Standardization of qualitative and quantitative polymerase chain reaction methods in transgenic Indica rice

Abstract

Rice crop gets damaged by pests and insects, and to overcome this problem globally many transgenic rice lines are being developed. Simultaneously, a need to detect and quantitate the transgene with special reference to copy number estimation also became evident. The primary objective of this study, was to successfully apply a multiplex Polymerase Chain Reaction (PCR) assay to detect the transgene- Galanthus nivalis agglutinin (GNA), Sucrose phosphate synthase (SPS) an endogenous gene, 35S-Cauliflower mosaic Virus (CaMV) as a promoter and Nopaline synthase (NOS) as terminator gene in Genetically Modified (GM) rice to differentiate from normal cultivar (NC) in a single step. Further a Sybr green based quantitative Real Time PCR (qPCR) assay was adapted to quantify the transgene (GNA) and its copy number estimation. This Sybr green based assay is economical simply utilizes specific primers for targeted genes being used in conventional PCR. The sensitivity and specificity of the protocols was also determined in terms of Limit of detection (LOD) 0.01%, and Limit of quantitation (LOQ) 0.1%. A dilution series of the genomic DNA from GM rice was used to generate a standard curve for relative quantification of the GNA and SPS gene. An added advantage is a quick and economical screening in early stages of GM rice lines and accurate determination of the transgene copy number in rice lines, processed foods and contaminated food products.