基于重组酶聚合酶扩增结合测流层析试纸条的猪细小病毒核酸快速检测方法的建立

Establishment of a rapid nucleic acid detection method for porcine parvovirus based on recombinant enzyme polymerase amplification-lateral flow dipstick (RPA-LFD)

摘要:

【背景】 猪细小病毒(porcine parvovirus, PPV)是危害猪群生殖健康的重要病原之一，给养猪业带来重大经济损失，对猪源性产品的质量安全造成严重威胁。【目的】 建立一种重组酶聚合酶扩增(recombinase polymerase amplification, RPA)结合侧流层析试纸条(lateral flow dipstick, LFD)方法，用于PPV核酸的快速、灵敏检测。【方法】 针对PPV的NS1基因保守序列设计特异性RPA引物，通过反应条件优化建立了PPV的RPA-LFD检测方法。【结果】 该方法在39 ℃条件下20 min内即可完成检测，最低检测限为10 copies/μL；与猪圆环病毒2型、猪伪狂犬病毒、猪瘟病毒、猪蓝耳病毒、口蹄疫病毒等无交叉反应；人工污染猪组织样品和模拟临床样品检测结果表明，该方法与常规PCR方法的检测结果一致。【结论】 本研究建立的RPA-LFD方法不依赖热循环仪，操作简单、灵敏度高、特异性强，为PPV核酸的现场快速检测提供了技术手段。

Abstract:

[Background] Porcine parvovirus (PPV) is one of the major pathogens threatening the reproductive health of swine herds, causing tremendous economic losses to the pig farming industry and posing a serious threat to the quality and safety of porcine products. [Objective] To establish a rapid and sensitive detection method for PPV nucleic acid based on recombinase polymerase amplification (RPA) combined with lateral flow dipstick (LFD) assay. [Methods] We designed specific primer pairs based on the conserved sequences of the NS1 gene in PPV. Then, we developed a RPA-LFD method for the detection of PPV by optimizing the reaction conditions. [Results] The RPA-LFD method can detect PPV nucleic acid within 20 min at 39 ℃, with a limit of detection of 10 copies/μL. The experimental results showed that only PPV was positively detected by this method, whereas no cross reaction with porcine circovirus type 2, pseudorabies virus, swine fever virus, porcine reproductive and respiratory syndrome virus or foot-and-mouth disease virus was observed. In addition, the porcine tissue samples artificially polluted with PPV and the simulated clinical samples were detected by RPA-LFD and traditional PCR, and the results of the two methods were consistent. [Conclusion] The developed RPA-LFD method does not rely on a thermal cycler and it is simple to operate and highly sensitive and specific, providing an effective strategy for the on-site rapid detection of PPV nucleic acid.