as early as from the 5th week of gestation, 16 opening the field for many applications of non-invasive prenatal testing such as fetal gender determination 16,17 or RHD genotyping, 18 chromosomal aneuploidies, [19][20][21][22][23] and an increasing number of single gene disorders. [24][25][26][27][28][29][30] In this work, we developed a novel and original strategy based on ddPCR combined with minisequencing, allowing the non-invasive prenatal diagnosis of achondroplasia.…”

“…Two additional healthy subjects, negative for FGFR3 mutation, were used as negative controls. Written informed consent was obtained prior to venipuncture, and the study was ethically approved by the Comité Consultatif sur le Traitement de l'Information en matière de Recherche dans le domaine de la Santé (CCTIRS -ref 13.386) and the Comité de Protection des Personnes (CPP ref 2014-janvier-13465). In all cases, confirmation of prenatal testing result was performed by conventional procedures, blindly to the results of non-invasive testing.…”

The evidence base for circulating tumour DNA blood-based biomarkers for the early detection of cancer: a systematic mapping review

“…Since the first description of circulating cfDNA in blood [ 5 , 6 ], it has become clear that total ctDNA levels rise in a number of disorders in addition to cancer including myocardial infarction [ 7 ], serious infections, and inflammatory conditions [ 8 ], as well as pregnancy where it can be used for prenatal diagnosis [ 9 ]. The source of this DNA appears to be mainly the result of cell death – either by necrosis or apoptosis [ 5 , 9 – 11 ].…”