STANDARDIZATION AND VALIDATION OF QUANTITATIVE RT-PCR TECHNIQUE FOR SPECIFIC DETECTION OF POTATO VIRUS A'

Abstract

ABSTRACT: We optimized a SYBR Green-based quantitative RT-PCR (qRT-PCR) assay technique to detect Potato virus A from potato leaf samples. Optimization was achieved by varying the components of PCR reaction mix and also by altering the PCR conditions specifically the annealing temperature of the primers where 60°C showed good amplification without any non-specific peak. To make the assay more feasible and economical for large scale detection of PVA in potato leaf samples, the final volume of the reaction mix was reduced from 50 to 20 µl without compromising its sensitivity. To evaluate the sensitivity and efficiency of the assay, a standard curve was generated by using known concentrations of 10-fold serially diluted pure RTPCR amplified coat protein gene of PVA which showed high efficiency of 93.419 % with high linearity (co-relation coefficient of R2 = 0.996). In our qRT-PCR analysis, the standard deviation of Ct values decreased with increase in DNA concentration and increased as concentration of DNA decreased. To overcome the problem of false results, elongation factor ef 1-α gene from potato was used which confirmed the reliability of the optimized assay. The assay was also validated by running across potato leaf samples from different geographical regions of India to detect PVA and found that it was sensitive and reliable as it showed consistent results. Hence, the optimized assay could be used in rapid screening of germplasm, large scale screening of planting material for virus freedom to ensure healthy seed potato production and in routine diagnostics laboratories as well.