鸡毒支原体和滑液囊支原体双重LFD-RPA快速检测方法的建立
Establishment of Dual LFD-RPA Rapid Detection Method for Mycoplasma gallisepticum and Mycoplasma synoviae
田兴苗 1王健霖 1郭磊 2司朵朵 1龚振兴 1李继东1
作者信息
- 1. 宁夏大学动物科技学院,银川 750021
- 2. 宁夏晓鸣农牧股份有限公司,银川 750011
- 折叠
摘要
[目的]鸡毒支原体(Mycoplasma gallisepticum,MG)和滑液囊支原体(Mycoplasma synoviae,MS)是对家禽危害最大的两种支原体,MG、MS的早期诊断是预防和控制禽支原体的关键环节.通过将重组酶聚合酶扩增技术(recombinase polymerase amplification,RPA)和测流层析试纸条(lateral flow dipstick,LFD)相结合,建立一种操作便捷、反应快速、结果可视化、可同时检测MG、MS两种病原的快速检测方法.[方法]基于MG的mgc2 基因、MS的vlhA基因设计特异性引物和探针,优化反应时间、反应温度及引物配比建立双重LFD-RPA快速检测方法,评价其灵敏度、特异性、稳定性,同时对 120 份临床样品进行检测.[结果]双重LFD-RPA检测方法在 37℃下反应 5-10 min即可完成扩增,其RPA-MG-F2/R2、RPA-MS-F1/R1 最佳引物配比为 0.6∶1.4.MG、MS的最低检出限分别为 3.75 copies/μL、3.46 copies/μL.用该方法与OIE推荐的PCR方法对 120 份样品进行检测,二者符合率为 93.3%.[结论]MG、MS双重LFD-RPA检测方法在恒温条件下即可完成扩增,其操作简单、反应快速、灵敏度高、特异性强、稳定性好,无需使用任何仪器即可观察到结果,适用于基层MG、MS单独感染或混合感染的现场快速检测.
Abstract
[Objective]Mycoplasma gallisepticum(MG)and Mycoplasma synoviae(MS)are the two most harmful mycoplasmas to poultry.Early diagnosis of MG and MS was a key to prevent and control avian mycoplasma.Through combining recombinase polymerase amplification(RPA)technology with lateral flow dipstick(LFD),a rapid detection method for MG and MS with convenient operation,rapid response and visual results was established.[Method]Specific primers and probes were designed based on MG mgc2 gene and MS vlhA gene,and a dual LFD-RPA rapid detection method was established by optimizing reaction time,reaction temperature and primer ratio,and its sensitivity,specificity and stability were evaluated.Meanwhile,120 clinical samples were tested.[Result]The amplification was done by dual LFD-RPA detection method at 37℃for 5-10 min,and the optimal primer ratio of RPA-MG-F2/R2 and RPA-MS-F1/R1 was 0.6∶1.4.The minimum detection limits of MG and MS were 3.75 copies/μL and 3.46 copies/μL,respectively.This method and the PCR method recommended by OIE were used to detect 120 samples,and the coincidence rate was 93.3%.[Conclusion]The amplification can be done by the dual LFD-RPA detection method of MG and MS under constant temperature conditions.It has simple operation,rapid response,high sensitivity,strong specificity and good stability,and the results can be observed without using any instrument.It is suitable for the field rapid detection of MG and MS infection alone or mixed infection at the primary level.
关键词
鸡毒支原体/滑液囊支原体/重组酶聚合酶扩增技术/测流层析试纸条Key words
Mycoplasma gallisepticum/Mycoplasma synoviae/recombinase polymerase amplification/lateral flow dipstick引用本文复制引用
基金项目
宁夏回族自治区科技创新团队建设项目(2022BSB03107)
宁夏银川市校企联合创新专项(2022XQ009)
宁夏大学产教融合研究生联合培养示范基地建设项目()
出版年
2024
国家科技期刊平台
NSTL
万方数据