Real-Time Quantitative PCR Assay Analysis, Evaluation and optimization for the Cocoa Swollen Shoot Virus

Abstract

Cocoa swollen shoot virus (CSSV) sequence was used to design a TaqMan probe-based quantitative polymerase chain reaction (PCR) assay from the nuclear SSR marker (mTcCIR25) in order to determine the cocoa cell number. Artificial fragment from the CSSV genome and microsatellite were used to generate calibration curves from 10¹ to 10⁵ fragments per µl which were included in each real-time PCR assay. Generally, there were variations between given concentrations and observed DNA copy concentration of CSSV TaqMan assay decreasing from standard solutions 5 to 1. The accuracy of copy number detection for mTcCIR25 assay was poor at low concentrations (50 to 500 copies) compared with CSSV assay. The closeness of the amplification plots in the mTcCIR25 amplification graphs, the low Ct standard deviation values and the high correlation coefficient of the standard curves indicated that real-time PCR was robust and reproducible for the quantification of the CSSV. This holds great promises for future applications not only in quantitative real-time PCR for CSSV but may be applied to other pathogens.