唐菖蒲伯克霍尔德氏菌椰毒假单胞菌酵米面亚种环介导等温扩增荧光方法的建立

A novel method for detecting Burkholderia gladioli pv.cocovenenans by fluorescent probe-based loop-mediated isothermal amplification

【目的】建立一个基于环介导等温扩增与荧光探针结合的唐菖蒲伯克霍尔德氏菌椰毒假单胞菌酵米面亚种(Burkholderia gladioli pv.cocovenenans)特异性检测方法。【方法】通过多重序列比对分析,选择B.gladioli pv.cocovenenans共有的bonA为靶位点,设计环介导等温扩增的特异性引物和荧光探针,通过19株B.gladioli和3株非目标菌株,验证该方法的特异性。将该方法的检测特异性与现有的基于实时荧光定量PCR鉴定B.gladioli pv.cocovenenans的方法进行比较,并对建立的方法进行灵敏度探究。【结果】建立的环介导等温扩增荧光鉴定方法对B.gladioli pv.cocovenenans具有特异性,能在30 min内得出结果,且该方法能进行可视化直接观测。该方法的基因组DNA最低检测限度为1.98 pg/μL,检测灵敏度为2.7×102 CFU/mL。另外,该方法能应用到食品样品中B.gladioli pv.cocovenenans的检测。【结论】本研究建立了一个快速、可视化、特异性强的检测方法,能有效用于B.gladioli pv.cocovenenans的快速筛查,为保障食品安全提供了一种快速、准确的分子检测手段。

[Objective]To establish a fluorescent probe-based loop-mediated isothermal amplification method for the specific detection of Burkholderia gladioli pv.cocovenenans.[Methods]According to the results of the multiple sequence alignment analysis,a conserved sequence,bonA,was selected for the specific detection of B.gladioli pv.cocovenenans.The primers and fluorescent probes were designed according to the conserved sequence for the establishment of a fluorescent probe-based loop-mediated isothermal amplification method.The established method was employed to detect 19 strains of B.gladioli and 3 non-target strains,and thus the specificity of this method was examined.Furthermore,the specificity of this method was compared with that of the real-time fluorescent PCR.Additionally,we investigated the limit of detection of this method for genomic DNA.[Results]The established method exhibited specificity towards B.gladioli pv.cocovenenans and could obtain the result within 30 min.Moreover,this method could visualize the detection results.The limits of detection for the genomic DNA and bacterial suspension were 1.98 pg/μL and 2.7×102 CFU/mL,respectively.In addition,this method can be applied to the detection of B.gladioli pv.cocovenenans in food samples.[Conclusion]A rapid,visualizable,and specific method was established for detecting B.gladioli pv.cocovenenans and can serve as a rapid and accurate molecular detection method for ensuring food safety.