Event-Specific Detection of Stacked Genetically Modified Maize Bt11 × GA21 by UP-M-PCR and Real-Time PCR

Abstract

More and more stacked GMOs have been developed for more improved functional properties and/or a stronger intended characteristic, such as antipest, improved product efficiency etc. Bt11 x GA21 is a new kind of stacked GM maize developed by Monsanto Company. Since there are no unique flanking sequences in stacked GMOs, up to now, no appropriate method has been reported to accurately detect them. In this passage, a novel universal primer multiplex PCR (UP-M-PCR) was developed and applied as a rapid screening method for the simultaneous detection of five target sequences (NOS, 35S, Bt11 event, GA21 event, and IVR) in maize Bt11 x GA21. This method overcame the disadvantages rooted deeply in conventional multiplex PCR such as complex manipulation, lower sensitivity, self-inhibition and amplification disparity resulting from different primers. What's more, it got a high specificity and had a detection limit of 0.1% (approximates to 38 haploid genome copies). Furthermore, real-time PCR combined with multivariate statistical analysis was used for accurate quantification of stacked GM maize Bt11 x GA21 in 100% GM maize mixture (Bt11 x GA21, Bt11 and GA21). Detection results showed that this method could accurately validate the content of Bt11, GA21 and Bt11 x GA21 in 100% GM mixture with a detection limit of 0.5% (approximates to 200 haploid genome copies) and a low relative standard deviation <5%. All the data proved that this method may be widely applied in event-specific detection of other stacked GMOs in GM-mixture.

A Novel Universal Primer-Multiplex-PCR Method with Sequencing Gel Electrophoresis Analysis

“…Thus visualization is from the primary source and does not suffer any degradation or blurring that can accompany secondary imaging devices which involve fluorescence, autoradiography, focusing lenses, film development or digital image processing. (III) Silver staining offers similar sensitivity to autoradiography, but avoids radioactive handling, delays from development times and waste disposal issues [19]. Although this is an advantage in terms of scope, it nevertheless means that the protocol must be applied with due care; almost any other biological impurity such as stray human fingerprints incorporated into or onto the gel matrix on the gel surface will stain with perfect detail.…”

“…The UP-M-PCR method was originally developed by our lab and it firstly applied to the detection of stacked GM events Bt11×GA21 [19]. In this study we used the same PCR system and combined it with sequencing gel electrophoresis analysis to simultaneously detect 15 target genes in the GM crops successfully, but the sequence of UP is different from before, which just showed the flexibility of this novel PCR method.…”

“…A novel u niversal p rimer- m ultiplex PCR (UP-M-PCR) method was devised at the basis of the problem originating from conventional multiplex PCR, and up to now, it has been used to simultaneously detect four meat species including chicken, cattle, pig and horse [17], to simultaneously detect Escherichia coli , Listeria monocytogenes , and Salmonella spp. in food samples [18], and used in the event-specific detection of stacked genetically modified maize Bt11×GA21 [19]. In the present study, we also use this novel universal primer-multiplex PCR method to simultaneously detect nine commonly used selectable marker and reporter genes, including hpt , gus , nptII , aadA , pat , 35s , bar , nos , uidA , as well as six endogenous genes Pa , Ivr , Lec , sps , sad1 and FatA of six most common GM crops papaya, maize, soybean, rice, cotton and canola, respectively, which could overcome the shortcomings of conventional multiplex PCR described previously, and significantly simplify the procedure of identification for GMOs.…”

Soft Fruit Traceability in Food Matrices using Real-Time PCR

“…To discriminate between different fruit species, it is necessary to detect sequence-specific amplification products, as in the detection of GMOs [24] or other raw materials (hazelnut, olive oil, solanaceae) [25,26,27]. Both primer pairs used in these analyses detected polymorphic profiles that were able to discriminate between our species.…”

Teknik PCR Kualitatif untuk Deteksi Produk Rekayasa Genetika Jagung Event BT11 dan GA21

“…Sebagai alternatif, teknik PCR secara multipleks dapat digunakan dalam meningkatkan efisiensi teknik deteksi PRG yang mengandung lebih dari satu gen interes dalam suatu genom atau multi event dalam suatu sampel, baik tanaman, makanan, maupun pakan ternak, secara simultan. Teknik PCR secara dupleks atau multipleks telah digunakan oleh beberapa peneliti untuk deteksi PRG baik yang berupa event tunggal atau stacked (Babekova et al, 2008;Hedreyda dan Roxas, 2010;Xu et al, 2009). Namun demikian, teknik PCR secara multipleks biasanya memerlukan optimasi kondisi PCR untuk mengatasi rendahnya sensitivitas produk amplifikasi atau amplikon yang dihasilkan oleh primer yang berbedabeda.…”