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Qualitative and Quantitative PCR Methods for Event-specific Detection of Genetically Modified Cotton Mon1445 and Mon531

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Abstract

Based on the DNA sequences of the junctions between recombinant and cotton genomic DNA of the two genetically modified (GM) cotton varieties, herbicide-tolerance Mon1445 and insect-resistant Mon531, event-specific primers and probes for qualitative and quantitative PCR detection for both GM cotton varieties were designed, and corresponding detection methods were developed. In qualitative PCR detection, the simplex and multiplex PCR detection systems were established and employed to identify Mon1445 and Mon531 from other GM cottons and crops. The limits of detection (LODs) of the simplex PCR were 0.05% for both Mon1445 and Mon531 using 100 ng DNA templates in one reaction, and the LOD of multiplex PCR analysis was 0.1%. For further quantitative detection using TaqMan real-time PCR systems for Mon1445 and Mon531, one plasmid pMD-ECS, used as reference molecule was constructed, which contained the quantitative amplified fragments of Mon1445, Mon531, and cotton endogenous reference gene. The limits of quantification (LOQs) of Mon1445 and Mon531 event-specific PCR systems using plasmid pMD-ECS as reference molecule were 10 copies, and the quantification range was from 0.03 to 100% in 100 ng of the DNA template for one reaction. Thereafter, five mixed cotton samples containing 0, 0.5, 0.9, 3 and 5% Mon1445 or Mon531 were quantified using established real-time PCR systems to evaluate the accuracy and precision of the developed real-time PCR detection systems. The accuracy expressed as bias varied from 1.33 to 8.89% for tested Mon1445 cotton samples, and from 2.67 to 6.80% for Mon531. The precision expressed as relative standard deviations (RSD) were different from 1.13 to 30.00% for Mon1445 cotton, and from 1.27 to 24.68% for Mon531. The range of RSD was similar to other laboratory results (25%). Concluded from above results, we believed that the established event-specific qualitative and quantitative PCR systems for Mon1445 and Mon531 in this study are acceptable and suitable for GM cotton identification and quantification.

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Abbreviations

35S:

Cauliflower Mosaic Virus 35S promoter

aad:

Aminoglycoside adenyltransferase gene from Transposon Tn7

AmpR :

Ampicillin resistance gene

CMoVb:

Modified Figwort Virus 35S promoter

CP4-EPSPS:

5-Enolpyruvylshikimate−3-phosphate synthase gene from Agrobacterium tumefaciens CP4

Cry1Ac:

Cry1Ac delta-endotoxin from Bacillus thuringiensis subsp. kurstaki (Btk)

CTP2:

Chloroplast Transit Peptide sequence from Arabidopsis thaliana

CV :

Coefficient Value

E35S:

Cauliflower Mosaic Virus (CaMV) 35S promoter with duplicated enhanced region

E9 3′:

3′ Polyadenylation signal of Pisum sativum ribulose−1,5-bisphosphate carboxylase small subunit

GM:

Genetically modified

LOD:

Limit of detection

LOQ:

Limit of quantization

NOS 3′:

3′ nontranslated polyadenylation signal of Agrobacterium tumefaciens nopaline synthase gene

NptII:

Nopaline synthase gene from Agrobacterium tumefaciens

oriV:

Origin of plasmid replication in Agrobacterium tumefaciens

RB:

Right border from Agrobacterium tumefaciens

RSD:

Relative standard deviation

Sad1:

Stearoyl–Acyl Carrier Protein Desaturase gene from Gossypium hirsutum

SD:

Standard deviation

UNG:

Amperase uracil N-glycosylase gene

References

  • E Anklam F Gadani P Heinze H Pijnenburg G Eede ParticleVan den (2002) ArticleTitleAnalytical methods for detection and determination of genetically modified organism in agricultural crops and plant-derived food products Eur Food Res Technol 214 3–26 Occurrence Handle10.1007/s002170100415 Occurrence Handle1:CAS:528:DC%2BD38XhsFersLc%3D

    Article  CAS  Google Scholar 

  • K Arumuganathan ED Earle (1991) ArticleTitleNuclear DNA content of some important plant species Plant Mol Biol Rep 9 208–218 Occurrence Handle1:CAS:528:DyaK38XhslSjurY%3D

    CAS  Google Scholar 

  • Beazleyn KA, Hillyard JR, Pang S and Roberts JK (2004) Cotton event PV-GHBK04 (757) and compositions and methods for detection thereof. US Patent. Pub. No.: US2004/0045054 A1

  • KG Berdal A Holst-Jensen (2001) ArticleTitleRoundup Ready® soybean event-specific real-time quantitative PCR assay and estimation of the practical detection and quantification limits in GMO analyses Eur Food Res Technol 213 432–438 Occurrence Handle10.1007/s002170100403 Occurrence Handle1:CAS:528:DC%2BD38XlsVCg

    Article  CAS  Google Scholar 

  • J Ding J Jia L Yang H Wen C Zhang W Liu D Zhang (2004) ArticleTitleValidation 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 3372–3377 Occurrence Handle10.1021/jf049915d Occurrence Handle15161200 Occurrence Handle1:CAS:528:DC%2BD2cXjslOhs7w%3D

    Article  PubMed  CAS  Google Scholar 

  • InstitutionalAuthorNameEuropean Commission, (2003a) ArticleTitleCommission Regulation (EC) No. 1829/2003 of September 22, 2003, Concerning on genetically modified food and feed Off. J. Eur. Commun L268 1–23

    Google Scholar 

  • InstitutionalAuthorNameEuropean Commission, (2003b) ArticleTitleCommission Regulation (EC) No. 1830/2003 of September 22, 2003, Concerning the traceability and labelling of genetically modified organisms and the traceability of food and feed products produced from genetically modified organisms and amending Directive 2001/18/EC Off. J. Eur. Commun L268 24–28

    Google Scholar 

  • G Gaskell MW Bauer J Durant NC Allum (1999) ArticleTitleWorlds apart? The reception of genetically modified foods in Europe and the U. S Science 285 384–386 Occurrence Handle10.1126/science.285.5426.384 Occurrence Handle10411496 Occurrence Handle1:CAS:528:DyaK1MXkvVGhtL0%3D

    Article  PubMed  CAS  Google Scholar 

  • JL Harwood (1988) ArticleTitleFatty acid metabolism Ann Rev Plant Physiol Plant Mol Biol 39 101–138 Occurrence Handle1:CAS:528:DyaL1cXltVOltrs%3D Occurrence Handle10.1146/annurev.pp.39.060188.000533

    Article  CAS  Google Scholar 

  • M Hernández A Río T Esteve S Prat M Pla (2001) ArticleTitleA rapeseed-specific gene, Acetyl-CoA Carboxylase, can be used as a reference for qualitative and real-Time quantitative PCR detection of transgenes from mixed food samples J Agric Food Chem 49 3622–3627 Occurrence Handle11513638 Occurrence Handle10.1021/jf010173n Occurrence Handle1:CAS:528:DC%2BD3MXkvFSjsrs%3D

    Article  PubMed  CAS  Google Scholar 

  • M Hernández M Pla T Esteve S Prat P Puigdomenech A Ferrando (2003) ArticleTitleA specific real-time quantitative PCR system for event MON810 in Maize YieldGard® based on the 3′-transgene integration sequence Transgenic Res 12 179–189 Occurrence Handle12739886

    PubMed  Google Scholar 

  • A Holck M Vaitilingom L Didierjean K Rudi (2002) ArticleTitle5′-Nuclease PCR for quantitative event-specific detection of the genetically modified Mon810 MaisGard maize Eur Food Res Technol 214 449–454 Occurrence Handle10.1007/s00217-001-0473-y Occurrence Handle1:CAS:528:DC%2BD38XkvVCgsrw%3D

    Article  CAS  Google Scholar 

  • HY Huang TM Pan (2004) ArticleTitleDetection of genetically modified maize MON810 and NK603 by multiplex and real-time polymerase chain reaction methods J Agric Food Chem 52 3264–3268 Occurrence Handle15161180 Occurrence Handle1:CAS:528:DC%2BD2cXjs1KgtL4%3D

    PubMed  CAS  Google Scholar 

  • J Huang S Rozelle C Pray Q Wang (2002) ArticleTitlePlant biotechnology in China Science 295 674–677 Occurrence Handle10.1126/science.1067226 Occurrence Handle11809972 Occurrence Handle1:CAS:528:DC%2BD38XptF2ktw%3D%3D

    Article  PubMed  CAS  Google Scholar 

  • E Ido M Hayami (1997) ArticleTitleConstruction of T-tailed vectors derived from a pUC plasmid: a rapid system for direct cloning of unmodified PCR products Biosci Biotechnol Biochem 61 1766–1767 Occurrence Handle9362125 Occurrence Handle1:CAS:528:DyaK2sXntVyjsL4%3D Occurrence Handle10.1271/bbb.61.1766

    Article  PubMed  CAS  Google Scholar 

  • James C (2005) Global status of commercialized Biotech/GM crops. ISAAA Briefs No. 32

  • D James AM Schmidt E Wall M Green S Masri (2003) ArticleTitleReliable detection and identification of genetically modified maize, soybean, and canola by multiplex PCR analysis J Agric Food Chem 51 5829–5834 Occurrence Handle10.1021/jf0341159 Occurrence Handle13129280 Occurrence Handle1:CAS:528:DC%2BD3sXmsVegt7o%3D

    Article  PubMed  CAS  Google Scholar 

  • A Jankiewicz H Broll J Zagon (1999) ArticleTitleThe official method for the detection of genetically modified soybeans (German Food Act LMBG § 35): a semi-quantitative study of sensitivity limits with glyphosate-tolerant soybeans (Roundup Ready) and insect-resistant Bt maize (Maximizer) Eur Food Res Technol 209 77–82 Occurrence Handle10.1007/s002170050461 Occurrence Handle1:CAS:528:DyaK1MXksFakt7w%3D

    Article  CAS  Google Scholar 

  • H Kuribara Y Shindo T Matsuoka K Takubo S Futo N Aoki T Hirao H Akiyama Y Goda M Toyoda A Hino (2002) ArticleTitleNovel reference molecules for quantitation of genetically modified maize and soybean J AOAC Int 85 1077–1189 Occurrence Handle12374407 Occurrence Handle1:CAS:528:DC%2BD38XnsFekt7k%3D

    PubMed  CAS  Google Scholar 

  • Q Liu SP Singh PJ Sharp AG Green DR Marshall (1996) ArticleTitleNucleotide sequence of a cDNA from Gossypium hirsutum encoding a stearoyl-acyl carrier protein desaturase Plant Physiol 110 1436

    Google Scholar 

  • Matsuoka T (2001) GMO labeling and detection methods in Japan. APEC-JIRCAS Joint Symposium and Workshop on Agricultural Biotechnology

  • Ministry of Agriculture and Forestry (2000) Guidelines for Labeling of Genetically Modified Agricultural Products. MAF Notification No. 31

  • Monsanto Company (2002a) Safety Assessment of Roundup Ready® Cotton Event 1445

  • Monsanto Company (2002b) Safety Assessment of Bollgard® Cotton Event 531

  • A Pan D Zhang L Pan J Chen Z Yuan W Liang (2003) ArticleTitleA rapid PCR method for detection of Glyphosate resistant transgenic rape Agric Sci China 36 856–860

    Google Scholar 

  • D Proudnikov V Yuferov Y Zhou KS LaForge A Ho MJ Kreek (2003) ArticleTitleOptimizing primer-probe design for fluorescent PCR J Neurosci Methods 123 31–45 Occurrence Handle10.1016/S0165-0270(02)00325-4 Occurrence Handle12581847 Occurrence Handle1:CAS:528:DC%2BD3sXhtVGhsLs%3D

    Article  PubMed  CAS  Google Scholar 

  • Rangwala TS and Ye M (2002) Cotton event PV-GHGT07 (1445) and compositions and methods for detection thereof. International Patent Pub. No.: WO 02/34946 A2

  • JK Rho T Lee SI Jung TS Kim YH Park YM Kim (2004) ArticleTitleQualitative and quantitative PCR methods for detection of three lines of genetically modified potatoes J Agric Food Chem 52 3269–3274 Occurrence Handle10.1021/jf0499020 Occurrence Handle15161181 Occurrence Handle1:CAS:528:DC%2BD2cXjsleguro%3D

    Article  PubMed  CAS  Google Scholar 

  • SB Rønning KG Berdal A Holst-Jensen (2003) ArticleTitleEvent specific real-time quantitative PCR for genetically modified Bt11 maize (Zea mays) Eur Food Res Technol 216 347–354

    Google Scholar 

  • DS Smith PW Maxwell SH Boer ParticleDe (2004) ArticleTitleMethod for the detection of synthetic cry3A in transgenic potatoes J Agric Food Chem 52 809–815 Occurrence Handle14969535 Occurrence Handle1:CAS:528:DC%2BD2cXlvFyisQ%3D%3D

    PubMed  CAS  Google Scholar 

  • I Taverniers P Windels E Bockstaele ParticleVan M Loose ParticleDe (2001) ArticleTitleUse of cloned DNA fragments for event-specific quantification of genetically modified organisms in pure and mixed food products Eur Food Res Technol 213 417–424 Occurrence Handle10.1007/s002170100405 Occurrence Handle1:CAS:528:DC%2BD38XlsVCi

    Article  CAS  Google Scholar 

  • C Terry N Harris (2001) ArticleTitleEvent-specific detection of Roundup Ready Soya using two different real time PCR detection chemistries Eur Food Res Technol 213 425–431 Occurrence Handle10.1007/s002170100404 Occurrence Handle1:CAS:528:DC%2BD38XlsVCh

    Article  CAS  Google Scholar 

  • H Weng A Pan L Yang C Zhang Z Liu D Zhang (2004) ArticleTitleEstimating number of transgene copies in transgenic rapeseed by real-time PCR assay With HMG I/Y as an endogenous reference gene Plant Mol Biol Rep 22 289–300 Occurrence Handle1:CAS:528:DC%2BD2MXhs1yqsbg%3D

    CAS  Google Scholar 

  • H Weng L Yang Z Liu J Ding A Pan D Zhang (2005) ArticleTitleA novel reference gene, highmobility-group protein I/Y, can be used in qualitative and real-time quantitative PCR detection of transgenic rapeseed cultivars J AOAC Int 88 577–584 Occurrence Handle15859086 Occurrence Handle1:CAS:528:DC%2BD2MXjtVCnt7s%3D

    PubMed  CAS  Google Scholar 

  • P Windels S Bertrand A Depicker W Moens E Bockstaele ParticleVan M Loose ParticleDe (2003) ArticleTitleQualitative and event-specific PCR real-time detection methods for StarLink™ maize Eur Food Res Technol 216 259–263 Occurrence Handle1:CAS:528:DC%2BD3sXitVSjurY%3D

    CAS  Google Scholar 

  • C Wolf M Scherzinger A Wurz U Pauli P Hubner J Luthy (2000) ArticleTitleDetection of cauliflower mosaic virus by the polymerase chain reaction: testing of food components for false-positive 35S promoter screening results Eur Food Res Technol 210 367–372 Occurrence Handle10.1007/s002170050565 Occurrence Handle1:CAS:528:DC%2BD3cXjsVKksr8%3D

    Article  CAS  Google Scholar 

  • www.agbios.com (2005)

  • www.biotech.org.cn (2005)

  • LT Yang JX Chen H Cheng SR Jia YH Liu LW Pan DB Zhang (2005a) ArticleTitleValidation of a cotton specific gene, Sad1, used as an endogenous reference gene in qualitative and real-time quantitative PCR detection of transgenic cottons Plant Cell Rep 24 237–245 Occurrence Handle10.1007/s00299-005-0929-9 Occurrence Handle1:CAS:528:DC%2BD2MXlt1Wmtbg%3D

    Article  CAS  Google Scholar 

  • LT Yang AH Pan JW Jia JY Ding JX Chen H Cheng CM Zhang DB Zhang (2005b) ArticleTitleValidation of a tomato specific gene, LAT52, used as an endogenous reference gene in qualitative and real-time quantitative PCR detection of transgenic tomatoes J Agric Food Chem 53 183–190 Occurrence Handle1:CAS:528:DC%2BD2cXhtVyqurnO

    CAS  Google Scholar 

  • LT Yang JY Ding CM Zhang JW Jia HB Weng WX Liu DB Zhang (2005c) ArticleTitleEstimating the copy number of transgenes in transformed rice by real-time quantitative PCR Plant Cell Rep 23 759–763 Occurrence Handle10.1007/s00299-004-0881-0 Occurrence Handle1:CAS:528:DC%2BD2MXitVaht7o%3D

    Article  CAS  Google Scholar 

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Authors and Affiliations

  1. School of life Science and Biotechnology, Shanghai Jiao Tong University, 800 Dongchuan Road, 200240, Shanghai, P.R. China

    Litao Yang, Bingjun Qian & Dabing Zhang

  2. Department of Biological Science and Technology, Nanjing University, 22 Hankou Road, 210093, Nanjing, P.R. China

    Litao Yang, Jianxiu Chen & Cheng Huang

  3. Key Laboratory of Agricultural Genetics and Breeding, Agro-biotech Research Center, Shanghai Academy of Agricultural Sciences, 2901 Beidi Road, 201106, Shanghai, P.R. China

    Aihu Pan, Kewei Zhang, Changsong Yin & Dabing Zhang

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  1. Litao Yang
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  2. Aihu Pan
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  3. Kewei Zhang
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  8. Dabing Zhang
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Correspondence to Dabing Zhang.

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Yang, L., Pan, A., Zhang, K. et al. Qualitative and Quantitative PCR Methods for Event-specific Detection of Genetically Modified Cotton Mon1445 and Mon531. Transgenic Res 14, 817–831 (2005). https://doi.org/10.1007/s11248-005-0010-z

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  • DOI: https://doi.org/10.1007/s11248-005-0010-z

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