Objective: To establish a recombinase-aided isothermal amplification (RAA) assay for the nucleic acid detection of Angiostrongylus cantonensis.
Methods: The internal transcribed spacer-1 (ITS1) gene sequence of A. cantonensis was used as the detection target sequence, and the specific primers and probes were designed and synthesized, followed by screening of the primers and probes with the highest specificity, to establish the basic and fluorescent RAA assay for nucleic acid detection of A. cantonensis. The sensitivity of the fluorescent RAA assay was evaluated by using the target gene fragment sequence-contained recombinant plasmids at various copy numbers and the genomic DNA from A. cantonensis as the template DNA samples, and the specificity of the fluorescent RAA assay was evaluated by using the genomic DNA from A. cantonensis, Schistosoma mansoni, Ascaris lumbricoides, Clonorchis sinensis, Echinococcus granulosus and Ancylostoma duodenale, as well as Pomacea canaliculata and Biomphalaria straminea snail tissues as the template DNA samples.
Results: A fluorescent RAA assay was successfully established for nucleic acid detection of A. cantonensis, which achieved real-time amplification of the specific DNA fragment of A. cantonensis within 20 min at 37 ℃. By using the target gene fragment sequence-contained recombinant plasmids at various copy numbers and the genomic DNA from A. cantonensis as the DNA templates, the lowest detection limits of the fluorescent RAA assay were 10 copies/μL of recombinant plasmids and 100 pg/μL of genomic DNA, respectively. The fluorescent RAA assay was negative for detection of the genomic DNA from A. cantonensis, S. mansoni, A. lumbricoides, C. sinensis, E. granulosus, A. duodenale, and P. canaliculata and B. straminea snail tissues.
Conclusions: A simple, rapid fluorescent RAA assay has been successfully established, which has a high sensitivity and specificity for the nucleic acid detection of A. cantonensis.
［摘要］ 目的 建立一种基于重组酶介导等温扩增技术 (RAA) 的广州管圆线虫核酸检测方法。方法 选择广州管圆线虫内转录间隔区1 (ITS1) 基因序列作为靶基因序列, 设计、合成并筛选出特异性最强的引物和探针, 构建用于广州管圆线虫核酸检测的基础及荧光RAA检测方法。分别以含检测靶基因片段序列的不同拷贝数重组质粒以及不同浓度广州管圆线虫基因组DNA为模板进行荧光RAA扩增, 评价其检测敏感性; 分别以广州管圆线虫、曼氏血吸虫、似蚓蛔线虫、华支睾吸虫、细粒棘球绦虫、十二指肠钩口线虫及小管福寿螺和藁杆双脐螺螺体基因组DNA为模板进行荧光RAA扩增, 评价其检测特异性。结果 成功建立了用于广州管圆线虫核酸检测的荧光RAA法, 该方法可在37 ℃ 20 min内对广州管圆线虫特异性DNA片段实现实时扩增。以含靶基因片段序列的不同拷贝数重组质粒和不同浓度广州管圆线虫基因组DNA为模板时, 该法最低检出限分别为10拷贝/μL重组质粒和100 pg/μL基因组DNA; 以曼氏血吸虫、似蚓蛔线虫、华支睾吸虫、细粒棘球绦虫、十二指肠钩口线虫及小管福寿螺和藁杆双脐螺螺体基因组DNA为模板, 检测结果均为阴性。结论 成功建立了一种可用于广州管圆线虫核酸检测的荧光RAA法, 其检测简便快速, 具备较好的敏感性和特异性。.
Keywords: Angiostrongylus cantonensis; Detection efficiency; Isothermal amplification; Nucleic acid detection; Recombinase.