부산대학교 도서관

• dPCR protocol investigated measuring hCMV whole virus panels over four years. • First reference measurement procedure for human pathogenic DNA virus quantification. • High reproducibility and trueness confirmed Method for value assignment of viral materials for calibration and quality assurance. • Method has the potential for harmonization of routine hCMV load testing. • Method has the potential to harmonization for routine hCMV load testing. A candidate digital PCR (dPCR)-based reference measurement procedure for quantification of human cytomegalovirus (hCMV) was evaluated in 10 viral load comparison schemes (seven external quality assessment (EQA) and three additional training schemes) organized by INSTAND e.V. over four years (between September 2014 and March 2018). Four metrology institutes participated in these schemes using the same extraction method and dPCR measurement procedure for the hCMV specific target sequence of UL54 gene. The calibration independent reference measurement procedure results from the metrology institutes were compared to the results of the clinical diagnostic laboratories applying hCMV qPCR measurement procedures calibrated to reference materials. While the criteria for the acceptable deviation from the target value interval for INSTAND's EQA schemes is from −0.8 log 10 to +0.8 log 10 , the majority of dPCR results were between −0.2 log 10 to +0.2 log 10. Only 4 out of 45 results exceeded this interval with the maximum deviation of −0.542 log 10. In the training schemes containing samples with lower hCMV concentrations, more than half of the results deviated less than ±0.2 log 10 from the target value, while more than 95% deviated less than ±0.4 log 10 from the target value. Evaluation of intra- and inter-laboratory variation of dPCR results confirmed high reproducibility and trueness of the method. This work demonstrates that dPCR has the potential to act as a calibration independent reference measurement procedure for the value assignment of hCMV calibration and reference materials to support qPCR calibration as well as ultimately for routine hCMV load testing. [ABSTRACT FROM AUTHOR]