Detection and identification of genetically modified EE-1 brinjal (Solanum melongena) by single, multiplex and SYBR® real-time PCR.
BACKGROUND
Brinjal is an important vegetable crop. Major crop loss of brinjal is due to insect attack. Insect-resistant EE-1 brinjal has been developed and is awaiting approval for commercial release. Consumer health concerns and implementation of international labelling legislation demand reliable analytical detection methods for genetically modified (GM) varieties.


RESULTS
End-point and real-time polymerase chain reaction (PCR) methods were used to detect EE-1 brinjal. In end-point PCR, primer pairs specific to 35S CaMV promoter, NOS terminator and nptII gene common to other GM crops were used. Based on the revealed 3' transgene integration sequence, primers specific for the event EE-1 brinjal were designed. These primers were used for end-point single, multiplex and SYBR-based real-time PCR. End-point single PCR showed that the designed primers were highly specific to event EE-1 with a sensitivity of 20 pg of genomic DNA, corresponding to 20 copies of haploid EE-1 brinjal genomic DNA. The limits of detection and quantification for SYBR-based real-time PCR assay were 10 and 100 copies respectively.


CONCLUSIONS
The prior development of detection methods for this important vegetable crop will facilitate compliance with any forthcoming labelling regulations.
Department of Food Safety and Analytical Quality Control Laboratory, CSIR-Central Food Technological Research Institute, Mysore 570 020, India.
Rajashekhar V
RV
Ballari
Asha
A
Martin
Lalitha R
LR
Gowda
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D052160:Benzothiazoles; D018556:Crops, Agricultural / Q000235:genetics / Q000378:metabolism*; D003959:Diamines; D004731:Endotoxins / Q000235:genetics / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000737:chemistry; D005513:Food Inspection / Q000379:methods* / Q000592:standards; D005515:Food Labeling / Q000331:legislation & jurisprudence; D030841:Food, Genetically Modified / Q000009:adverse effects; D006460:Hemolysin Proteins / Q000235:genetics / Q000378:metabolism*; D007194:India; D007881:Legislation, Food; D057230:Limit of Detection; D060885:Multiplex Polymerase Chain Reaction; D009930:Organic Chemicals / Q000737:chemistry; D010572:Pest Control, Biological; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D014674:Plant Proteins, Dietary / Q000235:genetics / Q000378:metabolism; D030821:Plants, Genetically Modified / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011804:Quinolines; D012333:RNA, Messenger / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D032323:Solanum melongena / Q000235:genetics / Q000378:metabolism*; D013728:Terminator Regions, Genetic
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D052160:Benzothiazoles; D003959:Diamines; D004731:Endotoxins; D005456:Fluorescent Dyes; D006460:Hemolysin Proteins; D009930:Organic Chemicals; D010940:Plant Proteins; D014674:Plant Proteins, Dietary; D011804:Quinolines; D012333:RNA, Messenger; C030956:insecticidal crystal protein, Bacillus Thuringiensis; C098022:SYBR Green I