屋尘螨过敏原Der p 1基因逆转录环介导等温扩增可视化检测方法的建立

Establishment of visual detection method for house dust mite allergn Der p 1 by reverse transcriptional loop-mediated isothermal amplification

目的 建立一种能快速、准确检测环境中屋尘螨过敏原Der p 1基因的逆转录环介导等温扩增可视化(RT-LAMP)技术。方法 用Primer Explore 4.0对Der p 1基因进行RT-LAMP引物设计与筛选,提取屋尘螨、粉尘螨、热带无爪螨、腐食酪螨总RNA,利用优化的RT-LAMP反应体系分别扩增4个螨种的Der p 1基因,评价方法的特异度和灵敏度。采集74份哮喘患儿的床尘样品,提取总RNA进行Der p 1基因RT-PCR和RT-LAMP检测。结果 利用优化的RT-LAMP方法检测4种螨的Der p 1基因,只有屋尘螨阳性,琼脂糖凝胶电泳检测扩增产物呈现特征性梯形条带,肉眼和紫外灯下均观察到绿色荧光。建立的RT-LAMP对Der p 1基因的最小检测量为1.0×10^(-6 )μg,敏感性比常规RT-PCR高10倍;检测74份床尘样品,阳性率85.1%,与RT-PCR阳性率70.3%比较差异具有统计学意义(χ^(2)=4.72,P<0.05)。结论 建立的屋尘螨过敏原Der p 1基因RT-LAMP较常规RT-PCR灵敏度和特异性更高,可用于屋尘螨过敏原污染的快速检测。

Objective To develop a method to detect the concentration of Der p 1 in the environmentdust samples based on reverse transcription loop-mediated isothermal amplification(RT-LAMP) visualization technology.Methods RT-LAMP Primers were designed and screened with the Primer Explore 4.0.The total RNA were extracted from Dermatophagoides pteronyssinus,Dermatophagoides farinae,Blomia tropicalis and Tyrophagus putrescentiae separately,which were used for detection of Der p 1 with optimized RT-LAMP reaction system to evaluate specificity and sensitivity of the method.Bed dust samples from 74 children with asthma were collected,and the total RNA was extracted from these 74 dust sample separately for the detection of Der p 1 with RT-PCR and RT-LAMP,respectively.Results The optimized RT-LAMP method was used to detect Der p 1 from the total RNA isolated from the four species of mites,and only Dermatophagoides pteronyssinus were positive.Agarose gel electrophoresis(2%) showed characteristic trapezoidal strips,and green fluorescence was observed under both naked eye and ultraviolet lamp.The minimum detection amount for Der p 1 was 1.0×10^(-6) μg with RT-LAMP assay and the sensitivity was 10 times higher than that of conventional RT-PCR.Among the 74 bed dust samples,the positive rate of RT-LAMP was 85.1%,which was higher than that of RT-PCR(70.3%),and the difference between the two mechods was statistical significance(P<0.05).Conclusion A visual method for detection of Der p 1 was successfully established with RT-LAMP.Compared with conventional RT-PCR,RT-LAMP was more sensitive,rapid and specific in detecting dust mite allergens.