Zhao S, Liu Y H, Ye Y Y, Li W, Zhang J F, Guo L C, Ying Q J, Yang H T, Yang K
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, Wuxi 214064, China.
Jiangsu Qitian Gene Technology Co., Ltd., China.
Zhongguo Xue Xi Chong Bing Fang Zhi Za Zhi. 2020 Jun 30;32(4):335-339. doi: 10.16250/j.32.1374.2020058.
To establish a recombinase-aided isothermal amplification (RAA) assay for nucleic acid detection of .
The 121 bp highly-repeated sequence of was selected as the target gene fragment to be detected. The primers and fluorescent probes were designed using the Amplfix software, and a fluorescent RAA assay was established and optimized. The fluorescent RAA assay was performed to detect gradient diluent recombinant plasmids containing target gene fragment and different concentrations of genomic DNA to determine the sensitivity, and this assay was applied to detect the genomic DNA of , , and to evaluate the specificity.
A fluorescent RAA assay was successfully established, which was effective to amplify the specific gene fragments of within 20 min at 39 ℃. The minimum detectable limit of the fluorescent RAA assay was 10 copies/μL using recombinant plasmids as templates and 0.1 fg/μL using genomic DNA samples as templates. The fluorescent RAA assays were all negative for detecting the genomic DNA from , , and .
A novel fluorescent RAA assay is successfully established, which is simple, rapid, sensitive and specific to detect genomic DNA of .
建立一种用于[具体检测对象]核酸检测的重组酶辅助等温扩增（RAA）方法。
选择[具体检测对象]的121 bp高度重复序列作为待检测的靶基因片段。使用Amplfix软件设计引物和荧光探针，建立并优化荧光RAA检测方法。进行荧光RAA检测以检测含靶基因片段的梯度稀释重组质粒和不同浓度的[具体检测对象]基因组DNA，确定灵敏度，并将该检测方法应用于检测[具体检测对象1]、[具体检测对象2]、[具体检测对象3]和[具体检测对象4]的基因组DNA，评估特异性。
成功建立了荧光RAA检测方法，该方法在39℃下20分钟内可有效扩增[具体检测对象]的特异性基因片段。以重组质粒为模板时，荧光RAA检测的最低检测限为10拷贝/μL，以[具体检测对象]基因组DNA样本为模板时为0.1 fg/μL。检测[具体检测对象1]、[具体检测对象2]、[具体检测对象3]和[具体检测对象4]的基因组DNA时，荧光RAA检测均为阴性。
成功建立了一种新型荧光RAA检测方法，该方法用于检测[具体检测对象]的基因组DNA具有简单、快速、灵敏和特异的特点。
