Summary

내부 양성 대조군과 양적 재조합 효소 중합 효소 증폭 분석 개발

Published: March 30, 2015
doi:
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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Disclosures

The authors have nothing to disclose.

Acknowledgements

Materials

qRPA Assay
HIV-1 forward primerIntegrated DNA Technologiescustom DNA oligos5’-TGG CAG TAT TCA TTC ACA ATT TTA AAA GAA AAG G-3’ 
HIV-1 reverse primerIntegrated DNA Technologiescustom DNA oligos5’-CCC GAA AAT TTT GAA TTT TTG TAA TTT GTT TTT G-3’ 
HIV-1 probeBioSearch Technologies custom DNA oligos5’- TGC TAT TAT GTC TAC TAT TCT TTC CCC [SIMA/HEX] GC [THF] C [dT-BHQ1] GTA CCC CCC AAT CCC C -3’ 
IPC probeBioSearch Technologies custom DNA oligos5’-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 acetateTwistDxTwistAmp exo
PCR tube stripsBioRadTLS0801
PCR flat cap tube stripsBioRadTCS0803
Micro-seal adhesiveBioRad558/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 DNAApplied Biosystems360486
96 well cold-blockCole ParmerEW-36700-48
Thermal cyclerBioRadCFX96
MiniFugeVWR93000-196
VortexVWR58816-121
Tris buffer 1.0 M, pH 8.0EMD Millipore648314
EDTA 0.5 M, pH 8.0PromegaV4321
Nuclease free waterAmbionAM9937
IPC Development
Cryptosporidium parvum IPC templateWaterborne IncP102CIt 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 primerIntegrated DNA Technologiescustom DNA oligos5’-TGG CAG TAT TCA TTC ACA ATT TTA AAA GAA AAG G/ ATC TAA GGA AGG CAG CAG GC-3’
PCR long reverse primerIntegrated DNA Technologiescustom DNA oligos5’- CCC GAA AAT TTT GAA TTT TTG TAA TTT GTT TTT G/ TGC TGG AGT ATT CAA GGC ATA -3’
Phusion High-Fidelty DNA PolymeraseNew England BiolabsM0530S
Qiaquick Gel Extraction KitQiagen28704
TAE 10X bufferEMD Millipore574797
AgaroseSigma AldrichA9539
Microscope Experiments
Upright fluorescence microscopeZeissZeiss Imager.J1
Stage heaterBioscience ToolsTC-GSH
1-Channel Precision High Stability Temperature ControllerBioscience ToolsTC-1100S
FAM/GFP filter cubeZeissfilter set 38 (000000-1031-346)excitation BP 470/40 nm, emission BP 520/50 nm
HEX filter cubeChroma49014excitation BP 530/30 nm, emission BP 575/40 nm
Laser cutterEngraver's NetworkVLS3.60
1/8" black acrylicMcMaster Carr8505K113
1.5 mm clear acrylicMcMaster CarrPD-72268940 
Super glueOffice DepotDuro super glue 
PCR grade mineral oilSigma AldrichM8662-5VL
Data Analysis
Microsoft ExcelMicrosoft
MATLABMATLAB
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 IllustratorAdobe
JoVE_qRPA_well.aiIncluded in SI
JoVE_qRPA_base.aiIncluded in SI
AxioVision softwareZeiss
JoVE_AxioVision_Script.ziscriptIncluded in SI

References

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Development of a Quantitative Recombinase Polymerase Amplification Assay with an Internal Positive Control

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Cite This Article
Crannell, Z. A., Rohrman, B., Richards-Kortum, R. Development of a Quantitative Recombinase Polymerase Amplification Assay with an Internal Positive Control. J. Vis. Exp. (97), e52620, doi:10.3791/52620 (2015).

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