A low-cost and open-source protocol to produce key enzymes for molecular detection assays

Mendoza-Rojas, Gabriel; Sarabia-Vega, Vanessa; Sanchez-Castro, Ana Elena; Tello, Lesia; Cabrera-Sosa, Luis; Nakamoto, Jose A.; Peñaranda, Katherin; Adaui, Vanessa; Alcántara, Roberto; Milón, Pohl

doi:10.1016/j.xpro.2021.100899

Cited by 5 publications

(3 citation statements)

References 20 publications

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“…LbCas12a-based detection reactions were performed as described previously ( Chen et al, 2018 ; Broughton et al, 2020 ) with some modifications ( Alcántara et al, 2021a ). The recombinant LbCas12a protein was expressed and purified as described ( Mendoza-Rojas et al, 2021 ). A single-stranded DNA (ssDNA) fluorophore quencher (FQ)-labeled reporter probe (5′ Cy3/TTATT/BHQ-2 3′) was selected and synthesized commercially (Macrogen Inc., Seoul, South Korea).…”

Section: Methodsmentioning

confidence: 99%

Novel CRISPR-based detection of Leishmania species

Dueñas

Nakamoto²,

Cabrera-Sosa³

et al. 2022

Front. Microbiol.

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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.

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Section: Methodsmentioning

confidence: 99%

Novel CRISPR-based detection of Leishmania species

Dueñas

Nakamoto²,

Cabrera-Sosa³

et al. 2022

Front. Microbiol.

Self Cite

View full text Add to dashboard Cite

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“…This open hardware device facilitates PCR reactions through precise, easily adjustable temperature cycles, and can be powered by a standard USB/USB-C charger, making it ideal for use outside traditional laboratory settings. Concurrently, better access to public domain DNA plasmids encoding for diagnostic enzymes has surged during the pandemic [30][31][32][33][34]. For instance, the FreeGenes project and ReClone network have engaged researchers around the globe who share DNA plasmids encoding these off-patent enzymes and protocols for their local production [35].…”

Section: Introductionmentioning

confidence: 99%

“…We focus on detecting the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) for two main reasons: first, the students' motivation for learning about the virus causing the COVID19 pandemic; and second, the availability of low-cost and open-access RT-PCR reagents for SARS-CoV-2 detection, a result of ongoing local initiatives (e.g. [30][31][32][33][34]). The crucial role of SARS-CoV-2 RNA detection in COVID-19 diagnostics has underscored the relevance of this technique.…”

mentioning

confidence: 99%

Open Educational Resources for distributed hands-on teaching in molecular biology

Cerda,

Aravena,

Zapata

et al. 2024

Preprint

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Open Educational Resources (OER), freely accessible learning, teaching and research materials, have been proposed as key enabling tools to achieve inclusive knowledge societies and equitable access to education. Here, we describe novel OER consisting of low cost and locally produced public domain biological reagents, open source hardware and free software collaborative notebooks to teach LAMP DNA amplification, RT-PCR RNA detection, enzyme kinetics and fluorescence imaging. These resources have been distributed nationwide to students' homes as a lab-in-a-box, i.e. remote teaching during the pandemic lockdowns, as well as in the form of personalized learning environments during in-person teaching after the opening of teaching laboratories. All the protocols and design files are available under open source licenses.

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UnCovid: A versatile, low-cost, and open-source protocol for SARS-CoV-2 RNA detection

Alcántara¹,

Peñaranda²,

Mendoza-Rojas³

et al. 2021

STAR Protocols

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Here, we describe a detailed step-by-step protocol to detect SARS-CoV-2 RNA using RT-PCR-mediated amplification and Crispr/Cas-based visualization. The optimized assay uses basic molecular biology equipment such as conventional thermocyclers and transilluminators for qualitative detection. Alternatively, a fluorescence plate reader can be used for quantitative measurements. The protocol detects two regions of the SARS-CoV-2 genome in addition to the human RNaseP sample control. Aiming to reach remote regions, this work was developed to use the portable molecular workstation from BentoLab.

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A low-cost and open-source protocol to produce key enzymes for molecular detection assays

Cited by 5 publications

References 20 publications

Novel CRISPR-based detection of Leishmania species

Novel CRISPR-based detection of Leishmania species

Open Educational Resources for distributed hands-on teaching in molecular biology

UnCovid: A versatile, low-cost, and open-source protocol for SARS-CoV-2 RNA detection

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