[基于荧光重组酶辅助等温扩增的核酸检测方法的开发用于检测]
[Development of a fluorescent recombinase-aided isothermal amplification-based nucleic acid assay for detection of ].
作者信息
Lin H, Zhao S, Liu Y H, Shao L, Ying Q J, Yang K
机构信息
Jiangsu Province Blood Center, Nanjing 210042, China.
Key Laboratory of National Health Commission on Parasitic Disease Control and Prevention, Jiangsu Provincial Key Laboratory on Parasite and Vector Control Technology, Jiangsu Institute of Parasitic Diseases, China.
出版信息
Zhongguo Xue Xi Chong Bing Fang Zhi Za Zhi. 2021 Oct 27;33(5):452-456. doi: 10.16250/j.32.1374.2021187.
OBJECTIVE
To develop a fluorescent recombinase-aided isothermal amplification (RAA)-based nucleic acid assay for detection of .
METHODS
Specific primers and probes were designed targeting internal transcribed spacer 1 () gene for RAA assay, and a fluorescent RAA assay was developed for detection of following screening of primer pairs and optimization of primer and probe concentrations. The sensitivity of RAA assay for detection of was evaluated using recombinant plasmid containing gene sequences at different copies and genomic DNA at different concentrations as templates, and the specificity of RAA assay for detection of was evaluated using the genomic DNA of transfusion-transmitted parasites, including , , , , , , and .
RESULTS
After the optimal primer pair was screened from 9 pairs of primer combinations, the final primer and probe concentrations were optimized as 0.3 μmol/L and 0.08 μmol/L, respectively. Nucleic acid detection of was completed by the fluorescent RAA assay at an isothermal temperature of 39 °C within 20 min. Remarkable florescent signals were seen within 5 min following RAA detection of genomic DNA of and , and no cross-reactions were observed with , , , , or . The lowest limitation of detection of the fluorescent RAA assay was 10 copies/μL recombinant plasmid containing gene sequences and 1 fg/μL genomic DNA.
CONCLUSIONS
A rapid, simple, sensitive and specific fluorescent RAA assay is successfully developed for detection of and , which is effective for field screening of leishmaniasis.
目的
开发一种基于荧光重组酶辅助等温扩增（RAA）的核酸检测方法用于检测……
方法
针对……的内转录间隔区1（ITS1）基因设计特异性引物和探针用于RAA检测，在筛选引物对并优化引物和探针浓度后，开发了一种用于检测……的荧光RAA检测方法。以含有不同拷贝数……基因序列的重组质粒和不同浓度的……基因组DNA为模板，评估RAA检测方法检测……的灵敏度，以包括……、……、……、……、……、……、……和……在内的输血传播寄生虫的基因组DNA评估RAA检测方法检测……的特异性。
结果
从9对引物组合中筛选出最佳引物对后，最终将引物和探针浓度分别优化为0.3 μmol/L和0.08 μmol/L。通过荧光RAA检测方法在39℃等温温度下20分钟内完成了……的核酸检测。在对……和……的基因组DNA进行RAA检测后5分钟内可见明显的荧光信号，但与……、……、……、……或……未观察到交叉反应。荧光RAA检测方法的最低检测限为含有……基因序列的重组质粒10拷贝/μL和……基因组DNA 1 fg/μL。
结论
成功开发了一种用于检测……和……的快速、简单、灵敏且特异的荧光RAA检测方法，对利什曼病的现场筛查有效。
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