Doğantürk Yağmur Eylül, Dağ-Güzel Aylin, Kuşkucu Mert Ahmet
Department of Medical Microbiology, İstanbul University-Cerrahpaşa School of Medicine, İstanbul, Türkiye.
Department of Medical Microbiology, İstanbul Aydın University School of Medicine, İstanbul, Türkiye.
Infect Dis Clin Microbiol. 2023 Dec 29;5(4):353-366. doi: 10.36519/idcm.2023.255. eCollection 2023 Dec.
Digital polymerase chain reaction (dPCR) assay is an advanced PCR technique that allows for the simultaneous detection and absolute quantification of diverse pathogens.Commercially validated kits available for detecting all subtypes of human adenovirus (HAdV) are limited. This study aimed to demonstrate the development of an in-house nanoplate-based dPCR assay with high sensitivity, even at low copy numbers.
In this methodological study, the standardized HAdV DNA was prepared by amplifying the specific hexon gene region with real-time PCR and purifying the HAdV DNA using magnetic beads from HAdV-positive extractions. Dilutions were tested in triplicate during three independent runs to determine the dynamic range, the limit of detection (LoD), the limit of quantification (LoQ), precision, and reproducibility. The primer and probe sequences used in the study were selected based on a literature review to ensure the detection of all HAdV serotypes in a single run. The selected primers were verified using the US National Center for Biotechnology Information (NBCI) nBLAST tools, and the target sequence was determined using the BioEdit software. The DNA concentration of the stock solution was measured using a Qubit fluorometer. The estimated copy number of the stock solution per milliliter was calculated based on the length of the amplified base sequence and fluorometer measurement.
The dynamic range of the test was determined to be from 770.4 to 0.9476 cp/μl, with the LoD and LoQ values both being 0.9476 cp/μl. The coefficient of determination (r ) value of the test was 0.9986.
The results demonstrated that the dPCR method could be an ideal tool for the diagnosis and absolute quantification of human adenoviruses, especially in low copy numbers. In order to determine the reproducibility of the test and validate the method for field use, it needs to be developed and adapted in various laboratories and supported by clinical studies.
数字聚合酶链反应（dPCR）检测是一种先进的PCR技术，可同时检测多种病原体并进行绝对定量。可用于检测人类腺病毒（HAdV）所有亚型的商业验证试剂盒有限。本研究旨在证明开发一种基于纳米板的高灵敏度内部dPCR检测方法，即使在低拷贝数情况下也能检测。
在本方法学研究中，通过实时PCR扩增特定六邻体基因区域制备标准化的HAdV DNA，并使用磁珠从HAdV阳性提取物中纯化HAdV DNA。在三次独立运行中对稀释液进行一式三份测试，以确定动态范围、检测限（LoD）、定量限（LoQ）、精密度和重现性。本研究中使用的引物和探针序列是根据文献综述选择的，以确保一次运行就能检测所有HAdV血清型。使用美国国家生物技术信息中心（NBCI）的nBLAST工具验证所选引物，并使用BioEdit软件确定目标序列。使用Qubit荧光计测量储备溶液的DNA浓度。根据扩增碱基序列的长度和荧光计测量结果计算每毫升储备溶液的估计拷贝数。
测试的动态范围确定为770.4至0.9476拷贝数/微升，LoD和LoQ值均为0.9476拷贝数/微升。测试的决定系数（r²）值为0.9986。
结果表明，dPCR方法可能是诊断和绝对定量人类腺病毒的理想工具，尤其是在低拷贝数情况下。为了确定测试的重现性并验证该方法在现场使用的有效性，需要在各个实验室进行开发和调整，并得到临床研究的支持。
