2种免疫层析技术用于4种传染病及其病原体检测的研究

Detecting 4 kinds of Infectious Diseases and the Pathogens by Two Immunochromatography Assays

[目的]寻求能够快速定性／定量检测4种传染病及其病原体的试验方法。[方法]应用双抗体夹心法，建立胶体金免疫层析法、上转磷光（UCP）免疫层析法检测炭疽杆菌芽孢（以下简称炭疽芽孢）、鼠疫杆菌、肠出血性大肠埃希菌O157：H7（以下简称EHEC O157），评价方法的灵敏性、特异性。通过分别检测模拟掺入3种菌／芽孢的“白色粉末”和检测3种细菌的强毒株，评价在实际环境样品检测中的应用。应用双抗原夹心法，建立胶体金免疫层析法、上转磷光（UCP）免疫层析法检测鼠疫F1抗体、SARS病毒抗体，检测239份青海鼠疫疫区人员血清、1236份新疆鼠疫疫源地鼠血清，检测18份恢复期SARS病人血清和9份健康对照者血清，以及206份出入境健康人员血清，考核2种检测抗体的方法在实际样品检测中的应用。通过比较炭疽杆菌、鼠疫杆菌、大肠埃希菌O157以及鼠疫杆菌抗体、SARS病毒抗体的胶体金免疫层析方法、UCP免疫层析方法，比较我们建立的2套系统与加拿大RAMP系统检测炭疽杆菌的能力。[结果]UCP免疫层析方法能在35min内完成检测，炭疽芽孢检测掺入“白色粉末”的样品灵敏性为1×10^4cfu／test，炭疽芽孢最低检测限为1×10^5cfu／ml（原溶液芽孢浓度），多种不同的芽孢菌及其它细菌共38种54株检测说明该法特异性良好，仅与腊样芽孢杆菌有交叉反应，特异性为97％；鼠疫杆菌检测灵敏性为5×10^4cfu／ml，最低检测菌数为5×10^3cfu／test，在10^7cfu／ml水平上未发现与30余种细菌有交叉反应，包括最近缘假结核耶尔森菌；EHEC O157检测灵敏性为1×10^3cfu／ml，仅与弗氏枸橼酸杆菌有交叉反应，特异性为98％。胶体金免疫层析方法能在10min内完成检测，炭疽芽孢、鼠疫杆菌、EHEC O157检测灵敏度分别为1×10^6cfu／ml、1×10^5cfu／ml、1×10^5cfu／ml，“白色粉末”等环境样品最低可检测敏感性为1×10^5cfu，1×10^4cfu，1×10^4cfu／test。胶体金免疫层析方法分别应用于炭疽、鼠疫强毒株的检测，检测限为5×10^4cfu／test和7×10^4cfu／test。在实际血清样品检测中，与间接血凝方法对比，鼠疫抗体UCP和胶体金免疫层析方法有更好的敏感性。血清样品检测显示，SARS抗体的胶体金和UCP免疫层析方法均有较好的特异性和敏感性。[结论]2种免疫层析方法都有快速、敏感、特异的特点：上转磷光免疫层析方法可实现定量检测，敏感性优于胶体金免疫层析法；炭疽检测与加拿大RAMP检测系统相当；胶体金免疫层析法具有简便、直观、易行的特点。2种免疫层析方法均适合于炭疽芽孢芽孢、鼠疫杆菌、EHEC O157、鼠疫杆菌抗体、SARS抗体的现场检测和筛查。

Objective To seek a qualitative/quantitative method for four infectious diseases and their pathogens detection. Method The colloid gold immunochromatography assay was established and Up - converting Phosphor （UCP） immunochromatography assay was used based on double antibody sandwiched assay to detect Bacillus anthracis, Yersinia Pestis and Escherichia Coli O157： H7（O157）. The sensitivity and specificity of the method were evaluated by detecting ＂the white power＂ which had been mixed with 3 kinds of bacterium/spores and their cultures, respectively in order to evaluate the application in detecting the real samples. Establish colloid gold immunity chromatography assay and UCP immunity chromatography assay based on double antigen sandwiched assay to detect F1 antibodies of Yersinia Pestis, antibodies of SARS. Two hundred and thirty nine people serum in plague epidemic area of Qinghai, 1236 mice serum in plague epidemic area of Xinjiang, 18 serum of SARS patients at recovering stage, 9 serum of healthy control people, and 206 serum of entry - exit healthy people were evaluated for their application in detecting the real samples. The colloid gold immunity chromatography assay and UCP immunity chromatography assay were compared with RAMP developed by Canadian in the detection of Bacillus anthracis, Yersinia Pestis, Escherichia Coli O157, and SARS antibodies. Results The method of UCP immunity chromatography assay was finished in 35 minutes. The sensitivity of detecting Bacillus anthracis in ＂the white power＂ was approximately 1×10^4cfu/test CFU/test and the detection limit was about 1×10^5cfu/ml. The results of detecting spore forming bacteria（38 kinds and 54 strains） showed the good specificity which was 97%. We only found the cross - reactivity with bacillus cereus. The sensitivity of detecting Yersinia Pestis was 5 x 104 cfu/ml and the detection limit was 5×10^3 cfu/test. We didn＇ t find any cross - reactivity at the level of detection 10^7cfu/ml of Yersinia Pestis even with the most related yersinia pseutotuberculosis. The sensitivity of detecting Escherichia Coli O157 was 1×10^3cfu/ml, cross - reactivity only with Citmbacter freundii. The specificity was 98%. The colloid gold immunity chromatography assay was finished in 10 minutes. The sensitivity of detecting Bacillus anthracis, Yersinia Pestis, Escherichia Coli 0,57 separately was respectively 1×10^6 cfu/ ml,1×10^5cfu/ml, 1×10^5cfu/ml. The lowest detection limit in environmental sample, such as white power, was respectively 1×10^5cfu, 1×10^4 cfu, 1×10^4 cfu/test. The detection limits of velogenic and mesogenic strains in Bacillus anthracis and Yersinia Pestis were 5×10^4 cfu/test and 7×10^4 cfu/test. Compared with indirect hemagglutination test in serum samples, the colloid gold immunity chromatography assay and UCP immunity chromatography assay showed the better sensitivity. The detection of SARS antibodies by colloid gold immunity chromatography assay and UCP immunity chromatography assay showed the better specificity and sensitivity. Conclusion The two immunity chromatography assays show the characteristics of rapidity, sensitivity and specificity. The UCP immunity chromatography assay can achieve the quantitative detection, the sensitivity of which was better than colloid gold immunity chromatography assay, and the capability of detecting Bacillus anthracis was corresponded with the RAMP system of Canada. The colloid gold immunity chromatography assay has the characteristics of convenient, intuitionistic and facile. The two methods are suitable for field inspection and screening in Bacillus anthracis, Yersinia Pestis, Escherichia COli O157, Yersinia Pestis antibodies, and SARS antibodies.