crispr cas12a trans cleavage mixture  (New England Biolabs)

Journal: Frontiers in Plant Science

Article Title: Bursaphelenchus xylophilus detection and analysis system based on CRISPR – Cas12

doi: 10.3389/fpls.2022.1075838

Figure Lengend Snippet: (A) Schematic illustration of loop-mediated isothermal amplification (LAMP). (B) Schematic illustration of CRISPR-Cas12a detection. Step 1: LAMP products are obtained. Protospacer adjacent motif (PAM) sites guide the CRISPR/Cas12a-gRNA complex to recognize target sites. Step 2: Cas12a effectors are activated. Step 3: The activated effectors nonspecifically cleave single-stranded DNA reporter molecules by trans-cleavage. (C) Schematic illustration of the LAMP-CRISPR/Cas12a assay workflow. The LAMP-CRISPR/Cas12a assay involves three closely linked steps: rapid template preparation (step 1), LAMP reaction (step 2), and CRISPR-Cas12a cleavage and signal detection (step 3).

Article Snippet: For lateral flow detection, 100 µL HybriDetect Assay Buffer and 10 μl aliquot of products from the CRISPR-Cas12a trans-cleavage mixture (250 nM Cas12a, 500 nM crRNA, 400 nM ssDNA-lateral flow biosensor reporter, 2 µL LAMP product, and 2.5 µL NEBuffer 2.1) were added to a reaction tube.

Techniques: Amplification, CRISPR

Journal: Frontiers in Plant Science

Article Title: Bursaphelenchus xylophilus detection and analysis system based on CRISPR – Cas12

doi: 10.3389/fpls.2022.1075838

Figure Lengend Snippet: (A) Fluorescence changes of three crRNA reactions for 1 h detected by CRISPR-Cas12a fluorescence assays. Values are shown in the graph as means ± SD (n = 3). (B) Fluorescence curves at different reaction temperatures. (C) Fluorescence curves of different crRNA concentrations. (D) Effects of different LAMP reaction times on the Cas12a reaction. NTC: no template control.

Article Snippet: For lateral flow detection, 100 µL HybriDetect Assay Buffer and 10 μl aliquot of products from the CRISPR-Cas12a trans-cleavage mixture (250 nM Cas12a, 500 nM crRNA, 400 nM ssDNA-lateral flow biosensor reporter, 2 µL LAMP product, and 2.5 µL NEBuffer 2.1) were added to a reaction tube.

Techniques: Fluorescence, CRISPR

Journal: Frontiers in Plant Science

Article Title: Bursaphelenchus xylophilus detection and analysis system based on CRISPR – Cas12

doi: 10.3389/fpls.2022.1075838

Figure Lengend Snippet: Specificity of the CRISPR-Cas12a enhanced fluorescent assay evaluated by its ability to distinguish base mismatches. Protospacer adjacent motif (PAM) sequences are shown in red; base mismatches are shown in green.

Article Snippet: For lateral flow detection, 100 µL HybriDetect Assay Buffer and 10 μl aliquot of products from the CRISPR-Cas12a trans-cleavage mixture (250 nM Cas12a, 500 nM crRNA, 400 nM ssDNA-lateral flow biosensor reporter, 2 µL LAMP product, and 2.5 µL NEBuffer 2.1) were added to a reaction tube.

Techniques: CRISPR, Fluorescence

Journal: Frontiers in Plant Science

Article Title: Bursaphelenchus xylophilus detection and analysis system based on CRISPR – Cas12

doi: 10.3389/fpls.2022.1075838

Figure Lengend Snippet: (A) Specificity of the LAMP-CRISPR/Cas12a assay evaluated by its ability to distinguish B xylophilus strains and related species. B.C: Botrytis cinerea ; B.M: B mucronatus ; QY: B xylophilus (from Liaoning); NJ: B xylophilus (from Nanjing); CQ: B xylophilus (from Chongqing); HS: B xylophilus (from Anhui); B.D: B doui ; NTC: negative control (ddH2O). (B) End-point fluorescence visualization of the specificity test. (C) Detection of base changes or deletions in target sequences by LAMP-CRISPR/Cas12a assay by measuring fluorescence intensity under blue-light irradiation 1 h after the reaction. Panel 1: From left to right, 0–5 base changes; the last tube is the negative control. At four base changes, the fluorescence intensity dropped significantly. Panel 2: From left to right, 0–5 base deletions. the last tube is the negative control. At five base deletions, the fluorescence intensity dropped significantly.

Article Snippet: For lateral flow detection, 100 µL HybriDetect Assay Buffer and 10 μl aliquot of products from the CRISPR-Cas12a trans-cleavage mixture (250 nM Cas12a, 500 nM crRNA, 400 nM ssDNA-lateral flow biosensor reporter, 2 µL LAMP product, and 2.5 µL NEBuffer 2.1) were added to a reaction tube.

Techniques: CRISPR, Negative Control, Fluorescence, Irradiation

Journal: Frontiers in Plant Science

Article Title: Bursaphelenchus xylophilus detection and analysis system based on CRISPR – Cas12

doi: 10.3389/fpls.2022.1075838

Figure Lengend Snippet: (A) Sensitivity of the LAMP-CRISPR/Cas12a assay for B xylophilus detection. Purified B xylophilus genomic DNA was diluted by 10 −1 , 10 −2 , 10 −3 , 10 −4 , 10 −5 , 10 −6 , 10 −7 , and 10 −8 to give DNA concentrations of 66.4 ng/µL, 6.64 ng/µL, 664 pg/µL, 66.4 pg/µL, 6.64 pg/µL, 0.664 pg/µL, 66.4 fg/µL, and 6.64 fg/µL. Different concentrations of DNA were detected by LAMP-CRISPR/Cas12a assay and visualized by blue light. (B) Results of the LAMP-CRISPR/Cas12a assay visualized by lateral flow biosensor. (C) Real-time fluorescence profiles of DNA at different concentrations in LAMP-CRISPR/Cas12a assay. (D) Sensitivity of the PCR assay for B xylophilus detection. The image shows amplification bands targeting the SYG-2 gene. Lanes 1–6 lanes are B xylophilus genomic DNA dilutions 664 ng/μL, 66.4 ng/µL, 6.64 ng/µL, 664 pg/µL, 66.4 pg/µL, and 6.64 pg/µL (3 replicates per concentration). (E, F) Different concentrations of DNA were detected by traditional LAMP and visualized by HNB and SYBRgreenI. (G) Real-time fluorescence curves of DNA with different concentrations in LAMP.

Article Snippet: For lateral flow detection, 100 µL HybriDetect Assay Buffer and 10 μl aliquot of products from the CRISPR-Cas12a trans-cleavage mixture (250 nM Cas12a, 500 nM crRNA, 400 nM ssDNA-lateral flow biosensor reporter, 2 µL LAMP product, and 2.5 µL NEBuffer 2.1) were added to a reaction tube.

Techniques: CRISPR, Purification, Fluorescence, Amplification, Concentration Assay

Journal: Frontiers in Plant Science

Article Title: Bursaphelenchus xylophilus detection and analysis system based on CRISPR – Cas12

doi: 10.3389/fpls.2022.1075838

Figure Lengend Snippet: Comparison of the LAMP-CRISPR/Cas12a Assay with Traditional LAMP for detection of B. xylophilus .

Article Snippet: For lateral flow detection, 100 µL HybriDetect Assay Buffer and 10 μl aliquot of products from the CRISPR-Cas12a trans-cleavage mixture (250 nM Cas12a, 500 nM crRNA, 400 nM ssDNA-lateral flow biosensor reporter, 2 µL LAMP product, and 2.5 µL NEBuffer 2.1) were added to a reaction tube.

Techniques:

Journal: Frontiers in Plant Science

Article Title: Bursaphelenchus xylophilus detection and analysis system based on CRISPR – Cas12

doi: 10.3389/fpls.2022.1075838

Figure Lengend Snippet: Processing scheme for the LAMP-CRISPR/Cas12a assay includes the following steps: DNA extraction, isothermal amplification reactions, and CRISPR-Cas12 detection of the B xylophilus by fluorescence.

Article Snippet: For lateral flow detection, 100 µL HybriDetect Assay Buffer and 10 μl aliquot of products from the CRISPR-Cas12a trans-cleavage mixture (250 nM Cas12a, 500 nM crRNA, 400 nM ssDNA-lateral flow biosensor reporter, 2 µL LAMP product, and 2.5 µL NEBuffer 2.1) were added to a reaction tube.

Techniques: CRISPR, DNA Extraction, Amplification, Fluorescence