Development and Evaluation of a Duplex Real-Time PCR Assay With a Novel Internal Standard for Precise Quantification of Plasma DNA.
- Author:
Dan CHEN
1
;
Shiyang PAN
;
Erfu XIE
;
Li GAO
;
Huaguo XU
;
Wenying XIA
;
Ting XU
;
Peijun HUANG
Author Information
- Publication Type:Original Article
- Keywords: Plasma DNA; Duplex PCR; Quantification; Internal standard; Reference interval; Trauma
- MeSH: Adult; DNA/*blood/standards; Female; Healthy Volunteers; Humans; Male; Middle Aged; Real-Time Polymerase Chain Reaction/*methods/standards; Reference Values; Wounds and Injuries/blood
- From:Annals of Laboratory Medicine 2017;37(1):18-27
- CountryRepublic of Korea
- Language:English
- 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.
