牛支原体环介导等温扩增技术结合横向流动试纸条检测方法的建立

Loop-mediated isothermal amplification (LAMP) combined with lateral flow dipstick (LFD) for detection of Mycoplasma bovis

摘要:

【背景】牛支原体(Mycoplasma bovis)是引起牛乳房炎、关节炎及呼吸道疾病等的重要病原体之一，严重危害养牛业的健康发展。【目的】建立一种便捷、快速、灵敏和特异的环介导等温扩增(loop-mediated isothermal amplification, LAMP)技术结合横向流动试纸条(lateral flow dipstick, LFD)方法用于牛支原体的检测。【方法】对牛支原体P48特异性基因序列进行对比，选择保守性区域。运用Primer Explorer V5在线软件设计引物，并通过荧光染料法与以oppD/F、oppD、urvC为靶基因所设计的LAMP引物进行比较。对筛选出的最佳P48引物组的内引物(FIP/BIP)用生物素和6-羧基荧光素标记；利用单一控制变量法对反应温度、时间与引物浓度比进行优化；将LAMP检测方法与LFD相结合。最后评价该方法的敏感性、特异性、重复性及临床应用效果。【结果】P48 LAMP引物组荧光信号更强，C_t值较小，扩增效率较高，优于已报道的LAMP引物组；当反应温度为60 ℃、引物(F3/B3:FIP/BIP)浓度比为1:4、反应时间为40 min时最佳；最低检测浓度为17.28 fg/µL，比行业标准PCR检测方法灵敏1 000倍；与多杀性巴氏杆菌、牛疱疹病毒等9种引起牛呼吸道疾病相关病原体均无交叉反应；批间与批内试验均一致；运用该方法对39份临床鼻拭子的检出率为28.21%，高于行业标准的PCR法(检出率23.07%)。【结论】成功建立一种敏感、特异以及便于基层使用的牛支原体LAMP-LFD检测方法，为防控牛支原体病提供了技术支持。

Abstract:

[Background]Mycoplasma bovis is one of the major pathogens causing bovine mastitis, arthritis, and respiratory diseases, seriously endangering the healthy development of the cattle industry. [Objective] To establish a convenient, rapid, sensitive, and specific method based on loop-mediated isothermal amplification (LAMP) combined with lateral flow dipstick (LFD) for the detection of M.bovis. [Methods] The conserved region was selected by comparison of the P48 specific sequence of M.bovis and used to design primers by Primer Explorer V5 online. The designed primers were compared with the LAMP primers designed with oppD/F,oppD, and urvC as target genes by the fluorescent dye method. The optimal internal primers (FIP/BIP) designed based on P48 were labeled with biotin and 6-carboxyfluorescein, respectively. The reaction temperature, time, and primer concentration ratio were optimized by the single factor method. LAMP was combined with LFD for the detection of M.bovis. Finally, the sensitivity, specificity, repeatability, and clinical application effect of the established method were evaluated. [Results] The LAMP primers designed based on P48 had stronger fluorescence signal, lower Ct value, and higher amplification efficiency, outperforming the reported LAMP primers. The reaction conditions were optimized as 60 ℃, primer concentration ratio (F3/B3:FIP/BIP) of 1:4, and 40 min. The lower limit of detection of the established method was 17.28 fg/μL, which was 1 000 times lower than that of the PCR method, the standard method in the industry. There was no cross-reaction with 9 pathogens causing bovine respiratory diseases, such as Pasteurella multocida and bovine herpes virus (BHV). The results of inter-batch and intra-batch tests were consistent. The detection rate of 39 clinical nasal swabs by the established method was 28.21%, which was higher than that (23.07%) of the PCR method. [Conclusion] A sensitive, specific, and easy-to-use LAMP-LFD method for detecting M.bovis was successfully established, which provided technical support for the prevention and control of M.bovis.