Development and Evaluation of a Duplex Real-Time PCR Assay With a Novel Internal Standard for Precise Quantification of Plasma DNA

Abstract

Background: Circulating levels of cell-free DNA increase in many pathologic conditions. However, notable discrepancies in the quantitative analysis of cell-free DNA from a large number of laboratories have become a considerable pitfall, hampering its clinical application.

Methods: We designed a novel recombinant DNA fragment that could be applied as an internal standard in a newly developed and validated duplex real-time PCR assay for the quantitative analysis of total cell-free plasma DNA, which was tested in 5,442 healthy adults and 200 trauma patients.

Results: Compared with two traditional methods, this novel assay showed a lower detection limit of 0.1 ng/mL, lower intra- and inter-assay CVs, and higher accuracy in the recovery test. The median plasma DNA concentration of healthy males (20.3 ng/mL, n=3,092) was significantly higher than that of healthy females (16.1 ng/mL, n=2,350) (Mann-Whitney two-sample rank sum test, P<0.0001). The reference intervals of plasma DNA concentration were 0-45.8 ng/mL and 0-52.5 ng/mL for healthy females and males, respectively. The plasma DNA concentrations of the majority of trauma patients (96%) were higher than the upper normal cutoff values and were closely related to the corresponding injury severity scores (R²=0.916, P<0.0001).

Conclusions: This duplex real-time PCR assay with a new internal standard could eliminate variation and allow for more sensitive, repeatable, accurate, and stable quantitative measurements of plasma DNA, showing promising application in clinical diagnosis.

Keywords: Duplex PCR; Internal standard; Plasma DNA; Quantification; Reference interval; Trauma.

Fig. 1. The schematic concepts of duplex real-time PCR with internal standard for quantitative detection of plasma DNA. The whole process includes 4 steps: (A) A 41-bp artificial double-stranded DNA sequence corresponding to the human β–actin gene is cloned and inserted to pMD18-T vector, which is then linearized by restriction enzyme digestion. (B) The recombinant plasmid DNA is added into the cell-free plasma sample with known concentration and extracted together with endogenous nucleic acids. (C) The internal standard and target gene are then amplified simultaneously in the same tube with the common reverse primer focusing on the 41-bp corresponding sequence by duplex real-time PCR, where fluorescence signals are detected separately (Applied Biosystems 7500 Sequence Detector). (D) The plasma DNA concentrations are calculated according to the internal standard by the equation.

Fig. 2. Evaluation of the specificity of the duplex real-time PCR assay with internal standard. Real-time PCR amplifications of plasma DNA, internal standard DNA, and the mixture of both in a parallel PCR reaction with their corresponding primers and probes are performed in preliminary experiments to confirm the specificity of this novel duplex PCR assay (A). The target β–actin gene and internal standard are amplified by their corresponding primers for 91-bp and 99-bp PCR products respectively (B) and Taqman probes for fluorescent signals released from JOE and FAM reports and detected in channels 2 and 1 of the ABI 7500 Sequence Detector (C).

Fig. 3. Evaluation of the sensitivity of the three different plasma DNA quantitative assays. The statistically significant correlations of DNA levels of 10-fold serial diluted plasma samples (X-axis, common logarithmic scale) with quantitative results (Y-axis, common logarithmic scale) determined by the novel duplex real-time PCR assay with (A) internal standard, (B) the single real-time PCR assay with external standards, and (C) the dye PicoGreen assay.

Fig. 4. Quantitative analysis of plasma DNA in the plasma of 400 healthy adults by three methods. While the median plasma DNA concentration of 400 healthy adults determined by duplex real time PCR with internal standard (18.1 ng/mL) was significantly higher than those determined by real time PCR and PicoGreen assay with external standards (8.2 ng/ml and 13.3 ng/ml; Wilcoxon matched-pairs signed-rank test, P=0.0000) (A), there were statistically significant but weak positive correlations among plasma DNA levels determined by these three different methods (B-D).

Fig. 5. Quantitative analysis of plasma DNA in 5,443 healthy adults and 200 trauma patients by the duplex real time PCR assay with internal standard. (A) The histogram shows a positively skewed distribution of populations in the different levels of plasma DNA. (B) Box-plots of plasma DNA in healthy adults and trauma patients with different genders shows the reference intervals of plasma DNA concentration are 0-45.8 ng/mL and 0-52.5 ng/mL for healthy females and males, respectively, and the plasma DNA concentrations of most trauma patients (96%) are higher than the upper reference ranges. (C) A statistically significant positive correlation is revealed between plasma DNA concentrations in trauma patients with their ISS values (Spearman rank correlation test, R²=0.916, P<0.0001).