Using Pooled Recombinant Plasmids as Control Materials for Diagnostic Real-Time PCR.

Area of Science:

• Molecular Biology
• Clinical Diagnostics
• Quality Control

Background:

• Limited documentation exists on using plasmid DNA as reference materials for PCR quality control (QC) in clinical diagnostic laboratory-developed tests (LDTs).
• This study addresses the need for robust QC materials in molecular diagnostics.
• Plasmid DNA offers potential advantages as a stable, non-infectious reference standard.

Purpose of the Study:

• To detail an approach for preparing and titering plasmid DNA controls.
• To explore and present the practicality of using these plasmid controls in a clinical diagnostic setting.
• To evaluate the utility of plasmid DNA for routine QC in multiplex PCR assays.

Main Methods:

• Amplification and TA-cloning of 11 target DNA fragments.
• Verification of DNA fragment identity via DNA sequencing.
• Real-time PCR using TaqMan probes on RGQ and CFX-96 thermal cyclers.

Main Results:

• Successful cloning of all 11 target DNA fragments into E. coli vectors.
• Plasmid pools generated stable, reproducible positive controls for multiplex PCR.
• Plasmid controls provided meaningful data for routine QC monitoring and quality assurance (QA) when spiked into clinical specimens.

Conclusions:

• Plasmid pools serve as valuable, inexhaustible reference materials for routine QC in diagnostic laboratories.
• The use of plasmid controls enhances laboratory-developed test (LDT) reliability.
• Plasmid controls offer a safe alternative to live organisms for quality assurance (QA).