Establishment and evaluation of the detection method of Cryptosporidium specific DNA fragment by recombinase aided isothermal amplification
10.16250/j.32.1374.2019168
- VernacularTitle:重组酶介导的隐孢子虫属特异性等温核酸扩增方法的建立及评价
- Author:
Bi-Xian NI
1
,
2
;
Xiao-Min WU
1
,
2
;
Yan-Hong LIU
3
;
Xiang-Zhen XU
1
,
2
;
Qing-Jie YING
3
;
Jun CAO
1
,
2
;
Yang DAI
1
,
2
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, Wuxi 214064, China
2. Public Health Research Center, Jiangnan University, China
3. Jiangsu Qitian Gene Technology Co., Ltd, China
- Publication Type:Journal Article
- Keywords:
Cryptosporidium;
Nucleic acid detection;
Recombinase-aided isothermal amplification assay;
Fluorescent probe;
Diagnostic efficiency
- From:
Chinese Journal of Schistosomiasis Control
2019;31(4):388-392
- CountryChina
- Language:Chinese
-
Abstract:
Objective To establish a recombinase-aided isothermal amplification (RAA) assay for detection of Cryptosporidium. Methods Based on Cryptosporidium-specific 18S rRNA selected as the target gene to be detected, and the primer sequences and fluorescent probes designed using the software Amplfix, and a fluorescent RAA assay was established and optimized. The fluorescent RAA assay was performed to detect 18S RNA target sequence-contained recombinant plasmids at various copies, genomic DNA of Cryptosporidium oocysts at various concentrations, and genomic DNA extracted from various numbers of Cryptosporidium oocysts to assess the sensitivity of the assay, and to detect genomic DNA extracted from Cryptosporidium oocysts, Giardia lamblia cysts, Schistosoma japonicum eggs, Ascaris lumbricoides eggs, Clonorchis sinensis eggs, Salmonella and Shigella to determine the specificity of the assay. Results A fluorescent RAA assay was successfully established, which was effective to amplify the specific 18S RNA gene fragments of Cryptosporidium within 20 min at 39 ℃. The lowest limits of the fluorescent RAA assay were 102 copies/μL for detection of 18S RNA target sequence-contained recombinant plasmids at various copies, 1 pg/μL for detection of genomic DNA of Cryptosporidium oocysts at various concentrations, and one Cryptosporidium oocyst/μL for detection of genomic DNA extracted from various numbers of Cryptosporidium oocysts, and the fluorescent RAA assay was all negative for detection of genomic DNA from G. lamblia cysts, S. japonicum eggs, A. lumbricoides eggs, C. sinensis eggs, Salmonella and Shigella. Conclusion A novel fluorescent RAA assay is successfully established, which is simple, rapid, sensitive and specific to detect genomic DNA of Cryptosporidium oocysts.
