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
Dans Anglais
| 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.
Texte intégral:
Disponible
Indice:
WPRIM (Pacifique occidental)
Sujet Principal:
Valeurs de référence
/
Plaies et blessures
/
ADN
/
Réaction de polymérisation en chaine en temps réel
/
Volontaires sains
Limites du sujet:
Adulte
/
Femelle
/
Humains
/
Mâle
langue:
Anglais
Texte intégral:
Annals of Laboratory Medicine
Année:
2017
Type:
Article
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