环介导等温扩增技术检测马尔尼菲青霉菌的研究

Application of loop-mediated isothermal amplification in detection of Penicillium marneffei

目的应用环介导等温扩增(LAMP)技术建立马尔尼菲青霉菌(PM)快速检测方法,探讨该方法的特异性、敏感性等,评价其临床应用价值。方法针对马尔尼菲青霉菌的特异性基因MP1基因设计4条特异引物,反应前加入钙黄绿素荧光试剂,63℃恒温反应60min,结束后通过目视检测荧光及浊度仪检测产物浊度值判断结果;评价所建立LAMP方法的特异性和敏感性,建立标准曲线,并用LAMP法及PCR法检测样本,比较样本检出情况。结果该方法特异性良好,PM出现特异性LAMP扩增反应,而烟曲霉菌、腐皮镰刀菌、棘状外瓶酶菌、大肠埃希菌、嗜水气单胞菌、哈氏孤菌、创伤孤菌、水等均未出现扩增;检测限约为1.7×10-7 ng/μl,为PCR检测方法的10倍;以LAMP反应时间X轴,浓度的负次方数为Y轴,构建的标准曲线方程为y=0.4145x-8.9968,相关系数R2=0.9963,呈良好的线性关系;样本检测结果显示,LAMP法较PCR法获得更高的敏感性。结论 LAMP检测方法具有特异性高、敏感性高、反应时间短的特点,简便易行,适合于基层对PM的快速检测。

OBJECTIVE To establish the rapid detection method of Penicillium marneffei（PM）based on the loopmediated isothermal amplification（LAMP）and explore the specificity and sensitivity of the method so as to determine the clinical application value.METHODS Totally four specific primers were designed according to the consensus sequences of MP1 gene of PM,the calcein fluorescent reagent was added before the LAMP assay was performed at 63°C for 60 min,the result was interpreted through visual inspection of fluorescence and by reading the optical density of the real-time turbidity meter.The sensitivity and specificity of the LAMP method were evaluated,and the formula for the standard curve was built.The samples were detected by using the LAMP method and PCR method,and the detection rates of samples were compared.RESULTS The method had high specificity,and the PM showed specific LAMP amplification reaction,however,the Aspergillus fumigatus,Fusarium solani,Escherichia coli,Aeromonas hydrophila,Vibrio harrington,Vibrio vulnificus,and water did not show any amplification.The LAMP was 10-folds more sensitive than PCR with detection limits of 1.7×10-7 ng/μl DNA.Specificity test showed no amplifications were observed in the reference strains.A formula for the time period that the LAMP reaction time took（X）and the number of negative power of the PM DNA concentration took（Y）was built as y=0.4145x-8.9968,and the correlation index R2 was 0.9963,showing good linear relationship.The results of detection of samples indicated that the LAMP method was more sensitive than the PCR method.CONCLUSION The LAMP method is characterized by the high specificity,high sensitivity,and short reaction time,and simple operation;it is suitable for the rapid detection of PM.