Detection and copy number estimation of the transgenic nucleotide sequences in an unknown GM event of Oryza sativa

Ali M. Sajjad, Tanzeela Bashir, Shaina Saeed, Emmad Ahmad


The present study was designed to establish a qualitative detection method based on conventional and real time PCR assay to screen the commonly grown rice varieties for the presence of the cry1Ac gene. The detection of genetically modified rice in the screening process would necessitate accurate assay development and precise qualitative PCR tests complying with established procedures for the detection and characterization of transgenes in food grains. Such assay would not only enable the monitoring of transgene flow in local agricultural environment but also the characterization of different plant species produced with this transgene and its regulatory components. Thus, a reliable and quick screening assay was established for the qualitative detection of the transgene along with the promoter and selectable marker gene in genetically modified rice. By conventional PCR, a fragment of 215 bp was amplified with gene specific primers of cry1Ac. Primers for other transgenes such as gna and bar were also employed; however, no amplification was detected. The presence of the p35s, sps, and nptII genes was confirmed by qualitative real-time PCR. The specificity of the respective PCR products was checked through melt peak curve analysis. Sharp and precise melting temperatures indicated the presence of a single kind of PCR product in correspondence to each of the primers used. Moreover, the copy number of cry1Ac was estimated by ∆∆CT method. It is proposed that the primer sets and experimental conditions used in this study will be sufficient to meet the requirements for molecular detection and characterization of the cry1Ac transgene and affiliated sequences in sorting out conventional rice varieties from the ones which are genetically modified. It will also help to monitor the ecological flow of these transgenes and other biosafety factors.


GM rice; real time PCR; ∆∆CT method

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Vidhya S, Ramanjini Gowda PH, Yogendra KN, Ningaraju TM, Salome T. Detection of genetically modified cotton seeds using PCR and real-time PCR. Indian J Biotechnol. 2012;11:176–181.

Wang LB, Liu LJ, Yan HM. Advances on transgenic Bt rice and bio-security and strategies. Life Science Research. 2009;13(2):182−188.

Chen H, Lin YJ, Zhang QF. Review and prospect of transgenic rice research. Chin Sci Bull. 2009;54:4049–4068.

Sikha D, Sharmistha B. Overview on current status of biotechnological interventions on yellow stem borer Scirpophaga incertulas (Lepidoptera: Crambidae) resistance in rice. Biotechnol Adv. 2009;28(1):70–81.

Saha P, Majumder P, Dutta I, Ray T, Roy SC, Das S. Transgenic rice expressing Allium sativum leaf lectin with enhanced resistance against sap-sucking insect pests. Planta. 2006;23:1329–1343.

Alcantara EP, Aguda RM, Curtiss A, Dean DH, Cohen MB. Bacillus thuringiensis δ-endotoxin binding to brush border membrane vesicles of rice stem borers. Arch Insect Biochem Physiol. 2004;55:169–177.

Huang J, Hu R, Rozelle S, Pray C. Insect-resistant GM rice in farmer fields: assessing productivity and health effects in China. Science. 2005;30:688–690.

Khan MH, Hamid R, Zahoor AS, Zubeda C. Agrobacterium mediated transformation to build resistance against bacterial blight in rice. Pak J Bot. 2007;39(4):1285–1292.

Amna N, Zubeda C, Hamid R, Bushra M. Evaluation of resistance of rice varieties against bacterial blight caused by Xanthomonas oryzae pv. oryzae. Pak J Bot. 2006;38(1):193–203.

Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real time quantitative PCR and the 2[−Delta Delta C(T)] method. Methods. 2001;25(4):402–408.

Babb VM, Haigler CH. Sucrose phosphate synthase activity rises in correlation with high-rate cellulose synthesis in three heterotrophic systems. Plant Physiol. 2001;127:1234–1242.

Haigler CH, Bir S, Zhang DH, Hwang SJ, Wu CF, Cai WX, et al. Transgenic cotton over-producing spinach sucrose phosphate synthase showed enhanced leaf sucrose synthesis and improved fiber quality under controlled environmental conditions. Plant Mol Biol. 2007;63:815–832.

Wang L, Cui N, Zhang KY, Fan HY, Li TL. Research advance of sucrose phosphate synthase (SPS) in higher plant. Int J Agric Biol. 2013;15:1221–1226.

Reiting R, Grohmann L, Mäde D. A testing cascade for the detection of genetically modified rice by real-time PCR in food and its application for detection of an unauthorized rice line similar to KeFeng6. Journal für Verbraucherschutz und Lebensmittelsicherheit. 2010;5:185–188.

Mahmood R, Rao AQ, Batool F, Azam S, Shahid AA, Husnain T. Transgene copy number and phenotypic variations in transgenic basmati rice. The Journal of Animal and Plant Sciences. 2012;22(4):1004–1013.

Mason G, Provero P, Vaira AM, Accotto GP. Estimating the number of integrations in transformed plants by quantitative real-time PCR. BMC Biotechnol. 2002;2:20.

Qiu YW, Gao XJ, Qi BR, Li Lu, Zhen Z. Establishment of Taqman real-time quantitative PCR assay for foreign gene copy numbers in transgenic soybean. J Northeast Agric Univ. 2012;19(4):48–52.

Register JC. Approaches to evaluating the transgenic status of transformed plants. Trends Biotechnol. 1997;15:141–146.

Cervera M, Pina JA, Juárez J, Navarro L, Peña L. A broad exploration of a transgenic population of citrus: stability of gene expression and phenotype. Theor Appl Genet. 2000;100:670–677.

McCabe MS, Mohapatra UB, Debnath SC, Power JB, Davey MR. Integration, expression and inheritance of two linked T-DNA marker genes in transgenic lettuce. Mol Breed. 1999;5:329–344.

Bashir K, Husnain T, Fatima T, Riaz N, Makhdoom R, Riazuddin S. Novel Indica basmati line (B-370) expressing two unrelated genes of Bacillus thuringiensis is highly resistant to two lepidopteran insects in the field. Crop Prot. 2005;24(10):870–879.

Riaz N, Husnain T, Fatima T, Makhdoom R, Bashir K, Masson L, et al. Development of Indica basmati rice harboring two insecticidal genes for sustainable resistance against lepidopteran insects. S Afr J Bot. 2006;72:217–223.

Husnain T, Asad J, Maqbool SB, Datta SK, Riazuddin S. Variability in expression of insecticidal Cry1Ab gene in Indica basmati rice. Euphytica. 2002;128(1):121–128.

Khan MH, Rashid H, Swati ZA, Chaudhry A. Agrobacterium mediated transformation to build resistance against bacterial blight in rice. Pak J Bot. 2007;39(4):1285–1292.

GMO Detection Method Database [Internet]. 2016 [cited 2016 Oct 10]. Available from:

Pandey V, Rao KV, Shukla PK, Dwivedi UN, Goel SK. Detection and quantitation of gna transgene in GM rice using real time PCR. J Environ Biol. 2013;35:167–172.