DSpace Repository

Novel CRISPR-based detection of Leishmania species

Show simple item record

dc.contributor.author Duenas, Eva
dc.contributor.author Nakamoto, Jose A.
dc.contributor.author Cabrera-Sosa, Luis
dc.contributor.author Huaihua, Percy
dc.contributor.author Cruz, Maria
dc.contributor.author Arévalo Zelada, Jorge Luis
dc.contributor.author Milon, Pohl
dc.contributor.author Adaui, Vanessa
dc.date.accessioned 2022-11-15T23:04:40Z
dc.date.available 2022-11-15T23:04:40Z
dc.date.issued 2022
dc.identifier.uri https://hdl.handle.net/20.500.12866/12578
dc.description.abstract 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 × 100 (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. en_US
dc.language.iso eng
dc.publisher Frontiers Media
dc.relation.ispartofseries Frontiers in Microbiology
dc.rights info:eu-repo/semantics/restrictedAccess
dc.rights.uri https://creativecommons.org/licenses/by-nc-nd/4.0/deed.es
dc.subject tegumentary leishmaniasis en_US
dc.subject Leishmania en_US
dc.subject CRISPR-Cas en_US
dc.subject nucleic acid detection en_US
dc.subject molecular diagnostics en_US
dc.subject kDNA en_US
dc.subject 18S rDNA en_US
dc.subject invasive and non-invasive clinical specimens en_US
dc.title Novel CRISPR-based detection of Leishmania species en_US
dc.type info:eu-repo/semantics/article
dc.identifier.doi https://doi.org/10.3389/fmicb.2022.958693
dc.relation.issn 1664-302X


Files in this item

Files Size Format View

There are no files associated with this item.

This item appears in the following Collection(s)

Show simple item record

info:eu-repo/semantics/restrictedAccess Except where otherwise noted, this item's license is described as info:eu-repo/semantics/restrictedAccess

Search DSpace


Browse

My Account

Statistics