Development of a droplet digital PCR assay for the detection of BK polyomavirus

作者：Lu Ai, Yating Zhao, Chianru Tan, Lu Bai, Gang Huang, Ruizhi Wang, Hao Huang, Xuegao Yu, Yong Guo, Peisong Chen · 发表于：Microbiology Spectrum · 年份：2024 · DOI：10.1128/spectrum.01089-24 · 被引用次数：7 · 研究领域：Polyomavirus and related diseases、Energy Harvesting in Wireless Networks、Transplantation: Methods and Outcomes

ABSTRACT The objective of this study was to establish a more sensitive and specific diagnostic method for detecting plasma BK polyomavirus (BKPyV) DNA load in patients after renal transplantation using droplet digital polymerase chain reaction (ddPCR) and to validate the methodology. The linear range, lower limit of detection, accuracy, precision, and specificity of the detection system were evaluated by using the WHO BKPyV standard (7.2 log 10 IU/mL) as a reference, in accordance with the relevant documents of the Clinical and Laboratory Standards Institute. Plasma samples were collected from 74 renal transplantation patients with urinary BKPyV-DNA levels exceeding 7 log 10 copies/mL. Quantitative PCR (qPCR) and ddPCR were performed, and their diagnostic efficacy for BKPyV-DNA in the diagnosis of BK polyomavirus-associated nephropathy was evaluated using a receiver operating characteristic (ROC) curve. The coefficients of variation for the repeated detection of BKPyV standard DNA were 2.55 and 4.71 at concentrations of 6.2 and 3.2 log 10 IU/mL, respectively. The linear range was 2.2–6.2 log 10 IU/mL, and the lowest detection limit was 100 IU/mL. By utilizing histopathological examination of renal biopsy as the gold standard for BKPyV diagnosis, the area under the ROC curve of 74 post-transplantation plasma samples detected by the ddPCR system was found to be 0.875 (95% CI: 0.797–0.953, P < 0.01). The optimal threshold was 512.86 copies/mL, with a sensitivity of 90.0% and ...