| 张梦怡,陈小萍,孙秀丽,马学军,申辛欣,郭彦言.血液中念珠菌重组人甘露聚糖结合凝集素蛋白磁珠富集联合重组酶辅助聚合酶链式反应检测方法的建立[J].Chinese journal of Epidemiology,2023,44(5):823-827 |
| 血液中念珠菌重组人甘露聚糖结合凝集素蛋白磁珠富集联合重组酶辅助聚合酶链式反应检测方法的建立 |
| Establishment of a recombined mannose-binding lectin protein-magnetic beads—enriched binding recombinant enzyme-assisted polymerase chain reaction assay for Candida in blood samples |
| Received:February 15, 2023 |
| DOI：10.3760/cma.j.cn112338-20230215-00079 |
| KeyWord: 白色念珠菌 热带念珠菌 重组人甘露聚糖结合凝集素蛋白磁珠 念珠菌血症 重组酶辅助聚合酶链式反应 |
| English Key Word: Candida albicans Candida tropicalis Recombined mannose-binding lectin protein-magnetic beads Candidemia Recombinant enzyme-assisted polymerase chain reaction |
| FundProject:国家重点研发计划(2021YFC2301102);国家自然科学基金(82202593) |
| Author Name | Affiliation | E-mail | | Zhang Mengyi | College of Clinical Medicine, North China University of Science and Technology, Tangshan 063210, China
National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China
Clinical Laboratory, Tangshan Gongren Hospital, Tangshan 063000, China | | | Chen Xiaoping | National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China | | | Sun Xiuli | College of Clinical Medicine, North China University of Science and Technology, Tangshan 063210, China
National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China | | | Ma Xuejun | National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China | | | Shen Xinxin | National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China | x616815@126.com | | Guo Yanyan | Clinical Laboratory, Tangshan Gongren Hospital, Tangshan 063000, China | 13932581079@163.com |
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| Abstract: |
| 目的 建立重组人甘露聚糖结合凝集素蛋白（M1蛋白）磁珠富集病原体结合重组酶辅助聚合酶链式反应（RAP）巢式核酸扩增技术检测血液中白色念珠菌和热带念珠菌的方法，以实现对白色念珠菌血症和热带念珠菌血症的早期诊断。方法 针对白色念珠菌和热带念珠菌的内转录间隔区的高度保守区域的设计引物探针，建立检测白色念珠菌和热带念珠菌的RAP检测方法；用梯度稀释的标准菌株核酸和临床常见的血流感染病原体核酸检验灵敏度、重复性、特异性；用模拟样本进行M1蛋白磁珠富集血浆白色念珠菌和热带念珠菌RAP和实时荧光PCR的检测，并对结果进行比较。结果 建立的双重RAP方法灵敏度为2.4~2.8拷贝/反应，重复性好，特异性高；M1蛋白磁珠富集病原体结合双重RAP方法可在4 h内完成对血浆中白色念珠菌和热带念珠菌的检测，<10 CFU/ml的病原体富集后进行RAP检测样本数较PCR检测样本数多。结论 本研究建立了检测血液中白色念珠菌和热带念珠菌的双重RAP方法，该检测方法具备准确、快速、减少污染物的优点，在念珠菌血症快速检测中具有较好的应用潜力。 |
| English Abstract: |
| Objective To establish a nested recombinant enzyme-assisted polymerase chain reaction (RAP) technique combined with recombined mannose-binding lectin protein (M1 protein)-magnetic beads enrichment for the detection of Candida albicans (C. albicans) and Candida tropicalis (C. tropicalis) in blood samples for the early diagnosis of candidemia albicans and candidiemia tropicalis. Methods The primer probes for highly conserved regions of the internal transcribed spacerregions of C. albicans and C. tropicalis were deigned to establish RAP assays for the detections of C. albicans and C. tropicalis; The sensitivity and reproducibility of nucleic acid tests with gradient dilutions of standard strains and specificity of nucleic acid tests with common clinical pathogens causing bloodstream infection were condcuted. M1 protein-magnetic bead enriched plasma C. albicans and C. tropicalis were used for RAP and PCR in with simulated samples and the results were compared. Results The sensitivity of the established dual RAP assay was 2.4-2.8 copies/reaction, with higher reproducibility and specificity. M1 protein-magnetic bead enrichment of pathogen combined with the dual RAP assay could complete the detections of C. albicans and C. tropicalis in plasma within 4 hours. Fie the pathogen samples at concentration <10 CFU/ml, the number of the samples tested by RAP was higher than that tested by PCR after enrichment. Conclusion In this study, a dual RAP assay for the detections of C. albicans and C. tropicalis in blood sample was developed, which has the advantages of accuracy, rapidity, and less contaminants and has great potential for rapid detection of Candidemia. |
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