| 龙炫辉,杨永强,魏 涛,邓宁波,桑 晔,唐文志,曾朱君.实时荧光重组酶聚合酶扩增快速检测临床常见曲霉菌方法的建立[J].,2020,(16):3189-3192 |
| 实时荧光重组酶聚合酶扩增快速检测临床常见曲霉菌方法的建立 |
| Establishment of A Real-time Fluorescent Recombinase Polymerase Amplification Method for Rapid Detection of Common Clinical Aspergillus |
| 投稿时间：2020-03-17 修订日期：2020-04-11 |
| DOI：10.13241/j.cnki.pmb.2020.16.042 |
| 中文关键词: 实时荧光重组酶聚合酶扩增 曲霉菌 快速检测 |
| 英文关键词: Real-time RPA Aspergillus Rapid detection |
| 基金项目:广东省医学科学技术研究基金项目（A2018522） |
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| 中文摘要: |
| 摘要 目的：针对曲霉菌属转录间隔区ITS1设计引物、探针，利用实时荧光重组酶聚合酶扩增（Real-time RPA）技术建立一种快速、准确、经济的临床常见曲霉菌检测鉴定方法。方法：利用建立的实时荧光重组酶聚合酶扩增体系对标准菌株及临床标本提取的DNA进行扩增，验证该方法的性能。结果：本研究针对曲霉菌属转录间隔区ITS1设计引物、探针利用RPA试剂盒（荧光型）建立了Real-time RPA扩增体系，在15分钟内即可检测出临床常见的四种曲霉菌；特异性试验结果显示反应体系只对烟曲霉、黄曲霉、土曲霉、黑曲霉四种曲霉呈现出明显的扩增曲线，而其它细菌和真菌均无扩增曲线。灵敏性试验显示最低检出限为10-3 ng/μL。临床验证试验的12份曲霉菌均有较高的扩增效应。结论：本研究建立的Real-time RPA方法能快速、特异、灵敏地检出烟曲霉、黄曲霉、土曲霉、黑曲霉等临床常见曲霉菌，为曲霉菌的快速、现场检测提供了一种新的思路。 |
| 英文摘要: |
| ABSTRACT Objective: To design primers and probes for Aspergillus transcript spacer ITS1, and to use Real-time fluorescent recombinase polymerase amplification (Real-time RPA) technology to establish a rapid, accurate, and economical method for detection and identification of common clinical Aspergillus. Methods: The established Real-time fluorescent recombinase polymerase amplification system was used to amplify the DNA extracted from standard strains and clinical specimens to verify the performance of the method. Results: In this study, we designed primers and probes for the Aspergillus transcript spacer ITS1 using RPA kit (fluorescent type) to establish a Real-time RPA amplification system, and detected four clinically common Aspergillus species within 15 minutes; The specific test results show that the reaction system only has specific amplification for Aspergillus fumigatus, Aspergillus flavus, Aspergillus terrestris and Aspergillus niger, while there was no amplification for other bacteria and fungi; The sensitivity test showed that the detection limit was 10-3 ng/μL. 12 samples of Aspergillus in clinical validation test had high amplification effect. Conclusion: The Real-time RPA method established in this study can detect Aspergillus fumigatus, Aspergillus flavus, Aspergillus terreus, Aspergillus niger and other common clinical aspergillus quickly, specifically and sensitively. It provides a new idea for rapid and field detection of Aspergillus. |
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