呼吸道真菌感染核酸胶体金试纸条快速检测方法的建立和评价

Establishment and evaluation of nucleic acid colloidal gold test strip for rapid detection of respiratory tract fungal infection

目的:采用聚合酶链式反应(PCR)技术和胶体金技术建立一种呼吸道真菌感染核酸胶体金试纸条检测方法,并对其检测效果进行评价。方法:根据GenBank数据库选择真菌内转录间隔区(ITS)基因片段作为靶基因,应用DNAMAN软件对真菌ITS基因片段进行分析,并分别在真菌通用引物5'端用生物素(Biotin)和异硫氰酸荧光素(FITC)进行修饰标记;建立PCR体系,进行体系的最佳条件优化;确定胶体金免疫层析试纸条标记链霉亲和素的最佳标记量和质控线及检测线最佳包被物浓度,组装核酸胶体金试纸条,对试纸条的特异度、灵敏度、重复性和稳定性进行验证。结果:水煮法提取白假丝酵母菌、新生隐球菌和毛霉菌DNA,浓度为180μg·L^(-1)以上,纯度为1.60~2.10;在pH 7.0条件下,每100μL胶体金溶液中制备胶体金标记链霉亲和素最佳标记量为3.3μg,质控线包被Biotin化牛血清白蛋白(BSA-Biotin)浓度为2.00 g·L^(-1),检测线包被抗FITC抗体浓度为1.00 g·L^(-1)。核酸试纸条的特异度与电泳结果一致,仅白假丝酵母菌、新生隐球菌和毛霉菌出现阳性结果,与金黄色葡萄球菌、肺炎链球菌、乙型溶血性链球菌、铜绿假单胞菌、肺炎克雷伯菌、鲍曼不动杆菌和大肠埃希菌等常见呼吸道感染细菌无交叉反应。灵敏度检测,白假丝酵母菌、新生隐球菌和毛霉菌的DNA浓度分别为10-4、10-2和10-3 mg·L^(-1)时核酸试纸条仍可准确检出,而普通PCR电泳结果显示白假丝酵母菌、新生隐球菌和毛霉菌最低检测浓度分别为0.01、1.00和0.10 mg·L^(-1);重复性检测,在不同实验室不同操作人员对核酸试纸条进行验证,结果一致,重复性良好;稳定性检测,核酸试纸条在第6、9和12个月进行检测,结果符合预期,稳定性良好。结论:本研究建立的呼吸道真菌感染核酸胶体金试纸条可以检测白假丝酵母菌、新生隐球菌和毛霉菌等真菌,特异度和灵敏度均较高,操作简便且快速。

Objective:To establish a method of nucleic acid colloidal gold strip for rapid detection of respiratory tract fungal infection with polymerase chain reaction(PCR)technique and colloidal gold technique,and to evaluate the detection effect.Methods:The fungal internal transcribed spacer(ITS)gene fragment was selected as the target gene according to GenBank database,and the fungal ITS gene fragment was analyzed by DNAMAN software,and labeled with Biotin and fluorescein isothiocyanate(FITC)at the 5'end of fungal universal primers,respectively;the PCR system was established and the optimal conditions of the systerm were optimized.The best labeling amount of colloidal gold test strips to label streptavidin and the best coating concentration of quality control line and detection line were comfirmed,the nucleic acid colloidal gold test strip was assembled,and the specificity,sensitivity,repeatability and stability of the colloidal gold test strip were verified.Results:The DNA of Candida albicans,Cryptococcus neoformans and Mucor was extracted by boiling method,the concentrations were more than 180μg·L^(-1),and the purities were 1.60-2.10.Under the condition of pH 7.0,the optimal labeling amount of colloidal goldlabeled streptavidin was 3.3μg in 100μL of colloidal gold-solution,the concentration of biotinylated bovine serum albumin(BSA-Biotin)coated by quality control line was 2.00 g·L^(-1),and the concentration of anti-FITC antibody coated by detection line was 1.00 g·L^(-1).The specificity of nucleic acid test strip was consistent with the results of electrophoresis,and only Candida albicans,Cryptococcus neoformans and Mucor showed positive results.There were no cross reactions with Staphylococcus aureus,Streptococcus pneumoniae,Type B Hemolytic streptococcus,Pseudomonas aeruginosa,Klebsiella pneumoniae,Acinetobacter baumannii,Escherichia coli and other common respiratory tract infection bacteria.The sensitivity detection results showed that the nucleic acid test strips could still accurately detect the DNA of Candida albicans,Cryptococcus neoformans and Mucor when the DNA concentrations were of 10-4,10-2 and 10-3μg·L^(-1),respectively.The results of ordinary PCR electrophoresis showed that the lowest detection concentrations of Candida albicans,Cryptococcus neoformans and Mucor were 0.01,1.00,and 0.10μg·L^(-1),respectively.In repeatability test,nucleic acid test strips were verified by different operators in different laboratories,the results were consistent,and the reproducibility was good;in stability test,nucleic acid test strips were tested in 6,9 and 12 months and the results were as expected with good stability.Conclusion:The established method of nucleic acid colloidal gold test strip for respiratory tract fungal infection in this study can be used to detect Candida albicans,Cryptococcus neoformans,Mucor and other fungi with high specificity,sensitivity,simple and rapid operation.