Development of a fluorescent recombinase-aided isothermal amplification-based nucleic acid assay for detection of Leishmania
10.16250/j.32.1374.2021187
- VernacularTitle:基于荧光重组酶介导等温扩增技术的 利什曼原虫核酸检测方法的建立
- Author:
Hong LIN
1
;
Song ZHAO
2
;
Yan-Hong LIU
3
;
Lei SHAO
1
;
Qing-Jie YING
3
;
Kun YANG
2
Author Information
1. Jiangsu Province Blood Center, Nanjing 210042, China
2. 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, China
3. Jiangsu Qitian Gene Technology Co., Ltd., China
- Publication Type:Journal Article
- Keywords:
Leishmania donovani;
Leishmania infantum;
Recombinase-aided isothermal amplification;
Nucleic acid detection;
Internal transcribed spacer 1;
Detection efficiency
- From:
Chinese Journal of Schistosomiasis Control
2021;33(5):452-456
- CountryChina
- Language:Chinese
-
Abstract:
Objective To develop a fluorescent recombinase-aided isothermal amplification (RAA)-based nucleic acid assay for detection of Leshimania. Methods Specific primers and probes were designed targeting Leishmania internal transcribed spacer 1 (ITS1) gene for RAA assay, and a fluorescent RAA assay was developed for detection of Leishmania following screening of primer pairs and optimization of primer and probe concentrations. The sensitivity of RAA assay for detection of Leishmania was evaluated using recombinant plasmid containing Leishmania ITS1 gene sequences at different copies and Leshimania genomic DNA at different concentrations as templates, and the specificity of RAA assay for detection of Leishmania was evaluated using the genomic DNA of transfusion-transmitted parasites, including Babesia microti, Toxoplasma gondii, Plamodium vivax, P. ovale, P. falciparum, P. malariae, L. donovani and L. infantum. Results After the optimal primer pair was screened from 9 pairs of primer combinations, the final primer and probe concentrations were optimized as 0.3 μmol/L and 0.08 μmol/L, respectively. Nucleic acid detection of Leishmania was completed by the fluorescent RAA assay at an isothermal temperature of 39 °C within 20 min. Remarkable florescent signals were seen within 5 min following RAA detection of genomic DNA of L. donovani and L. infantum, and no cross-reactions were observed with B. microti, T. gondii, P. vivax, P. ovale, P. falciparum or P. malariae. The lowest limitation of detection of the fluorescent RAA assay was 10 copies/μL recombinant plasmid containing Leishmania ITS1 gene sequences and 1 fg/μL Leishmania genomic DNA. Conclusions A rapid, simple, sensitive and specific fluorescent RAA assay is successfully developed for detection of L. donovani and L. infantum, which is effective for field screening of leishmaniasis.
