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Development and Evaluation of a Duplex Real-Time PCR Assay With a Novel Internal Standard for Precise Quantification of Plasma DNA
Annals of Laboratory Medicine ; : 18-27, 2017.
Article in English | WPRIM | ID: wpr-72422
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.
Subject(s)

Full text: Available Index: WPRIM (Western Pacific) Main subject: Reference Values / Wounds and Injuries / DNA / Real-Time Polymerase Chain Reaction / Healthy Volunteers Limits: Adult / Female / Humans / Male Language: English Journal: Annals of Laboratory Medicine Year: 2017 Type: Article

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Full text: Available Index: WPRIM (Western Pacific) Main subject: Reference Values / Wounds and Injuries / DNA / Real-Time Polymerase Chain Reaction / Healthy Volunteers Limits: Adult / Female / Humans / Male Language: English Journal: Annals of Laboratory Medicine Year: 2017 Type: Article