Development of Calorimetric PCR Assay as Cost Effective, Direct Post PCR Readout Platform

Abstract

The sophisticated expensive molecular laboratories are commonly present for detection of molecular template in given sample. But cost effective point of care diagnostic methodology without much costlier instruments at field level is the need of hour for molecular diagnostics. Here in this study an attempt was made through calorimetric pcr as simple post pCR development strategy for reading out of results by naked eye. The calorimetric PCR assay was demonstrated by using Herpesvirus of Turkey (HVT) referral strain (one of the vaccine strain against Mareks disease virus) in this study.  HVT specific primers were designed by using primer 3.0 software targeting US3 gene-specific to  HVT strain. The forward primer of the US3 gene which is specific to HVT was integrated with HRPzyme sequence (566bp) and amplified during the PCR. The PCR product including HRPzyme produced the distinguished blue colour with chromogenic substrates which was visualized by the naked eye. From these observations, simple, rapid colorimetric reaction based post PCR analysis was performed without using agarose gel electrophoresis or real-time methods employing fluorescent dyes.