Evaluation of the Application of Thermastop in Reverse Transcription Real Time PCR Assay Non-Specific Amplification

Abstract

Non-specific amplification may occur during polymerase chain reaction (PCR) assays when the DNA polymerase binds cDNA targets at temperatures below 50°C. The outcome includes potential false positive results or aberrant amplification curves that reduces the specificity of the assay. Non-specific amplification during PCR occurs more often at low starting temperatures when non-specific binding may occur. ThermaGenix thermastop is a proprietary additive for PCR reactions that functions to bind the DNA polymerase at temperatures below 50°C and prevent non-specific binding to reduce false positive results and improve the specificity of the assay. Another special property of thermastop is to continue functioning at low temperatures during subsequent cycles of the PCR reaction after being denatured to prevent non-specific amplification as the reaction continues. The application of the thermostop product was evaluated at the Iowa State University Veterinary Diagnostic Laboratory (ISU VDL) to improve the results of assays with low specificity or issues with aberrant amplification. This included the following RT-rtPCR or rtPCR assays: Bovine respiratory multiplexed PCR panel (BCoV, BoHV-1, BRSV, BVDV), GPS (Glaesserella parasuis) genotyping multiplexed PCR, and the small ruminant abortion panel (Toxoplasma gondii, Campylobacter jejuni, Campylobacter fetus, Coxiella burnetii, Chalamydia abortus, and Caprine herpesvirus). Results after including thermastop at varying concentrations in the PCR master mix produced no evidence of assay improvement or substantial change in both real time and gel-based PCR for these assays. However, thermastop has been applied to Sanger sequencing assays and improved results during amplification. In the future, the ISU VDL will develop an oligonucleotide with ThermaGenix as a proprietary product for continued use in PCR assays that have demonstrated improvement in results when included.