Development and Evaluation of a Duplex Real-Time PCR Assay With a Novel Internal Standard for Precise Quantification of Plasma DNA.
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.
Department of Laboratory Medicine, the First Affiliated Hospital of Nanjing Medical University, Nanjing, China.|National Key Clinical Department of Laboratory Medicine, the First Affiliated Hospital of Nanjing Medical University, Nanjing, China.
Dan
D
Chen
Department of Laboratory Medicine, the First Affiliated Hospital of Nanjing Medical University, Nanjing, China.|National Key Clinical Department of Laboratory Medicine, the First Affiliated Hospital of Nanjing Medical University, Nanjing, China. sypan@njmu.edu.cn.
Shiyang
S
Pan
Department of Laboratory Medicine, the First Affiliated Hospital of Nanjing Medical University, Nanjing, China.|National Key Clinical Department of Laboratory Medicine, the First Affiliated Hospital of Nanjing Medical University, Nanjing, China.
Erfu
E
Xie
Department of Laboratory Medicine, the First Affiliated Hospital of Nanjing Medical University, Nanjing, China.|National Key Clinical Department of Laboratory Medicine, the First Affiliated Hospital of Nanjing Medical University, Nanjing, China.
Li
L
Gao
Department of Laboratory Medicine, the First Affiliated Hospital of Nanjing Medical University, Nanjing, China.|National Key Clinical Department of Laboratory Medicine, the First Affiliated Hospital of Nanjing Medical University, Nanjing, China.
Huaguo
H
Xu
Department of Laboratory Medicine, the First Affiliated Hospital of Nanjing Medical University, Nanjing, China.|National Key Clinical Department of Laboratory Medicine, the First Affiliated Hospital of Nanjing Medical University, Nanjing, China.
Wenying
W
Xia
Department of Laboratory Medicine, the First Affiliated Hospital of Nanjing Medical University, Nanjing, China.|National Key Clinical Department of Laboratory Medicine, the First Affiliated Hospital of Nanjing Medical University, Nanjing, China.
Ting
T
Xu
Department of Laboratory Medicine, the First Affiliated Hospital of Nanjing Medical University, Nanjing, China.|National Key Clinical Department of Laboratory Medicine, the First Affiliated Hospital of Nanjing Medical University, Nanjing, China.
Peijun
P
Huang
D000328:Adult; D004247:DNA / Q000097:blood* / Q000592:standards; D005260:Female; D064368:Healthy Volunteers; D006801:Humans; D008297:Male; D008875:Middle Aged; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012016:Reference Values; D014947:Wounds and Injuries / Q000097:blood
D004247:DNA
Duplex PCR; Internal standard; Plasma DNA; Quantification; Reference interval; Trauma