[Development of a fluorescent recombinase-aided isothermal amplification-based nucleic acid assay for detection of Leishmania].
Zhongguo Xue Xi Chong Bing Fang Zhi Za Zhi
; 33(5): 452-456, 2021 Oct 27.
Article
en Zh
| MEDLINE
| ID: mdl-34791841
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.Palabras clave
Texto completo:
1
Base de datos:
MEDLINE
Asunto principal:
Ácidos Nucleicos
/
Leishmania
Tipo de estudio:
Diagnostic_studies
Idioma:
Zh
Revista:
Zhongguo Xue Xi Chong Bing Fang Zhi Za Zhi
Año:
2021
Tipo del documento:
Article
País de afiliación:
China