Transgenic Research

, 14:817 | Cite as

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

  • Litao Yang
  • Aihu Pan
  • Kewei Zhang
  • Changsong Yin
  • Bingjun Qian
  • Jianxiu Chen
  • Cheng Huang
  • Dabing Zhang
Article

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.

Key words

genetically modified cotton Mon1445, Mon531 multiplex PCR reference molecule TaqMan real-time PCR 

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

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Copyright information

© Springer 2005

Authors and Affiliations

  • Litao Yang
    • 1
    • 2
  • Aihu Pan
    • 3
  • Kewei Zhang
    • 3
  • Changsong Yin
    • 3
  • Bingjun Qian
    • 1
  • Jianxiu Chen
    • 2
  • Cheng Huang
    • 2
  • Dabing Zhang
    • 1
    • 3
  1. 1.School of life Science and BiotechnologyShanghai Jiao Tong UniversityShanghaiP.R. China
  2. 2.Department of Biological Science and TechnologyNanjing UniversityNanjingP.R. China
  3. 3.Key Laboratory of Agricultural Genetics and Breeding, Agro-biotech Research CenterShanghai Academy of Agricultural SciencesShanghaiP.R. China

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