Establishment of a recombinase-aided isothermal amplification assay for nucleic acid detection of Angiostrongylus cantonensis
10.16250/j.32.1374.2020091
- VernacularTitle:基于重组酶介导等温扩增技术的广州管圆线虫核酸检测方法的建立
- Author:
Qiang ZHANG
1
;
Xin DING
1
;
Yan-Hong LIU
2
;
Jian-Feng LIU
1
;
Xiang-Zhen XU
1
;
Qing-Jie YING
2
;
Yang DAI
1
;
Jun CAO
1
Author Information
1. National Health Commission Key Laboratory of Parasitic Disease Control and Prevention, Jiangsu Provincial Key Laboratory on Parasite and Vector Control Technology, Jiangsu Institute of Parasitic Diseases, Public Health Research Center, Jiangnan University, Wuxi 214064, China
2. Jiangsu Qitian Gene Technology Co., Ltd., China
- Publication Type:Journal Article
- Keywords:
Angiostrongylus cantonensis;
Isothermal amplification;
Recombinase;
Nucleic acid detection;
Detection efficiency
- From:
Chinese Journal of Schistosomiasis Control
2020;32(4):350-354
- CountryChina
- Language:Chinese
-
Abstract:
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.
