Anhui Province Key Laboratory of Veterinary Pathobiology and Disease Control, College of Animal Science and Technology, Anhui Agricultural University, Hefei, 230036, China.
Innovation Team of Small Animal Infectious Disease, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences (CAAS), Shanghai, 200241, China.
J Virol Methods. 2021 Dec;298:114290. doi: 10.1016/j.jviromet.2021.114290. Epub 2021 Sep 17.
A rapid and accurate diagnosis of mixed viral infections is important for providing timely therapeutic interventions. The aim of this study was to develop a highly sensitive and specific method for the simultaneous detection of canine distemper virus (CDV), canine parvovirus (CPV) and canine coronavirus (CCV) in mixed infections by combining the high specificity of a dual priming oligonucleotide (DPO) primer system with the high sensitivity of a nanoparticle-assisted PCR (nanoPCR) assay. Under the optimised assay conditions, the multiplex DPO-nanoPCR assay developed using DPO primers was 100-fold more sensitive than the multiplex PCR assay using conventional primers. The detection limits of the multiplex DPO-nanoPCR assay for the recombinant plasmids containing the cloned CDV, CPV and CCV target sequences were 5.4 × 10, 6.5 × 10 and 1.6 × 10 copies in a 25 μL assay, respectively. No cross-reaction with other canine viruses was observed. This is the first reported use of a multiplex nanoPCR assay with the DPO primer system for the simultaneous detection of CDV, CPV and CCV in mixed infections. The high sensitivity and specificity of the assay indicated its potential for use in clinical diagnosis and field surveillance of animal epidemics.
快速准确地诊断混合病毒感染对于提供及时的治疗干预至关重要。本研究旨在通过结合双重引物寡核苷酸（DPO）引物系统的高特异性和纳米颗粒辅助 PCR（nanoPCR）检测法的高灵敏度，开发一种用于同时检测犬瘟热病毒（CDV）、犬细小病毒（CPV）和犬冠状病毒（CCV）在混合感染中的高度敏感和特异性方法。在优化的实验条件下，使用 DPO 引物开发的多重 DPO-nanoPCR 检测法比使用常规引物的多重 PCR 检测法灵敏 100 倍。该多重 DPO-nanoPCR 检测法对包含克隆 CDV、CPV 和 CCV 靶序列的重组质粒的检测限分别为在 25 μL 检测中为 5.4×10、6.5×10 和 1.6×10 拷贝。未观察到与其他犬病毒的交叉反应。这是首次报道使用 DPO 引物系统的多重 nanoPCR 检测法同时检测混合感染中的 CDV、CPV 和 CCV。该检测法的高灵敏度和特异性表明其在临床诊断和动物疫情的现场监测中有应用潜力。
