Journal: Poultry Science

Article Title: Field-deployable multiplex RAA-CRISPR/Cas12a platform rapidly and simultaneously detects seven Eimeria species in chickens

doi: 10.1016/j.psj.2026.106681

Figure Lengend Snippet: Screening of species-specific crRNAs and determination of fluorescence and visual detection thresholds for the E-MRC12a assay. (A-G) Real-time fluorescence curves of CRISPR/Cas12a reactions targeting the 18S rDNA of the seven Eimeria species using three candidate crRNAs per species: (A) E. maxima (EM1-EM3), (B) E. acervulina (EA1-EA3), (C) E. necatrix (EN1-EN3), (D) E. mitis (EI1-EI3), (E) E. praecox (EP1-EP3), (F) E.brunelti (EB1-EB3) and (G) E. tenella (ET1). (H) UV images of CRISPR/Cas12a reaction products for each species-specific crRNA. (I) Determination of the visual fluorescence threshold. NT: no-template control. Error bars represent mean ± SD (n = 3 per group).

Article Snippet: The CRISPR/Cas12a trans-cleavage assay was performed in a 20 μL reaction containing 2.5 μL of 10 × NEBuffer, 2 μL of LbCas12a protein (1 μM), 1 μL of crRNA (1 μM), 2 μL of ssDNA reporter (10 μM), 2 μL of RAA product, and ddH2O to volume.

Techniques: Fluorescence, CRISPR, Control

Journal: Poultry Science

Article Title: Field-deployable multiplex RAA-CRISPR/Cas12a platform rapidly and simultaneously detects seven Eimeria species in chickens

doi: 10.1016/j.psj.2026.106681

Figure Lengend Snippet: Evaluation of the specificity, sensitivity, and repeatability of the E-MRC12a assay . (A) Heatmap of fluorescence intensities from CRISPR/Cas12a reactions using species-specific crRNAs targeting genomic DNA from the seven Eimeria species, Escherichia coli, Salmonella spp ., and confirmed Eimeria -negative fecal samples. (B) Visual fluorescence under 254 nm UV light corresponding to (A). (C) Heatmap showing the sensitivity evaluation of the E-MRC12a assay. (D) Visual fluorescence under 254 nm UV light corresponding to (C). (E) Heatmap illustrating repeatability assessment of the E-MRC12a assay. NT: No-template control. For panels (A) and (C), different letters within a row indicate statistically significant differences ( P < 0.05); identical letters indicate no significant difference ( P > 0.05).

Article Snippet: The CRISPR/Cas12a trans-cleavage assay was performed in a 20 μL reaction containing 2.5 μL of 10 × NEBuffer, 2 μL of LbCas12a protein (1 μM), 1 μL of crRNA (1 μM), 2 μL of ssDNA reporter (10 μM), 2 μL of RAA product, and ddH2O to volume.

Techniques: Fluorescence, CRISPR, Control

Journal: Poultry Science

Article Title: Field-deployable multiplex RAA-CRISPR/Cas12a platform rapidly and simultaneously detects seven Eimeria species in chickens

doi: 10.1016/j.psj.2026.106681

Figure Lengend Snippet: Schematic view of the nucleic acid extraction and Eimeria –Multiplex RAA–CRISPR/Cas12a (E-MRC12a) typing assay.

Article Snippet: The CRISPR/Cas12a trans-cleavage assay was performed in a 20 μL reaction containing 2.5 μL of 10 × NEBuffer, 2 μL of LbCas12a protein (1 μM), 1 μL of crRNA (1 μM), 2 μL of ssDNA reporter (10 μM), 2 μL of RAA product, and ddH2O to volume.

Techniques: Extraction, Multiplex Assay, CRISPR

Journal: Frontiers in Microbiology

Article Title: A rapid isothermal RPA–CRISPR/Cas12a assay for detection of Rickettsia rickettsii

doi: 10.3389/fmicb.2026.1823193

Figure Lengend Snippet: Schematic overview of the RPA–Cas12a assay targeting the R. rickettsii vut gene.

Article Snippet: Cas12a detection was performed using a commercially available Cas12a enzyme (New England Biolabs, Ipswich, MA, USA; cat. no. M0653S) complexed with a target-specific crRNA designed for each amplicon (Set-1 or Set-2).

Techniques:

Journal: Frontiers in Microbiology

Article Title: A rapid isothermal RPA–CRISPR/Cas12a assay for detection of Rickettsia rickettsii

doi: 10.3389/fmicb.2026.1823193

Figure Lengend Snippet: Genomic localization of RPA primers and Cas12a crRNA targets within the R. rickettsii Sheila Smith genome ( CP121767.1 ). Forward RPA primers are shown in red, reverse primers in blue, and Cas12a crRNA spacer sequences are underlined. The Cas12a PAM motif is indicated in bold. Set-1 and Set-2 primer–crRNA combinations target distinct regions of the vut gene, generating amplicons of 104 bp and 92 bp, respectively.

Article Snippet: Cas12a detection was performed using a commercially available Cas12a enzyme (New England Biolabs, Ipswich, MA, USA; cat. no. M0653S) complexed with a target-specific crRNA designed for each amplicon (Set-1 or Set-2).

Techniques:

Journal: Frontiers in Microbiology

Article Title: A rapid isothermal RPA–CRISPR/Cas12a assay for detection of Rickettsia rickettsii

doi: 10.3389/fmicb.2026.1823193

Figure Lengend Snippet: Validation of the RPA–Cas12a assay using four R. rickettsii strains (Set 1 and Set 2). RPA was performed at 37 °C for 20 min, followed by Cas12a detection at 37 °C for 20 min. Representative endpoint reaction images are shown under UV, Cy2, and UV-blue light. Quantitative fluorescence values are shown as mean ± SD from triplicate reactions. Sample order (1–5): Sheila Smith, Morgan, Iowa, Hlp#2, and no-template control (NTC).

Article Snippet: Cas12a detection was performed using a commercially available Cas12a enzyme (New England Biolabs, Ipswich, MA, USA; cat. no. M0653S) complexed with a target-specific crRNA designed for each amplicon (Set-1 or Set-2).

Techniques: Biomarker Discovery, Fluorescence, Control

Journal: Frontiers in Microbiology

Article Title: A rapid isothermal RPA–CRISPR/Cas12a assay for detection of Rickettsia rickettsii

doi: 10.3389/fmicb.2026.1823193

Figure Lengend Snippet: Analytical sensitivity and observed limit of detection of the RPA–Cas12a assay targeting the R. rickettsii vut gene. Serial 10-fold dilutions of R. rickettsii gBlock DNA (10 −2 –10 −10 ) and a low-copy control (3 copies) were tested in two independent experimental sets (Set 1 and Set 2). RPA was performed at 37 °C for 20 min followed by Cas12a detection at 37 °C for 20 min. Reaction outputs are shown under UV, Cy2, and UV-blue light, with corresponding endpoint RFU measurements (mean ± SD; n = 3). The positivity threshold was defined as mean NTC + 3 SD; the observed LOD corresponds to the lowest input level positive in all replicates (3/3). Statistical significance was determined by one-way ANOVA with Dunnett’s multiple comparisons test (****, p < 0.0001).

Article Snippet: Cas12a detection was performed using a commercially available Cas12a enzyme (New England Biolabs, Ipswich, MA, USA; cat. no. M0653S) complexed with a target-specific crRNA designed for each amplicon (Set-1 or Set-2).

Techniques: Control

Journal: Frontiers in Microbiology

Article Title: A rapid isothermal RPA–CRISPR/Cas12a assay for detection of Rickettsia rickettsii

doi: 10.3389/fmicb.2026.1823193

Figure Lengend Snippet: Specificity of the RPA–Cas12a assay targeting R. rickettsii . Lanes: 1, R. rickettsii (Sheila Smith); 2, R. rickettsii (Morgan); 3, R. rickettsii (Iowa); 4, R. rickettsii (Hlp#2); 5, R. amblyommatis ; 6, R. montanensis ; 7, R. philipii (364D); 8, R. conorii ; 9, R. parkeri ; 10, Anaplasma marginale ; 11, A. phagocytophilum ; 12, Ehrlichia chaffeensis ; 13, E. canis ; 14, no-template control (NTC). Endpoint fluorescence values are shown as mean ± SD from triplicate reactions.

Article Snippet: Cas12a detection was performed using a commercially available Cas12a enzyme (New England Biolabs, Ipswich, MA, USA; cat. no. M0653S) complexed with a target-specific crRNA designed for each amplicon (Set-1 or Set-2).

Techniques: Control, Fluorescence

Journal: Frontiers in Microbiology

Article Title: A rapid isothermal RPA–CRISPR/Cas12a assay for detection of Rickettsia rickettsii

doi: 10.3389/fmicb.2026.1823193

Figure Lengend Snippet: Detection of R. rickettsii DNA in archived longitudinal canine blood DNA extracts using the RPA–Cas12a assay (Set 1 and Set 2). Archived DNA extracts from experimentally infected beagle dogs challenged with Sheila Smith strain (#SS1–#SS3; n = 3) or the Morgan strain (#M1–#M3; n = 3) were tested at the indicated days post-infection (DPI) using two independent primer–crRNA designs. Each time point was tested in triplicate reactions, and the plotted values represent mean endpoint fluorescence (RFU) ± SD from the Cas12a step. The dashed line represents the same fluorescence decision threshold used for the positivity calls (NTC mean + 3 SD).

Article Snippet: Cas12a detection was performed using a commercially available Cas12a enzyme (New England Biolabs, Ipswich, MA, USA; cat. no. M0653S) complexed with a target-specific crRNA designed for each amplicon (Set-1 or Set-2).

Techniques: Infection, Fluorescence