Title: Combinatory SYBR® Green Real-Time PCR Screening Approach for Tracing Materials Derived from Genetically Modified Rice
Citation: FOOD ANALYTICAL METHODS vol. 6 p. 361-369
Publisher: SPRINGER
Publication Year: 2013
JRC N°: JRC67739
ISSN: 1936-9751
URI: http://link.springer.com/content/pdf/10.1007%2Fs12161-012-9493-4
DOI: 10.1007/s12161-012-9493-4
Type: Articles in periodicals and books
Abstract: Two Real-time PCR approaches for the detection of genetically modified (GM) rice were tested: 1) a combination of SYBR®Green Real-time PCR methods detecting the 35S promoter (P-35S) of Cauliflower Mosaic Virus, the nopaline synthase terminator (T-nos) of Agrobacterium tumefasciens and the Bacillus thuringiensis (Bt) CryIAb/Ac toxins and 2) a P-35S/T-nos duplex Taqman® Real-time PCR system. Both systems correctly recognized their respective targets in control samples of Bt63, Kefeng6 and KMD1 insect-resistant and LLRice62 and LLRice601 herbicide resistant rice. Due to its high discriminating power and broad GMO coverage capacity, the SYBR®Green PCR approach was further tested in more detail. Melting curve, capillary and agarose-gel electrophoresis analyses indicated that the P-35S, T-nos and CryIAb/Ac targets in the GM rice events are similar to the corresponding targets present in many known genetically modified organisms (GMO). High Resolution Melting analysis showed that the CryIAb/Ac targets of the GM rice events Bt63 and Kefeng6 matched best the corresponding Bt11 CryIAb sequence. Digital PCR analysis on genomic DNA from the GM rice Bt63 and Kefeng6 events indicated that both GMO contained multiple inserts. Sensitivity tests showed that all SYBR®Green Real-time PCR methods could detect their targets at less than an estimated 5 copies/reaction. Finally, it was shown that these SYBR®Green PCR methods could detect low levels of their targets in rice consignments originating from China. Together, these results demonstrated that a 'P-35S & T-nos & CryIAb/Ac' combinatory SYBR®Green PCR screening is highly suited to trace the possible presence of Bt63, Kefeng6 and KMD1 GM rice materials in food products.
JRC Directorate:Institute for Health and Consumer Protection Historical Collection

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