单核细胞增生性李斯特菌LAMP-浊度/荧光检测方法的建立

Development of a LAMP-Turbidimeter/Fluorescence Test Method for Detecting Listeria monocytogenes

将环介导等温扩增技术(LAMP)分别与浊度信号检测系统(turbidimeter)和荧光信号检测系统(fluorescence)相结合,建立了LAMP-浊度/荧光单核细胞增生性李斯特菌检测方法。选择单核细胞增生性李斯特菌保守毒力基因iap序列,通过环介导等温扩增引物设计在线软件设计4条特异性引物,进行反应条件的优化,并对建立的LAMP的特异性和灵敏度进行评价分析。结果表明,构建的LAMP-浊度信号检测方法优化后的扩增温度为61℃,菌液最低检测浓度为22.1 CFU/mL,灵敏度是普通PCR扩增方法的10倍;LAMP-荧光信号检测方法优化后的扩增温度为64℃,菌液最低检测浓度为2.21 CFU/mL,灵敏度是普通PCR扩增方法的100倍;建立的两种LAMP方法的特异性良好,其中1株单核细胞增生性李斯特菌标准菌株和1株本试验分离保存的单核细胞增生性李斯特菌检测结果均为阳性,8株非单核细胞增生性李斯特菌检测结果均为阴性。利用两种LAMP方法对630份肉类及其制品、人工污染样品等进行检测,检出45个LAMP阳性,与国标法(GB)检测结果一致。结果表明,建立优化的单核细胞增生性李斯特菌LAMP-浊度/荧光检测方法具有快速、特异、灵敏等特点,特别适用于基层兽医、食品及口岸一线部门对单核细胞增生性李斯特菌快速筛查工作。

Combined of the loop-mediated isothermal amplification assay(LAMP)with Turbidimeter or Fluorescence a LAMP fluorescent/turbidimeter method for detecting Listeria monocytogenes was established.The four specific primers were designed by using the specific target gene iap and LAMP software(PrimerExplorer https://primerexplorer.jp/lampv5/index.html).The reaction temperature was optimized.The specificity and sensitivity of two methods were analyzed.The results showed that the ideal amplification temperature of LAMP-Turbidimeter method was 61℃,and the lowest content of colonies can reach 22.1 CFU/mL,which was 10-fold more than common PCR amplification,and the ideal amplification temperature of LAMP-Fluorescent method was 64℃,and the lowest content of colonies can reach 2.21 CFU/mL,which was 100-fold more than common PCR amplification.The specificity of the LAMP assay were confirmed,2 strains of Listeria monocytogenes were positive and 8 strains of non-Listeria monocytogenes were negative.In practice,45 pieces of samples detected from 630 pieces of meat and meat products,artificial contamination samples were positive using the LAMP method,which accorded with the detection result by GB.The results showed that the LAMP Fluorescence/Turbidimeter was rapid,sensitive and specific,it is practically applicable for the detecting Listeria monocytogenes in primary veterinary and port line departments.