摘要:
【目的】为基层快速检测新型番鸭细小病毒(New-genotype Muscovy duck Parvovirus,N- MDPV)提供可视化快速检测的技术手段。【方法】本研究以新型番鸭细小病毒VP3基因保守片段为靶点,利用重组酶聚合酶扩增技术(RPA)并设计特异性的RPA扩增引物,结合EXO荧光探针建立一种准确高效的新型番鸭细小病毒 RPA 恒温快速检测方法,对检测方法的灵敏度、特异性进行评价,并与传统PCR方法和病毒分离鉴定法进行比较。【结果】该检测方法可检测到10 fg /μL 的病毒核酸,具有较高的灵敏度; 只特异性地扩增新型番鸭细小病毒,与鸭腺病毒3型、禽腺病毒4型、鸭圆环病毒、鸭瘟病毒、鸭病毒性肝炎病毒、鸭坦布苏病毒和新型鸭呼肠孤病毒均未发生交叉反应,特异性良好; 利用本研究建立的RPA方法、前期建立的PCR方法和病毒分离鉴定方法对临床收集的38份鸭组织病料样品进行检测,结果显示阳性率分别为36.8%(14/38)、36.8%(14/38)和31.6%(12/38);且RPA检测的阳性样品经PCR方法检测和病毒分离鉴定方法检测均为阳性,符合率为100%。临床样品检测中与PCR符合率为100%。【结论】该方法可以很好地应用于缺乏相应检测设备的基层进行新型番鸭细小病毒的大规模临床样本检测,为新型番鸭细小病毒的可视化快速检测和流行病学调查提供技术手段。
Abstract:
【Objective】 Provide a visual and rapid detection technology for the rapid detection of the New-genotype Muscovy duck Parvovirus at the grassroots level.【Methods】 This study targets the conserved fragment of the VP3 gene of the New-genotype Muscovy duck Parvovirus. Recombinant enzyme polymerase amplification (RPA) technology was used and specific RPA amplification primers were designed. Combined with EXO fluorescent probes, an accurate and efficient isothermal rapid detection method for the New-genotype Muscovy duck Parvovirus RPA was established. The sensitivity and specificity of the detection method were evaluated, and compared with traditional PCR methods and virus isolation and identification methods.【Results】 This detection method can detect 10 fg/ μL The viral nucleic acid of L has high sensitivity; Only specific amplification of the New-genotype Muscovy duck Parvovirus was performed, and there was no cross reaction with duck adenovirus type 3, duck adenovirus type 4, duck circovirus, duck plague virus, duck viral hepatitis virus, duck tambusu virus, and new duck reovirus, indicating good specificity; Using the RPA method established in this study, the PCR method established earlier, and the virus isolation and identification method, 38 clinical collected duck tissue samples were tested, and the results showed positive rates of 36.8% (14/38), 36.8% (14/38), and 31.6% (12/38), respectively; And the positive samples detected by RPA were tested positive by PCR and virus isolation and identification methods, with a compliance rate of 100%. The compliance rate with PCR in clinical sample testing is 100%. 【Conclusion】 This method can be well applied to grassroots areas lacking corresponding detection equipment for large-scale clinical sample detection of the New-genotype Muscovy duck Parvovirus, providing technical means for visual rapid detection and epidemiological investigation of the novel muscovy duck parvovirus.