Jul 24, 2024
How to Improve the Reliability of qPCR Detection with Target Gene-optimized Internal Standards
- 1University of California, Riverside
Protocol Citation: Jonathan Phillips, Gregor Blaha 2024. How to Improve the Reliability of qPCR Detection with Target Gene-optimized Internal Standards. protocols.io https://dx.doi.org/10.17504/protocols.io.14egn6e7pl5d/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: July 21, 2024
Last Modified: July 24, 2024
Protocol Integer ID: 103799
Keywords: qPCR, Internal Standard, amplicon-specific, ddPCR
Funders Acknowledgements:
California Department of Food and Agriculture
Grant ID: 21-0001-056-SF
Abstract
Achieving robust qPCR results requires both sensitive and accurate detection of target genes. This is particularly challenging for pathogen detection in field samples because the absence of a detection signal does not necessarily indicate the absence of the pathogen. Inhibitors present in the sample can suppress the pathogen signal, leading to false-negative results. The presented protocol details the use of an optimized internal standard for detecting CLas in HLB-infected citrus samples.
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For permission to use the image or to contact the creator of the image, irod, please contact the lead author at gregor.blaha@ucr.edu
Protocol references
Anonymous (2010) ISO 4787.2010 Laboratory Glassware – Volumetric Instruments – Methods for Testing of Capacity and for Use.
Anonymous (2012) ASTM E969-02: Standard Specification for Glass Volumetric (Transfer) Pipets
Anonymous (2022) ASTM E542-22: Standard Practice for Gravimetric Calibration of Laboratory Volumetric Instruments
Maar D., et al. (2020) Transitioning Your Assay from Quantitative PCR to Droplet Digital PCR. Bio-Rad bulletin 7320.
Huggett J.F., et al.(2008) Differential susceptibility of PCR reactions to inhibitors: an important and unrecognised phenomenon. BMC Res Notes, 1, 70. PMID: 18755023. doi: 10.1186/1756-0500-1-70.