关键词: 呼吸道感染, 真菌, 胶体金, 核酸试纸条

Abstract:

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 180 μg·L^－1，and the purities were 1.60－2.10. Under the condition of pH 7.0， the optimal labeling amount of colloidal gold- labeled streptavidin was 3.3 μg in 100 μL 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^－3 μg·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 0.10^{μg·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.}

Key words: Respiratory tract infection, Fungi, Colloidal gold, Nucleic acid test strip