Development of a Synthetic Positive Control Which Also Detects Plasmid Contamination in Diagnostic Polymerase Chain Reaction

Abstract

This paper describes a technique for developing a positive control for use in a nested PCR to show that the PCR has functioned correctly with both outer and inner primers designed for the diagnostic amplification of 618 and 317 bp products, respectively. This positive control produces a larger product than the diagnostic sample that can be discriminated on an agarose gel. This technique is advantageous over traditional cloning of the diagnostic PCR product itself by: (1) making it visually easy to detect plasmid contamination and thus prevent false positives from the plasmid; (2) develop a positive control when the target organism is at a very low prevalence, so initial detection is not relied on for cloning positive controls (this will ensure the PCR is working correctly prior to diagnostic sampling, reducing false negatives); or (3) for developing a PCR and determining the sensitivity prior to the use of diagnostic samples. The methods used to produce this nested positive control demonstrate how to use large oligonucleotide primers in PCR without nonspecific binding occurring.