qRPA Assay | | | |
HIV-1 forward primer | Integrated DNA Technologies | custom DNA oligos | 5’-TGG CAG TAT TCA TTC ACA ATT TTA AAA GAA AAG G-3’ |
HIV-1 reverse primer | Integrated DNA Technologies | custom DNA oligos | 5’-CCC GAA AAT TTT GAA TTT TTG TAA TTT GTT TTT G-3’ |
HIV-1 probe | BioSearch Technologies | custom DNA oligos | 5’- TGC TAT TAT GTC TAC TAT TCT TTC CCC [SIMA/HEX] GC [THF] C [dT-BHQ1] GTA CCC CCC AAT CCC C -3’ |
IPC probe | BioSearch Technologies | custom DNA oligos | 5’-AGG TAG TGA CAA GAA ATA ACA ATA CAG GAC [FAM] T [THF] T [dT-BHQ1] GGT TTT GTA ATT GGA A -3’ |
RPA exo reagents (pellets, rehydration buffer, magnesium acetate | TwistDx | TwistAmp exo | |
PCR tube strips | BioRad | TLS0801 | |
PCR flat cap tube strips | BioRad | TCS0803 | |
Micro-seal adhesive | BioRad | 558/MJ | |
HIV-1 target (pHIV-IRES- eYFPΔEnvΔVifΔVpr) | | | custom plasmid, see: Segall, H. I., Yoo, E. & Sutton, R. E. Characterization and detection of artificial replication-competent lentivirus of altered host range. Molecular Therapy 8, 118–129, doi:10.1016/S1525-0016(03)00134-5 (2003). |
Human male genomic DNA | Applied Biosystems | 360486 | |
96 well cold-block | Cole Parmer | EW-36700-48 | |
Thermal cycler | BioRad | CFX96 | |
MiniFuge | VWR | 93000-196 | |
Vortex | VWR | 58816-121 | |
Tris buffer 1.0 M, pH 8.0 | EMD Millipore | 648314 | |
EDTA 0.5 M, pH 8.0 | Promega | V4321 | |
Nuclease free water | Ambion | AM9937 | |
IPC Development | | | |
Cryptosporidium parvum IPC template | Waterborne Inc | P102C | It is also possible to order a double stranded synthetic target from IDT if the user is unequipped to work with C. parvum (a BSL-2 infectious agent). PCR and RPA primers for C. parvum were designed using GenBank accession number AF115377.1 |
PCR long forward primer | Integrated DNA Technologies | custom DNA oligos | 5’-TGG CAG TAT TCA TTC ACA ATT TTA AAA GAA AAG G/ ATC TAA GGA AGG CAG CAG GC-3’ |
PCR long reverse primer | Integrated DNA Technologies | custom DNA oligos | 5’- CCC GAA AAT TTT GAA TTT TTG TAA TTT GTT TTT G/ TGC TGG AGT ATT CAA GGC ATA -3’ |
Phusion High-Fidelty DNA Polymerase | New England Biolabs | M0530S | |
Qiaquick Gel Extraction Kit | Qiagen | 28704 | |
TAE 10X buffer | EMD Millipore | 574797 | |
Agarose | Sigma Aldrich | A9539 | |
Microscope Experiments | | | |
Upright fluorescence microscope | Zeiss | Zeiss Imager.J1 | |
Stage heater | Bioscience Tools | TC-GSH | |
1-Channel Precision High Stability Temperature Controller | Bioscience Tools | TC-1100S | |
FAM/GFP filter cube | Zeiss | filter set 38 (000000-1031-346) | excitation BP 470/40 nm, emission BP 520/50 nm |
HEX filter cube | Chroma | 49014 | excitation BP 530/30 nm, emission BP 575/40 nm |
Laser cutter | Engraver's Network | VLS3.60 | |
1/8" black acrylic | McMaster Carr | 8505K113 | |
1.5 mm clear acrylic | McMaster Carr | PD-72268940 | |
Super glue | Office Depot | Duro super glue | |
PCR grade mineral oil | Sigma Aldrich | M8662-5VL | |
Data Analysis | | | |
Microsoft Excel | Microsoft | | |
MATLAB | MATLAB | | |
MATLAB script: "JoVE_qRPA_standard_curve.m” | Included in SI | | |
MATLAB script: "JoVE_qRPA_validation_and_quantification.m” | Included in SI | | |
MATLAB script: "JoVE_real_time_intensity_to_excel.m” | Included in SI | | |
Adobe Illustrator | Adobe | | |
JoVE_qRPA_well.ai | Included in SI | | |
JoVE_qRPA_base.ai | Included in SI | | |
AxioVision software | Zeiss | | |
JoVE_AxioVision_Script.ziscript | Included in SI | | |