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Updated: Mar 7, 2026
Development of a Quantitative Recombinase Polymerase Amplification Assay with an Internal Positive Control
Published on: March 30, 2015
Using Pooled Recombinant Plasmids as Control Materials for Diagnostic Real-Time PCR
Background:
Despite the potential usefulness of plasmids as reference materials for PCR, there are very few reports documenting the experience with using these materials in quality control (QC) of laboratory developed tests (LDTs), in a clinical diagnostic setting. In this study, an approach to preparing and titering such controls is detailed. The practicality of using plasmids as QC materials was explored and presented.
Methods:
Target DNA fragments (n = 11) were amplified from positive samples and cloned using TA-cloning. Identities of the fragments were ascertained using DNA sequencing. Real-time PCR was carried out using TaqMan probes on RGQ or CFX-96 thermal-cyclers.
Results:
All the 11 targeted DNA fragments were successfully cloned into E. coli vectors. Plasmid pools could be repeatedly reconstituted to generate stable and usable positive controls for multiplex PCR assays in a simple workflow. Importantly, plasmid controls generated meaningful run data that could be used in QC processes for monitoring routine runs. Further, plasmid material could be spiked into clinical specimens for quality assurance (QA) purposes, avoiding the culture of live infectious organisms, showing another routine usefulness of plasmids.
Conclusions:
Plasmid pools are useful and inexhaustible sources of reference materials for various routine QC uses in the diagnostic laboratory.
