Geng Yunyun, Wang Jianchang, Liu Libing, Lu Yan, Tan Ke, Chang Yan-Zhong
College of Life Sciences, Hebei Normal University, No.20, Road E. 2nd Ring South, Yuhua District, Shijiazhuang, Hebei Province, 050024, People's Republic of China.
Center of Inspection and Quarantine, Hebei Entry-Exit Inspection and Quarantine Bureau, No.318 Hepingxilu Road, Shijiazhuang, Hebei Province, 050051, People's Republic of China.
BMC Vet Res. 2017 Nov 6;13(1):311. doi: 10.1186/s12917-017-1232-z.
Canine parvovirus 2, a linear single-stranded DNA virus belonging to the genus Parvovirus within the family Parvoviridae, is a highly contagious pathogen of domestic dogs and several wild canidae species. Early detection of canine parvovirus (CPV-2) is crucial to initiating appropriate outbreak control strategies. Recombinase polymerase amplification (RPA), a novel isothermal gene amplification technique, has been developed for the molecular detection of diverse pathogens. In this study, a real-time RPA assay was developed for the detection of CPV-2 using primers and an exo probe targeting the CPV-2 nucleocapsid protein gene.
The real-time RPA assay was performed successfully at 38 °C, and the results were obtained within 4-12 min for 10-10 molecules of template DNA. The assay only detected CPV-2, and did not show cross-detection of other viral pathogens, demonstrating a high level of specificity. The analytical sensitivity of the real-time RPA was 10 copies/reaction of a standard DNA template, which was 10 times more sensitive than the common RPA method. The clinical sensitivity of the real-time RPA assay matched 100% (n = 91) to the real-time PCR results.
The real-time RPA assay is a simple, rapid, reliable and affordable method that can potentially be applied for the detection of CPV-2 in the research laboratory and point-of-care diagnosis.
犬细小病毒2型是一种线性单链DNA病毒，属于细小病毒科细小病毒属，是家犬和几种野生犬科动物的高度传染性病原体。早期检测犬细小病毒（CPV-2）对于启动适当的疫情控制策略至关重要。重组酶聚合酶扩增（RPA）是一种新型等温基因扩增技术，已被开发用于多种病原体的分子检测。在本研究中，开发了一种实时RPA检测方法，使用针对CPV-2核衣壳蛋白基因的引物和外切探针来检测CPV-2。
实时RPA检测在38°C下成功进行，对于10-10个模板DNA分子，在4-12分钟内获得结果。该检测仅检测到CPV-2，未显示对其他病毒病原体的交叉检测，表明具有高度特异性。实时RPA的分析灵敏度为标准DNA模板10拷贝/反应，比普通RPA方法灵敏10倍。实时RPA检测的临床灵敏度与实时PCR结果的匹配率为100%（n = 91）。
实时RPA检测是一种简单、快速、可靠且经济实惠的方法，有可能应用于研究实验室和即时诊断中CPV-2的检测。
