Validation of ST-LS1 as an Endogenous Reference Gene for Detection of AmA1 and cry1Ab Genes in Genetically Modified Potatoes using Multiplex and Real Time PCR

Abstract

Solanum tuberosum L. belonging to family Solanaceae being the most important tuberous vegetable crop, the development of genetically modified (GM) potato with improved traits is the need of the hour. PCR (polymerase chain reaction) assays are being widely used in GM detection to meet the regulatory and legislative requirements. Detection of target sequences along with plant species specific endogenous reference genes will help in developing reliable and precise PCR assays. In the present study, ST-LS1, a single copy gene from S. tuberosum, was validated as an endogenous reference gene for potato with the PCR assays after testing on different members of the Solanaceae family, viz., Solanum melongena, Solanum lycopersicon, Capsicum annum, Datura stramonium, Petunia hybrida and other GM crops that are under different stages of testing in field trials in India, viz., Gossypium hirsutum, Oryza sativa, Brassica oleracea var. botrytis, Abelmoschus esculentus and Zea mays. The primer pair for ST-LS1 gene of potato was designed and validated by amplifying 223 bp desired fragment of ST-LS1 in potato only, whereas no amplicon was detected in other crops taken up for the study. The specificity of designed primer pair was further validated on real time PCR using SYBR Green I system showing no fluorescent signals with any of the crops tested other than potato. Furthermore, multiplex PCR employing the validated ST-LS1 gene as an internal control was performed to detect AmA1 and cry1Ab genes in GM potatoes. The validated qualitative and quantitative PCR protocols are specific for identification of GM potato and further can be used for quantitative detection of transgenes. The developed protocols for amplification of ST-LS1 gene can also detect as low as 0.01 ng/μl potato DNA, which meets the regulatory requirements for establishment of a GM detection protocol for testing 0.01% contamination as per the Supreme Court of India’s directions.