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rpa amplification primers  (Biotechnology Information)
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Optimization of the <t>RPA-CRISPR/Cas13a</t> testing. ( A ) Schematic workflow of the RPA-CRISPR/Cas13a-based nucleic acid detection for MP. ( B ) Agarose gel electrophoresis analysis for the screening of RPA <t>amplification</t> primers. M: DNA marker; F1R1−F4R4: replicate amplifications of the plasmid DNA template (diluted to 10 −6 ). ( C ) Comparative evaluation of the RPA primers and matched CRISPR/Cas13a detection. Primer pair 3 combined with crRNA-3 demonstrated optimal performance for the detection of the plasmid template (diluted to 10 −7 ). ( D–I ) The end-point fluorescence intensity of the RPA-CRISPR/Cas13a assay for optimization of critical components, including crRNA concentrations of 4.86, 12.1, 24.3, 36.5, and 48.6 nM; T7 RNA polymerase concentrations of 0.25, 0.75, 1.5, 2.5, and 5.0 U/μL; rNTP concentrations of 0.25, 0.5, 1.0, 2.0, and 4.0 mM; primer concentrations of 200, 400, 600, 800, and 1,200 nM; Cas13a enzyme concentrations of 10, 20, 30, 40, and 50 nM; and Mg 2+ concentrations of 12, 13, 14, 15, and 16 mM. Average results from three biological replicates are shown. NTC, negative control; using nuclease-free water as a target DNA.
Rpa Amplification Primers, supplied by Biotechnology Information, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Optimization of the <t>RPA-CRISPR/Cas13a</t> testing. ( A ) Schematic workflow of the RPA-CRISPR/Cas13a-based nucleic acid detection for MP. ( B ) Agarose gel electrophoresis analysis for the screening of RPA <t>amplification</t> primers. M: DNA marker; F1R1−F4R4: replicate amplifications of the plasmid DNA template (diluted to 10 −6 ). ( C ) Comparative evaluation of the RPA primers and matched CRISPR/Cas13a detection. Primer pair 3 combined with crRNA-3 demonstrated optimal performance for the detection of the plasmid template (diluted to 10 −7 ). ( D–I ) The end-point fluorescence intensity of the RPA-CRISPR/Cas13a assay for optimization of critical components, including crRNA concentrations of 4.86, 12.1, 24.3, 36.5, and 48.6 nM; T7 RNA polymerase concentrations of 0.25, 0.75, 1.5, 2.5, and 5.0 U/μL; rNTP concentrations of 0.25, 0.5, 1.0, 2.0, and 4.0 mM; primer concentrations of 200, 400, 600, 800, and 1,200 nM; Cas13a enzyme concentrations of 10, 20, 30, 40, and 50 nM; and Mg 2+ concentrations of 12, 13, 14, 15, and 16 mM. Average results from three biological replicates are shown. NTC, negative control; using nuclease-free water as a target DNA.
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Optimization of the <t>RPA-CRISPR/Cas13a</t> testing. ( A ) Schematic workflow of the RPA-CRISPR/Cas13a-based nucleic acid detection for MP. ( B ) Agarose gel electrophoresis analysis for the screening of RPA <t>amplification</t> primers. M: DNA marker; F1R1−F4R4: replicate amplifications of the plasmid DNA template (diluted to 10 −6 ). ( C ) Comparative evaluation of the RPA primers and matched CRISPR/Cas13a detection. Primer pair 3 combined with crRNA-3 demonstrated optimal performance for the detection of the plasmid template (diluted to 10 −7 ). ( D–I ) The end-point fluorescence intensity of the RPA-CRISPR/Cas13a assay for optimization of critical components, including crRNA concentrations of 4.86, 12.1, 24.3, 36.5, and 48.6 nM; T7 RNA polymerase concentrations of 0.25, 0.75, 1.5, 2.5, and 5.0 U/μL; rNTP concentrations of 0.25, 0.5, 1.0, 2.0, and 4.0 mM; primer concentrations of 200, 400, 600, 800, and 1,200 nM; Cas13a enzyme concentrations of 10, 20, 30, 40, and 50 nM; and Mg 2+ concentrations of 12, 13, 14, 15, and 16 mM. Average results from three biological replicates are shown. NTC, negative control; using nuclease-free water as a target DNA.
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amplification primers  (Azenta)
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Optimization of the <t>RPA-CRISPR/Cas13a</t> testing. ( A ) Schematic workflow of the RPA-CRISPR/Cas13a-based nucleic acid detection for MP. ( B ) Agarose gel electrophoresis analysis for the screening of RPA <t>amplification</t> primers. M: DNA marker; F1R1−F4R4: replicate amplifications of the plasmid DNA template (diluted to 10 −6 ). ( C ) Comparative evaluation of the RPA primers and matched CRISPR/Cas13a detection. Primer pair 3 combined with crRNA-3 demonstrated optimal performance for the detection of the plasmid template (diluted to 10 −7 ). ( D–I ) The end-point fluorescence intensity of the RPA-CRISPR/Cas13a assay for optimization of critical components, including crRNA concentrations of 4.86, 12.1, 24.3, 36.5, and 48.6 nM; T7 RNA polymerase concentrations of 0.25, 0.75, 1.5, 2.5, and 5.0 U/μL; rNTP concentrations of 0.25, 0.5, 1.0, 2.0, and 4.0 mM; primer concentrations of 200, 400, 600, 800, and 1,200 nM; Cas13a enzyme concentrations of 10, 20, 30, 40, and 50 nM; and Mg 2+ concentrations of 12, 13, 14, 15, and 16 mM. Average results from three biological replicates are shown. NTC, negative control; using nuclease-free water as a target DNA.
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Optimization of the <t>RPA-CRISPR/Cas13a</t> testing. ( A ) Schematic workflow of the RPA-CRISPR/Cas13a-based nucleic acid detection for MP. ( B ) Agarose gel electrophoresis analysis for the screening of RPA <t>amplification</t> primers. M: DNA marker; F1R1−F4R4: replicate amplifications of the plasmid DNA template (diluted to 10 −6 ). ( C ) Comparative evaluation of the RPA primers and matched CRISPR/Cas13a detection. Primer pair 3 combined with crRNA-3 demonstrated optimal performance for the detection of the plasmid template (diluted to 10 −7 ). ( D–I ) The end-point fluorescence intensity of the RPA-CRISPR/Cas13a assay for optimization of critical components, including crRNA concentrations of 4.86, 12.1, 24.3, 36.5, and 48.6 nM; T7 RNA polymerase concentrations of 0.25, 0.75, 1.5, 2.5, and 5.0 U/μL; rNTP concentrations of 0.25, 0.5, 1.0, 2.0, and 4.0 mM; primer concentrations of 200, 400, 600, 800, and 1,200 nM; Cas13a enzyme concentrations of 10, 20, 30, 40, and 50 nM; and Mg 2+ concentrations of 12, 13, 14, 15, and 16 mM. Average results from three biological replicates are shown. NTC, negative control; using nuclease-free water as a target DNA.
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Optimization of the RPA-CRISPR/Cas13a testing. ( A ) Schematic workflow of the RPA-CRISPR/Cas13a-based nucleic acid detection for MP. ( B ) Agarose gel electrophoresis analysis for the screening of RPA amplification primers. M: DNA marker; F1R1−F4R4: replicate amplifications of the plasmid DNA template (diluted to 10 −6 ). ( C ) Comparative evaluation of the RPA primers and matched CRISPR/Cas13a detection. Primer pair 3 combined with crRNA-3 demonstrated optimal performance for the detection of the plasmid template (diluted to 10 −7 ). ( D–I ) The end-point fluorescence intensity of the RPA-CRISPR/Cas13a assay for optimization of critical components, including crRNA concentrations of 4.86, 12.1, 24.3, 36.5, and 48.6 nM; T7 RNA polymerase concentrations of 0.25, 0.75, 1.5, 2.5, and 5.0 U/μL; rNTP concentrations of 0.25, 0.5, 1.0, 2.0, and 4.0 mM; primer concentrations of 200, 400, 600, 800, and 1,200 nM; Cas13a enzyme concentrations of 10, 20, 30, 40, and 50 nM; and Mg 2+ concentrations of 12, 13, 14, 15, and 16 mM. Average results from three biological replicates are shown. NTC, negative control; using nuclease-free water as a target DNA.

Journal: Microbiology Spectrum

Article Title: Rapid, sensitive, and highly specific detection of Mycoplasma pneumoniae and its mutation using the RPA-CRISPR/Cas13a system

doi: 10.1128/spectrum.01793-25

Figure Lengend Snippet: Optimization of the RPA-CRISPR/Cas13a testing. ( A ) Schematic workflow of the RPA-CRISPR/Cas13a-based nucleic acid detection for MP. ( B ) Agarose gel electrophoresis analysis for the screening of RPA amplification primers. M: DNA marker; F1R1−F4R4: replicate amplifications of the plasmid DNA template (diluted to 10 −6 ). ( C ) Comparative evaluation of the RPA primers and matched CRISPR/Cas13a detection. Primer pair 3 combined with crRNA-3 demonstrated optimal performance for the detection of the plasmid template (diluted to 10 −7 ). ( D–I ) The end-point fluorescence intensity of the RPA-CRISPR/Cas13a assay for optimization of critical components, including crRNA concentrations of 4.86, 12.1, 24.3, 36.5, and 48.6 nM; T7 RNA polymerase concentrations of 0.25, 0.75, 1.5, 2.5, and 5.0 U/μL; rNTP concentrations of 0.25, 0.5, 1.0, 2.0, and 4.0 mM; primer concentrations of 200, 400, 600, 800, and 1,200 nM; Cas13a enzyme concentrations of 10, 20, 30, 40, and 50 nM; and Mg 2+ concentrations of 12, 13, 14, 15, and 16 mM. Average results from three biological replicates are shown. NTC, negative control; using nuclease-free water as a target DNA.

Article Snippet: Based on the principles of RPA primer design, the RPA amplification primers for the P1 and 23S rRNA genes of MP were designed using the National Center for Biotechnology Information Primer-BLAST website and Oligo software.

Techniques: CRISPR, Agarose Gel Electrophoresis, Amplification, Marker, Plasmid Preparation, Fluorescence, Negative Control

crRNA screening for MP drug-resistant mutations using the RPA-CRISPR/Cas13a. ( A ) RPA primer validation for the 23S rRNA A2063G mutation detection. Gel electropherogram shows the amplification products from five primer pairs (F3R1–F3R5) by using serially diluted 23S rRNA plasmid templates (10 −8 to 10 −5 ; lanes 1−4). M: DNA ladder. ( B ) First-round crRNA screening. The detection efficiency varied when the target mutation (A2063G) was positioned at different locations within the crRNA spacer. ( C ) Second-round crRNA screening. The detection specificity was further optimized by introducing additional mismatches flanking the target mutation. ( D ) Detection of selected crRNA in both wild-type and mutant MP samples. ns, P > 0.05; **** P < 0.0001.

Journal: Microbiology Spectrum

Article Title: Rapid, sensitive, and highly specific detection of Mycoplasma pneumoniae and its mutation using the RPA-CRISPR/Cas13a system

doi: 10.1128/spectrum.01793-25

Figure Lengend Snippet: crRNA screening for MP drug-resistant mutations using the RPA-CRISPR/Cas13a. ( A ) RPA primer validation for the 23S rRNA A2063G mutation detection. Gel electropherogram shows the amplification products from five primer pairs (F3R1–F3R5) by using serially diluted 23S rRNA plasmid templates (10 −8 to 10 −5 ; lanes 1−4). M: DNA ladder. ( B ) First-round crRNA screening. The detection efficiency varied when the target mutation (A2063G) was positioned at different locations within the crRNA spacer. ( C ) Second-round crRNA screening. The detection specificity was further optimized by introducing additional mismatches flanking the target mutation. ( D ) Detection of selected crRNA in both wild-type and mutant MP samples. ns, P > 0.05; **** P < 0.0001.

Article Snippet: Based on the principles of RPA primer design, the RPA amplification primers for the P1 and 23S rRNA genes of MP were designed using the National Center for Biotechnology Information Primer-BLAST website and Oligo software.

Techniques: CRISPR, Biomarker Discovery, Mutagenesis, Amplification, Plasmid Preparation