Novel CRISPR-based detection of Leishmania species
Tegumentary leishmaniasis, a disease caused by protozoan parasites of the genus Leishmania , is a major public health problem in many regions of Latin America. Its diagnosis is difficult given other conditions resembling leishmaniasis lesions and co-occurring in the same endemic areas. A combination...
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2022
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| author | Eva Dueñas Jose A. Nakamoto Luis Cabrera-Sosa Percy Huaihua María Cruz Jorge Arévalo Pohl Milón Vanessa Adaui |
| author_browse | Eva Dueñas Jorge Arévalo Jose A. Nakamoto Luis Cabrera-Sosa María Cruz Percy Huaihua Pohl Milón Vanessa Adaui |
| author_facet | Eva Dueñas Jose A. Nakamoto Luis Cabrera-Sosa Percy Huaihua María Cruz Jorge Arévalo Pohl Milón Vanessa Adaui |
| author_sort | Eva Dueñas |
| collection | Producción científica |
| description | Tegumentary leishmaniasis, a disease caused by protozoan parasites of the genus Leishmania , is a major public health problem in many regions of Latin America. Its diagnosis is difficult given other conditions resembling leishmaniasis lesions and co-occurring in the same endemic areas. A combination of parasitological and molecular methods leads to accurate diagnosis, with the latter being traditionally performed in centralized reference and research laboratories as they require specialized infrastructure and operators. Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas) systems have recently driven innovative tools for nucleic acid detection that combine high specificity, sensitivity and speed and are readily adaptable for point-of-care testing. Here, we harnessed the CRISPR-Cas12a system for molecular detection of Leishmania spp., emphasizing medically relevant parasite species circulating in Peru and other endemic areas in Latin America, with Leishmania (Viannia) braziliensis being the main etiologic agent of cutaneous and mucosal leishmaniasis. We developed two assays targeting multi-copy targets commonly used in the molecular diagnosis of leishmaniasis: the 18S ribosomal RNA gene (18S rDNA), highly conserved across Leishmania species, and a region of kinetoplast DNA (kDNA) minicircles conserved in the L . ( Viannia ) subgenus. Our CRISPR-based assays were capable of detecting down to 5 × 10 −2 (kDNA) or 5 × 10 0 (18S rDNA) parasite genome equivalents/reaction with PCR preamplification. The 18S PCR/CRISPR assay achieved pan- Leishmania detection, whereas the kDNA PCR/CRISPR assay was specific for L . ( Viannia ) detection. No cross-reaction was observed with Trypanosoma cruzi strain Y or human DNA. We evaluated the performance of the assays using 49 clinical samples compared to a kDNA real-time PCR assay as the reference test. The kDNA PCR/CRISPR assay performed equally well as the reference test, with positive and negative percent agreement of 100%. The 18S PCR/CRISPR assay had high positive and negative percent agreement of 82.1% and 100%, respectively. The findings support the potential applicability of the newly developed CRISPR-based molecular tools for first-line diagnosis of Leishmania infections at the genus and L . ( Viannia ) subgenus levels. |
| format | Article |
| id | oai:cris.upeu.edu.pe:20.500.15037/904 |
| institution | UPeU |
| language | English |
| publishDate | 2022 |
| publishDateRange | 2022 |
| publishDateSort | 2022 |
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| spelling | oai:cris.upeu.edu.pe:20.500.15037/9042026-10-30T16:14:58ZNovel CRISPR-based detection of Leishmania speciesEva DueñasJose A. NakamotoLuis Cabrera-SosaPercy HuaihuaMaría CruzJorge ArévaloPohl MilónVanessa AdauiCRISPRBiologyMinicircleLeishmaniaLeishmaniasisLeishmania braziliensisBartonellaCutaneous leishmaniasisVirologyPolymerase chain reactionGeneticsComputational biologyGeneParasite hostingWorld Wide WebComputer scienceCRISPRBiologyMinicircleLeishmaniaLeishmaniasisLeishmania braziliensisBartonellaCutaneous leishmaniasisVirologyPolymerase chain reactionGeneticsComputational biologyGeneParasite hostingHealth Sciences Medicine Public Health, Environmental and Occupational Health Research on Leishmaniasis StudiesLife Sciences Biochemistry, Genetics and Molecular Biology Molecular Biology CRISPR and Genetic EngineeringSocial Sciences Business, Management and Accounting Business and International Management Innovation and Socioeconomic DevelopmentTegumentary leishmaniasis, a disease caused by protozoan parasites of the genus Leishmania , is a major public health problem in many regions of Latin America. Its diagnosis is difficult given other conditions resembling leishmaniasis lesions and co-occurring in the same endemic areas. A combination of parasitological and molecular methods leads to accurate diagnosis, with the latter being traditionally performed in centralized reference and research laboratories as they require specialized infrastructure and operators. Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas) systems have recently driven innovative tools for nucleic acid detection that combine high specificity, sensitivity and speed and are readily adaptable for point-of-care testing. Here, we harnessed the CRISPR-Cas12a system for molecular detection of Leishmania spp., emphasizing medically relevant parasite species circulating in Peru and other endemic areas in Latin America, with Leishmania (Viannia) braziliensis being the main etiologic agent of cutaneous and mucosal leishmaniasis. We developed two assays targeting multi-copy targets commonly used in the molecular diagnosis of leishmaniasis: the 18S ribosomal RNA gene (18S rDNA), highly conserved across Leishmania species, and a region of kinetoplast DNA (kDNA) minicircles conserved in the L . ( Viannia ) subgenus. Our CRISPR-based assays were capable of detecting down to 5 × 10 −2 (kDNA) or 5 × 10 0 (18S rDNA) parasite genome equivalents/reaction with PCR preamplification. The 18S PCR/CRISPR assay achieved pan- Leishmania detection, whereas the kDNA PCR/CRISPR assay was specific for L . ( Viannia ) detection. No cross-reaction was observed with Trypanosoma cruzi strain Y or human DNA. We evaluated the performance of the assays using 49 clinical samples compared to a kDNA real-time PCR assay as the reference test. The kDNA PCR/CRISPR assay performed equally well as the reference test, with positive and negative percent agreement of 100%. The 18S PCR/CRISPR assay had high positive and negative percent agreement of 82.1% and 100%, respectively. The findings support the potential applicability of the newly developed CRISPR-based molecular tools for first-line diagnosis of Leishmania infections at the genus and L . ( Viannia ) subgenus levels.Articlehttps://pubmed.ncbi.nlm.nih.gov/36187950https://www.frontiersin.org/articles/10.3389/fmicb.2022.958693/pdfhttps://doi.org/10.3389/fmicb.2022.95869310.3389/fmicb.2022.958693https://hdl.handle.net/20.500.15037/904W4295942982enFrontiers in Microbiology1664-302X2022-09-152026-06-13T09:40:04Z2026-06-13T09:40:04Z |
| spellingShingle | CRISPR Biology Minicircle Leishmania Leishmaniasis Leishmania braziliensis Bartonella Cutaneous leishmaniasis Virology Polymerase chain reaction Genetics Computational biology Gene Parasite hosting World Wide Web Computer science CRISPR Biology Minicircle Leishmania Leishmaniasis Leishmania braziliensis Bartonella Cutaneous leishmaniasis Virology Polymerase chain reaction Genetics Computational biology Gene Parasite hosting Health Sciences Medicine Public Health, Environmental and Occupational Health Research on Leishmaniasis Studies Life Sciences Biochemistry, Genetics and Molecular Biology Molecular Biology CRISPR and Genetic Engineering Social Sciences Business, Management and Accounting Business and International Management Innovation and Socioeconomic Development Eva Dueñas Jose A. Nakamoto Luis Cabrera-Sosa Percy Huaihua María Cruz Jorge Arévalo Pohl Milón Vanessa Adaui Novel CRISPR-based detection of Leishmania species |
| title | Novel CRISPR-based detection of Leishmania species |
| title_full | Novel CRISPR-based detection of Leishmania species |
| title_fullStr | Novel CRISPR-based detection of Leishmania species |
| title_full_unstemmed | Novel CRISPR-based detection of Leishmania species |
| title_short | Novel CRISPR-based detection of Leishmania species |
| title_sort | novel crispr based detection of leishmania species |
| topic | CRISPR Biology Minicircle Leishmania Leishmaniasis Leishmania braziliensis Bartonella Cutaneous leishmaniasis Virology Polymerase chain reaction Genetics Computational biology Gene Parasite hosting World Wide Web Computer science CRISPR Biology Minicircle Leishmania Leishmaniasis Leishmania braziliensis Bartonella Cutaneous leishmaniasis Virology Polymerase chain reaction Genetics Computational biology Gene Parasite hosting Health Sciences Medicine Public Health, Environmental and Occupational Health Research on Leishmaniasis Studies Life Sciences Biochemistry, Genetics and Molecular Biology Molecular Biology CRISPR and Genetic Engineering Social Sciences Business, Management and Accounting Business and International Management Innovation and Socioeconomic Development |
| topic_facet | CRISPR Biology Minicircle Leishmania Leishmaniasis Leishmania braziliensis Bartonella Cutaneous leishmaniasis Virology Polymerase chain reaction Genetics Computational biology Gene Parasite hosting World Wide Web Computer science CRISPR Biology Minicircle Leishmania Leishmaniasis Leishmania braziliensis Bartonella Cutaneous leishmaniasis Virology Polymerase chain reaction Genetics Computational biology Gene Parasite hosting Health Sciences Medicine Public Health, Environmental and Occupational Health Research on Leishmaniasis Studies Life Sciences Biochemistry, Genetics and Molecular Biology Molecular Biology CRISPR and Genetic Engineering Social Sciences Business, Management and Accounting Business and International Management Innovation and Socioeconomic Development |
| url | https://pubmed.ncbi.nlm.nih.gov/36187950 https://www.frontiersin.org/articles/10.3389/fmicb.2022.958693/pdf https://doi.org/10.3389/fmicb.2022.958693 https://hdl.handle.net/20.500.15037/904 |
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