, 2010, Corbisier et al., 2005, Fernandez et al., 2005, Höhne et al., 2002, Joint Research Centre, 2011, Reiting et al., 2007 and Waiblinger et al., 2008). Concerning the t35S element of the pCAMBIA family vectors, its sequence was slightly different at the 5′ end compared to the authorised GMOs and LLPs events containing a t35S element (A2704-12, A5547-127, Bt11, Bt176, DAS59122, GHB119, LLRICE62, T25, TC1507 and Topas-19-2). CP-673451 purchase Therefore, this element was not detected by the t35S SYBR®Green detection method developed previously in-house (Broeders et al., 2012b; Personal communication). All these bioinformatics data were confirmed in vitro by qPCR SYBR®Green assay ( Table 3). In order to discriminate unauthorised GMOs containing
pCAMBIA family vectors, the selleckchem t35S pCAMBIA screening marker was developed. To this end, the sequence of the t35S pCAMBIA element was analysed. The majority of the pCAMBIA vectors (1200, 1201, 1281Z, 1291Z, 1300, 1301, 1302, 1303, 1304, 1380, 1381Xa, 1381Xb, 1381Xc, 1381Z, 1390, 1391, 1391Xa, 1391Xb, 1391Xc, 1391Z, 2200, 2300, 2301), except 0380 and 0390, possessed the t35S element. Its sequence was practically identical (slightly shorter by 5 bp at the 3′ end for 2200, 2201, 2300 and 2301). The t35S pCAMBIA a-R and t35S pCAMBIA c-F primers were designed manually in the conserved region of the pCAMBIA family vector
to discriminate exclusively this element (Tables Table 1 and Table 2). The specificity of this marker was tested initially in silico with the software wEMBOSS. To develop the t35S pCAMBIA marker, the specificity of t35S pCAMBIA c-F and a-R primers was tested in vitro selleck inhibitor on all authorised GMOs and LLPs events by the qPCR SYBR®Green
assay (Tables Table 1 and Table 2). As expected, only the Bt rice, containing a pCAMBIA cassette, was detected after 40 cycles with a Ct value at 22.70 and a Tm value at 73 °C, indicating that the screening marker was specific. All the other WT, GMO and LLP materials tested did not give a signal after 40 cycles. Then, the sensitivity of this marker was determined via the limit of detection with 6 repeats (LOD6). The LOD6 is defined as the amplicon copy number that affords a positive PCR result (expressed as Ct-value) upon six-fold measurement of the target sequence in the same DNA sample ( Table 4). To this end, DNA from 100% Bt rice was diluted to 4 ng/μl and 4 independent dilution series were prepared (in nuclease-free water) starting from this concentration. The dilution series (from 1 to 0.00005 ng/μl of DNA) were prepared prior to setting up each of the qPCR runs. For each assay, a range from 2000 to 0.1 HGEs was tested in a qPCR SYBR®Green assay. The HGE content of the DNA extracts was calculated according to the size of the rice genome (0.5 pg) ( Arumuganathan & Earle, 1991). The LOD6 was obtained at 5 HGEs (corresponding to 0.0025% of unauthorised GMOs) with a mean Ct value of 35.28 Ct and a mean Tm value of 72.52 °C.