Related Experiment Video
Updated: May 8, 2026
08:37
Development of a Quantitative Recombinase Polymerase Amplification Assay with an Internal Positive Control
Published on: March 30, 2015
A multi-target, non-infectious and clonable artificial positive control for routine PCR-based assays
Donna Ria J Caasi1, Mohammad Arif, Mark Payton
1National Institute for Microbial Forensics & Food and Agricultural Biosecurity, Oklahoma State University, Stillwater, OK 74078, USA; Department of Entomology and Plant Pathology, Oklahoma State University, Stillwater, OK 74078, USA.
Journal of Microbiological Methods
|September 10, 2013
Summary
Artificial positive controls (APCs) offer a reliable and safe alternative for PCR testing, especially for rare pathogens. Synthetically designed APCs demonstrated superior amplification rates compared to traditional methods.
Area of Science:
- Molecular Biology
- Plant Pathology
- Synthetic Biology
Background:
- Polymerase chain reaction (PCR) requires reliable positive controls for accurate results.
- Obtaining positive controls for rare or emerging pathogens is difficult and poses biosafety risks.
- Current positive controls often lack standardization and can be less efficient.
Purpose of the Study:
- To develop and evaluate artificial positive controls (APCs) for PCR.
- To assess the efficacy of synthetic DNA inserts as reliable and safe positive controls.
- To improve PCR reliability and biosafety for pathogen detection.
Main Methods:
- Designing de novo synthetic DNA inserts with primer sequences for target viruses and an internal control gene.
- In silico assessment of thermodynamic and folding parameters for 24 APC inserts.
- Cloning and testing two distinct APCs (optimal and sub-optimal) using endpoint PCR.
Main Results:
- The optimal APC achieved a 100% amplification rate.
- Virus-infected plant tissues, used as traditional controls, showed a 92% amplification rate.
- APCs accommodate diverse primer sequences, offering flexibility in target design.
Conclusions:
- Artificial positive controls (APCs) provide a highly reliable and safe alternative for PCR.
- APCs can streamline and standardize routine PCR assays.
- The development of APCs presents commercialization opportunities for synthetic positive control sequences.
