文献：

Establishment and Evaluation of a Rapid Detection Method for Respiratory Fungi Infection Nucleic Acid Colloidal Gold Test Strip

文献链接：

https://xueshu.baidu.com/usercenter/paper/show?paperid=141100e0ay0n02p0ec5m0g20p9638719&site=xueshu_se

作者：

姜菲菲; 宋伶俐; 潘蓓珍; 王岳峰; 李昊宇; 孙丽媛

相关产品：

荧光素-生物素 FITC-BIOTIN

原文摘要：

Establish a nucleic acid colloidal gold test strip detection method for respiratory fungal infections using polymerase chain reaction (PCR) technology and colloidal gold technology, and evaluate its detection effectiveness. Method: The fungal intratranscriptional spacer (ITS) gene fragment was selected as the target gene based on the GenBank database. The fungal ITS gene fragment was analyzed using DNAMAN software, and biotin and fluorescein isothiocyanate (FITC) were used to modify and label the 5 'end of the fungal universal primer; Establish a PCR system and optimize the optimal conditions for the system; Determine the optimal labeling amount, quality control line, and detection line concentration for streptavidin on colloidal gold immunochromatographic test strips, assemble nucleic acid colloidal gold test strips, and verify the specificity, sensitivity, repeatability, and stability of the test strips. Result: The DNA of Candida albicans, Cryptococcus neoformans, and Aspergillus was extracted by boiling method, with a concentration of 180 μ g · L ^ (-1) or higher and a purity of 1.60-2.10; Under pH 7.0 conditions, the optimal labeling amount for preparing colloidal gold labeled streptavidin per 100 μ L of colloidal gold solution is 3.3 μ g, the quality control line is coated with Biotinized bovine serum albumin (BSA Biotin) at a concentration of 2.00 g · L ^ (-1), and the detection line is coated with anti FITC antibody at a concentration of 1.00 g · L ^ (-1). The specificity of the nucleic acid test strip is consistent with the electrophoresis results, with only Candida albicans, Cryptococcus neoformans, and Trichoderma showing positive results. There is no cross reactivity with common respiratory infection bacteria such as Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus pyogenes, Pseudomonas aeruginosa, Klebsiella pneumoniae, Acinetobacter baumannii, and Escherichia coli. Sensitivity testing showed that the nucleic acid test strips could still accurately detect Candida albicans, Cryptococcus neoformans, and Trichoderma when the DNA concentrations were 10-4, 10-2, and 10-3 mg · L ^ (-1), respectively. However, ordinary PCR electrophoresis results showed that the lowest detection concentrations for Candida albicans, Cryptococcus neoformans, and Trichoderma were 0.01, 1.00, and 0.10 mg · L ^ (-1), respectively; Repeatability testing, where nucleic acid test strips are validated by different operators in different laboratories, with consistent results and good repeatability; Stability testing was conducted on nucleic acid test strips at months 6, 9, and 12, and the results met expectations, indicating good stability. Conclusion: The respiratory fungal infection nucleic acid colloidal gold test strip established in this study can detect fungi such as Candida albicans, Cryptococcus neoformans, and Aspergillus, with high specificity and sensitivity, simple and fast operation.

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Fungi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。

图为：荧光素-生物素 FITC-BIOTIN结构式

方法:根据GenBank数据库选择Fungi内转录间隔区(ITS)基因片段作为靶基因,应用DNAMAN软件对FungiITS基因片段进行分析,并分别在Fungi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*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*佳标记量和质控线及检测线*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*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)。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*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。