文章摘要
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].中华流行病学杂志,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
收稿日期:2023-02-15  出版日期:2023-05-13
DOI:10.3760/cma.j.cn112338-20230215-00079
中文关键词: 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
英文关键词: Candida albicans  Candida tropicalis  Recombined mannose-binding lectin protein-magnetic beads  Candidemia  Recombinant enzyme-assisted polymerase chain reaction
基金项目:国家重点研发计划(2021YFC2301102);国家自然科学基金(82202593)
作者单位E-mail
张梦怡 华北理工大学临床医学院, 唐山 063210
中国疾病预防控制中心病毒病预防控制所, 国家卫生健康委员会医学病毒和病毒病重点实验室, 北京 102206
唐山工人医院检验科, 唐山 063000 
 
陈小萍 中国疾病预防控制中心传染病预防控制所, 北京 102206  
孙秀丽 华北理工大学临床医学院, 唐山 063210
中国疾病预防控制中心病毒病预防控制所, 国家卫生健康委员会医学病毒和病毒病重点实验室, 北京 102206 
 
马学军 中国疾病预防控制中心病毒病预防控制所, 国家卫生健康委员会医学病毒和病毒病重点实验室, 北京 102206  
申辛欣 中国疾病预防控制中心病毒病预防控制所, 国家卫生健康委员会医学病毒和病毒病重点实验室, 北京 102206 x616815@126.com 
郭彦言 唐山工人医院检验科, 唐山 063000 13932581079@163.com 
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中文摘要:
      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。
英文摘要:
      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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