| 摘要: |
| 旨在建立一种以重组酶聚合酶扩增技术（RPA）为基础的快速检测方法，用于小鹅瘟病毒的快速检测。小鹅瘟是一种常见的水禽传染病，严重危害我国养鹅业的健康发展。为了快速准确对小鹅瘟进行诊断，减少该病的危害，本研究以小鹅瘟病毒VP3基因保守片段为靶点，利用重组酶聚合酶扩增技术（RPA）建立了一种准确高效的小鹅瘟病毒RPA恒温快速检测方法，并对检测方法的灵敏度、特异性进行评价，并与传统PCR和传统RT-PCR方法进行比较。结果表明，该检测方法具有较高的灵敏度，可检测到10 copies/μL的病毒核酸；具有良好的特异性，只特异性地扩增鹅细小病毒，而与鹅副黏病毒、鹅源鸭瘟病毒、小鹅流行性感冒病毒、鹅副伤寒病毒、大肠杆菌和曲霉菌均未发生交叉反应；同时该方法重复性检测的变异系数低于6%，具有很好的重复性。阳性符合试验表明该检测方法与荧光定量PCR符合率为99%。该方法可以很好地应用于小鹅瘟的大规模临床样本检测，为小鹅瘟病毒的高通量检测和流行病学调查提供技术手段。 |
| 关键词: 小鹅瘟病毒 重组酶聚合酶扩增技术(RPA) EXO探针 恒温快速检测 |
| DOI： |
| 投稿时间：2021-10-12修订日期：2022-03-03 |
| 基金项目:山东省重点研发计划(重大科技创新工程)项目（2019JZZY010720） |
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| Rapid Detection of Goose parvovirus based on Isothermal Recombinase Polymerase Amplification#$NBSAssay |
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| (Animal Husbandry Development Center of Rongcheng city) |
| Abstract: |
| The aim of this work is to develop a rapid recombinase polymerase amplification (RPA) method for detecting Goose parvovirus. Gosling parvovirus infection is a common disease of waterfowl, which seriously endangers the healthy development of goose industry in China. In order to develop a quick and accurate diagnostic method diagnose to reduce the harm of the disease, the conserved fragment of VP3 gene of gosling parvovirus was selected as the target. An accurate and efficient constant temperature rapid detection method of gosling parvovirus virus was established based on the recombinant enzyme polymerase amplification (RPA) technology. And the sensitivity and specificity of the detection method were evaluated and compared with traditional PCR and RT-PCR. Results showed that the method was highly sensitive with a detection limit of 10 copies/μL of viral nucleic acid. This method also exhibited a good specificity without any cross reaction with goose paramyxovirus, goose distemper virus, gosling influenza virus, goose paratyphoid virus, E. coli and the coefficient of variation of this method is less than 6%, which has good repeatability. The test of clinical samples showed that the coincidence rate between this method and real-time PCR was 99%. This method can be applied to the detection of gosling disease in large scale clinical samples, and provide a novel method for the high throughput detection and epidemiological investigation of gosling disease virus. |
| Key words: Goose parvovirus Recombinase polymerase amplification (RPA) Exo probe Isothermal and Rapid detection |
