快速诊断多房棘球蚴病胶体金免疫层析试条方法的建立与评价

Establishment and Evaluation of Colloid Gold Labeled Immunochromatographic Strip Test for Rapid Diagnosis of Alveolar Echinococcosis

目的建立快速、简便诊断多房棘球蚴病的胶体金免疫层析试条方法。方法提取多房棘球蚴原头节总RNA,通过RT-PCR获得编码Em18基因片段并克隆入pGEX-3X表达载体,异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达得到重组蛋白;采用柠檬酸三钠还原法制备胶体金,标记抗人IgG单克隆抗体;将重组Em18抗原包被于硝酸纤维素膜适当位置,制成检测特异抗体的胶体金免疫层析试条。用该试条检测多房棘球蚴病(56份)、细粒棘球蚴病(87份)、囊尾蚴病(30份)、日本血吸虫病(10份)和弓形虫病(10份)患者血清,以及健康人(50份)血清,以评价其检测的敏感性和特异性,同时用ELISA法进行平行检测,以评价该试条的诊断性能。结果以重组蛋白Em18为抗原胶体金免疫层析试条检测多房棘球蚴病患者血清,敏感性为92.9%(52/56)。与细粒棘球蚴病和囊尾蚴病患者血清分别存在9.2%(8/87)和3.3%(1/30)的交叉反应,与健康人血清存在8.0%(4/50)的假阳性率,与日本血吸虫病和弓形虫病患者血清则无交叉反应,特异性为93.0%(174/187)。ELISA法检测的敏感性和特异性分别为94.6%(54/56)和92%(172/187),与试条法比较两者差异均无统计学意义(P>0.05)。kappa分析结果显示,试条法与ELISA法检测多房棘球蚴病患者血清的结果高度一致(κ=0.98)。结论以重组Em18抗原建立的快速诊断胶体金免疫层析试条,检测多房棘球蚴病的敏感性和特异性均较高。

Objective To establish and evaluate a colloid gold immunochromatographic strip test for the diagnosis of alveolar echinococcosis.Methods Total RNA was prepared from Echinococcus multilocularis protoscoleces collected from Xinjiang Uygur Autonomous Region.Em18 gene was obtained by reverse transcription-polymerase chain reaction(RT-PCR).The PCR product was sequenced and cloned into pGEX-3X vector.The recombinant plasmid was expressed and induced by isopropyl-β-D-thiogalactopyranoside(IPTG)to obtain recombinant protein.The anti-human IgG monoclonal antibodies was conjugated with colloid gold as detecting reagent;the recombinant Em18 antigen and goat anti-mouse IgG were immobilized on nitrocellulose in proper position.The prepared immunochromatographic strip was evaluated using serum samples from patients with alveolar echinococcosis(56),cystic echinococcosis(87),cysticercosis(30),schistosomiasis japonica(10),toxoplasmosis(10)and healthy subjects(50).Comparison between the immunochromatographic strip test and ELISA was made by kappa statistics.Results Sensitivity detected by the immunochromatographic strip test was 92.9%(52/56).The cross-reactivity to cystic echinococcosis and cysticercosis was 9.2%(8/87)and 3.3%(1/30),respectively.There was no cross reactivity with schistosomiasis japonica and toxoplasmosis.4 samples out of 50 healthy people showed false positive reaction.The overall specificity was 93.0%(174/187).Sensitivity and specificity both showed no statistical difference between immunochromatographic strip test and ELISA.High degree of agreement was observed between the strip test and ELISA(κ=0.98).Conclusion The developed immunochromatographic strip test using recombinant Em18 antigen as coated antigen is a sensitive,specific,simple and rapid assay for diagnosing alveolar echinococcosis.