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.
About this article
References
[1] James, C. Executive summary of global status of commercialized biotech/GM crops. ISAAA Briefs 37. (2007)
[2] Premanandh, J. Global consensus-need of the hour for genetically modified organisms (GMO) labeling. Journal of Commercial Biotechnology, 17, (2010). 37-44.
[3] Gruere, G.P. & Rao, S. RA review of international labeling policies of genetically modified food to evaluate India’s proposed rule. AgBio Forum, 10, (2007)51-64.
[4] Mangal, M., Malik, K., & Randhawa, G.J. Import of transgenic planting material.National scenario. Current Science, 4, (2003) 454 -458.
[5] Lipp, M., Anklam, E., & Stave, J. W. Validation of an immunoassay for detection andquantitation of a genetically modified soyabean in food and food fractions using reference materials: inter laboratory study. Journal of AOAC International, 83, (2000) 919-927
View more references (24)
[6] Rogan, G. J., Dudin, Y. A., Lee, T. C., Magin, K. M., Astwood, J. D., Bhakta, N. S., et al.Immunodiagnostic methods for detection of 5-enolpyruvylshikimate-3-phosphate synthase in Round Ready soybeans. Journal of Food Control, 10, (1999) 407-414
[7] Stave, J. W., Magin, K., Schimmel, H., Lawruk, T. S., Wehling, P., & Bridges, A. AACCcollaborative study of a protein method for the detection of genetically corn. Cereal Foods World, 45, (2000) 497-501.
[8] Vollenhofer, S., Burg, K., Schmid, J., Kroath, H. genetically modified organisms in food screening and specific detection by polymerase chain reaction. J Agric Food Chem, 47, (1999) 5038-5043.
[9] Matsuoka, T., Kawashima, Y., Akiyama, H., Miura, H., Goda, Y., Kusakabe, Y., Isshiki, K.,Toyoda, M., Hino. A. A method of detecting recombinant DNAs from four lines of genetically modified maize. J Food Hyg Soc Jpn, 41, (2000) 137-143.
[10] Matsuoka, T., Hideo, K., Akiyama, H., Miura, H., Goda, 459 Y., Kusakabe, Y., Isshiki, K., Toyoda, M., Hino, A. A multiplex PCR method of detecting recombinant DNAs from five lines of genetically modified maize. J Food Hyg Soc Jpn. 42, 1, (2001) 24-32
[11] Hernandez, M., Rodriguez-Lazaro, D., Zhang, D., Esteve, T., Pla, M., Prat, S., Interlaboratory transfer of a PCR multiplex method for simultaneous detection of four genetically modified maize lines Bt11 MON810 T25 and GA21 J Agric Food Chem, 53, (2005) 3333-3337.
[12] Lee, S.H., Min, D.M., Kim, J., K. (2006) Qualitative and quantitative polymerase chain reaction analysis for genetically modified maize MON863 J Agric Food Chem, 54, 1124-1129.
[13] Vaitilingom, M., Pijnenburg, H., Gendre, F., Brignon, P. (1999) Real-Time PCR quantitative PCR detection of genetically modified Maximizer maize and Roundup Ready soybean in some representative foods. J Agric Food Chem, 47, 5261-5266.
[14] 14. Terzi, V., Ferrari, B., Finocchiaro, F., Fonzo, N.D., 490 Stanca, A.M., Lamacchia, C. (2003) TaqMan PCR for detection of genetically modified durum wheat J Cereal Sci, 37,157–163
[15] Chen, H., Wu, Y.H., Song, D.Y., Zhang, W. Dong X.Y., Li PW, On-line preconcentration and UV determination of DNA fragments by dynamic coating capillary electrophoresis and its application to detection of genetically modified oilseed rape based on PCR. Microchem J, (2007)17–22.
[16] Malliga, M., Nagarajan & Solke H.D.B. an oligonucleotide array to detect genetically modified events in potato. Plant Mol Biol Rep, 21, (2003) 259–270.
[17] Demeke, T., Ratnayaka, I Multiplex qualitative PCR assay for identification of genetically modified canola events and real-time event-specific PCR assay for quantification of the GT73canola event. Food Control, 19, (2008) 893–897.
[18] Ding, J., Jia, J., Yang, L., Wen, H., Zhang, C., Liu, W Validation of a rice specific gene, sucrose phosphate synthase, used as the endogenous reference gene for qualitative and real-time quantitative PCR detection of transgenes. J Agric Food Chem, 52, (2004) 3372–3377.
[19] Yang, L.T., Ding, J.Y., Zhang, C.M., Jia, J.W., Weng, H.B., Liu, W.X. Estimating the copynumber of transgenes in transformed rice by real-time quantitative PCR. Plant Cell Rep 23, (2005) 759–763.
[20] Jeong, S.C., Pack, I.S., Cho, E.Y., Youk, E.S., Park, S., Yoon, W.K. Molecular analysis and quantitative detection of a transgenic rice line expressing a bifunctional fusion TPSP. Food Control, 18, (2007) 1434–1442.
[21] Nagadhara, D., Ramesh, S., Pasalu, I.C., Kondala, Rao.Y., Krishnaiah, N.V., Sarma, N.P., Bown, D.P.,Gatehouse, J.A., Reddy, V.D., Rao, K.V. Transgenic indica rice resistant to sap sucking insects Plant Biotechnology Journal, (2003) 231-240.
[22] Shao-bin, Gu, Ying. Wu.,Shi-Chang, Li., LiLi, Jian-bo, Yang. Development of a real483 time multiplex PCR system for the quantitative detection of Chinese GM rice. Sci Food Agric, 89, (2009) 1101–1106.
[23] Mason,G., Provero, P., Vaira, A.M., Accotto, G.P. Estimating the number of integrationsin transformed plants by quantitative real-time PCR. BMC Biotechnol, 2, (2002) 20–30.
[24] Schmidt, M. A., Parrott, W.A Quantitative detection of transgenes in soybean Glycinemax (L.) Merrill and peanut (Arachis hypogaea L.) by real-time polymerase chain reaction. Plant Cell Rep, 20, (2001)422–428.
[25] Ingham, D. J., Beer, S., Money, S., Hansen, G. Quantitative real-time PCR assay fordetermining transgene copy number in transformed plants. Biotechniques, 31, (2001) 132–140.
[26] The Plant Cell, 14, 521, March (2002). www.plantcell.org.
[27] James, D., Schmidt AM, Wall E, Green M, Masri, S. Reliable Detection and identification of Genetically Modified Maize, Soybean, and Canola by Multiplex PCR Analysis.
[28] J Agric Food Chem, 51, (2003) 5829-5834 Ginzinger, D.G Gene quantification using real-time quantitative PCR an emergingtechnology hits the mainstream. Exp Hematol, 30, (2002).503–512.
[29] Goel, S.K., Tewari, S., lal, H., Mishra, P., Agarwal, S.K., Dwivedi, U.N., Kumar, M SYBR Green I chemistry based Real time PCR: Melting curve discrepancies. Current Trends in Biotechnology and Pharmacy. 2, (2008) 555 -561.