Establishment of a recombinase-aided isothermal amplification assay for nucleic acid detection of Echinococcus multilocularis and its preliminary application
10.16250/j.32.1374.2019284
- VernacularTitle:重组酶介导的等温核酸扩增技术检测多房棘球绦虫方法的建立及初步应用
- Author:
Hong-Rang ZHOU
1
,
2
,
3
,
4
,
5
;
Mu-Xin CHEN
1
,
2
,
3
,
4
,
5
;
Qing YU
1
,
2
,
3
,
4
,
5
;
Lin AI
1
,
2
,
3
,
4
,
5
;
Ying WANG
1
,
2
,
3
,
4
,
5
;
Qiu-Li XU
1
,
2
,
3
,
4
,
5
;
Ning XIAO
1
,
2
,
3
,
4
,
5
Author Information
1. National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention
2. Chinese Center for Tropical Diseases Research
3. WHO Collaborating Centre for Tropical Diseases
4. National Center for International Research on Tropical Diseases, Ministry of Science and Technology
5. Key Laboratory of Parasite and Vector Biology of National Health Commission, Shanghai 200025, China
- Publication Type:Journal Article
- Keywords:
Echinococcus multilocularis;
Recombinase-aided isothermal amplification assay (RAA);
Nucleic acid detection;
Diagnostic performance
- From:
Chinese Journal of Schistosomiasis Control
2020;32(2):168-173
- CountryChina
- Language:Chinese
-
Abstract:
Objective To establish a rapid nucleic acid detection technique for identification of Echinococcus multilocularis based on the recombinase aided isothermal amplification assay (RAA) and assess its diagnostic efficiency. Methods The mitochondrial gene sequence of E. multilocularis (GenBank accession number: AB018440) was used as a target sequence. The primers were designed according to the RAA reaction principle and synthesized, and RAA was performed using the generated primers. E. multilocularis genomic DNA at various concentrations and the pMD19-T (Simple) vector containing various copies of the target gene fragment were amplified using RAA to evaluate its sensitivity for detection of E. multilocularis, and RAA was em- ployed to detect the genomic DNA of E. granulosus G1 genotype, Taenia saginata, T. asiatica, T. multiceps, Dipylidium caninum, Toxocara canis, Trichuris trichiura, Giardia lamblia, Fasciola hepatica, Paragonimus westermani, Fasciola gigantica and Clonorchis sinensis to evaluate its specificity. In addition, the optimized RAA was employed to detect nine tissue specimens of E. granulosus-infected animals, 3 fecal samples from E. granulosus-infected dogs and 2 fecal samples from field infected dogs to examine its reliability and feasibility. Results The established RAA was able to detect the specific target gene fragment of E. multilocularis within 40 min. The lowest detect limit of RAA was 10 pg if E. multilocularis genomic DNA served as a template. If the re- combinant plasmid was used as a template, the minimally detectable copy number of RAA was 104. In addition, RAA was nega- tive for the genomic DNA of E. granulosus G1 genotype, T. saginata, T. asiatica, T. multiceps, D. caninum, T. canis, T. trichiura, G. lamblia, F. hepatica, P. westermani, F. gigantica and C. sinensis. The established RAA was positive for detection of the tissue specimens of infected animals, and simulated and field dog stool samples. Conclusion A rapid, sensitive and specific RAA is established, which shows promising values in identification of E. multilocularis and gene diagnosis of alveolar echinococcosis.
