FLEISCHWIRTSCHAFT international 1/2017

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44 Fleischwirtschaft international 1_2017 Testing Methods Fig. 1: In low-price-products soy flour is often used as an extender. Innovation in food control Duplex PCR opens new possibilities for the detection of GM soya in chicken sausages Up to now the detection of the GM-soy presence in chicken sausages was expensive and took some time. The successful merging of the procedures for endogene and transgene amplification in asingle tubemade it faster and less expensive. By Zoran T. Popovski, Elizabeta Miskoska-Milevska, Tome Nestorovski and ZlatkoPejkovski This study reports the screening for GMO presence in soy containg (Fig.1) chicken sausages (Fig. 2) in asingle step using duplex PCR. Previously,the screening was performed in two steps, one for revealing the soy DNA, and the second for detecting the presence of the construct that is present in GM soy.Anoptimization of the PCR conditions was performed focusing on the MgCl2 concentration and primers annealing temperature. The achieved data showed that aconcentration of 2.5 mM MgCl2 and atemperature of 60 °C are appropriate to amplify the both fragments in a single reaction. The results did not show any false positive or false negative data. They were wellmatched with those from the separately accomplished reactions. This kind of doubled PCR enables faster and cheaper detection of the presence of GM-soy.Itgives a possibility to eliminate too many negative samples before the quantification step with real-time PCR. How GMOs are detected Due to the fact that the nucleotide sequences of GMOs are determined, the detection of GMO presence in processed meat products commonly is performed using PCR based techniques (Mulis, 1987). One of the most applied genetic modifications among the crops is creating herbicide tolerant plants, especially against the roundup herbicide and that is the reason why those crops are named as “roundup ready” (Oxtoby et al., 1990). The first step in this procedure is to detect the presence of an “housekeeping” gene for the appropriate organism which was modified, and then to search for the presence of aconstruct that can be present in the sample (Windels, 2001).Asa“housekeeping” gene in the soy genome, usually,the gene for lectin synthesis is used, while for the detection of transgene, commonly,anamplification covering part of the promoter sequence and part of the inserted gene is performed (Holst-Jensen,2003). The screening of roundup ready soya (RRS) was performed in two ig. 2: Mostlychicken sausages are available for alow price. steps before, one for revealing soy DNA, and the second one for detecting the presence of the construct that is present in GM soy. (Meyer et al., 1997). The aim of this study was to simplify the procedure for GMO detection on DNA level developing anew duplex PCR in order to amplify the part of endogen and trans-
....................................... Fleischwirtschaft international 1_2017 45 Testing Methods Source: POPOVSKI et al. FLEISCHWIRTSCHAFT international 1_2017 Fig. 4A): MgCl2 gradient concentration from 0.5 Mm to 4.5 mM amplicons of 74 bp of the same sample on a2.5% agarose gel. #1–50 bp ladder #2–0.5 mM MgCl2 #3–1.0mM MgCl2 #4–1.5mMMgCl2 #5–2.0 mM MgCl2 #6–2.5 mM MgCl2 #7–3.0 mM MgCl2 #8–3.5 mM MgCl2 #9–4.0 mM MgCl2 and #10–4.5 mM MgCl2. B): Temperature gradient form 55 °C to 65 °C. Electrophoregram of 2.5% agarose gel. #1–50bpladder #2–55.8 °C #3–56.3 °C #4–57.1 °C #5–58.1°C#6–58.8 °C #7–59.6 °C #8–60.2 °C #9–61.6°C#10–62.2 °C #11–63.3 °C and#12–64.2°C gene in one reaction. It could serve as abasis to simplify the procedure for GMO detection. Materials and methods As starting material were used: certifi ed reference GMO free soybeans, for positive and negative control for RRS soybeans purchased from JRC-IRMM, soy free chicken sausages and chicken sausages that contained soy as an ingredient. The DNA isolation was performed using the CTAB method (DOYLE and DOYLE,1987) with some slight modifi cation (MISKOSKA- MILEVSKA et al., 2011)(Fig. 3). The soybean DNA detection was done using PCR to amplify the part of the lectin gene. The used forward primer 5’-GCC CTC TAC TCC ACC CCC ATC-3’ and reverse primer 5’ -GCC CATCTG CAA GCC TTT TTG TG -3’(POPPING, 2001),amplifi ed afragment with 118bpinlength. The reactions contained 3.0 mM MgCl2, 0.4 mM dNTP, 0.2 pM GM03/GM04, 1U Taqpolymerase and 100ngDNA. The annealing temperature was at 60 °C. GM soy DNA was detected with the second pair of primers RR1F(5’-CATTTG GAC AGG ACA CGC TGA-3’) and RR1R (5’-GAG CCA TGT TGT TAATTT GTG CC- 3’) (WEI et al., 2008). The amplicon with alength of 74 bp comprehended the end part of 35S promoter and the initial part of the CTP4 inserted gene. The reactions contain 2.5 mM MgCl2,0.4 mM dNTP, 0.2 mM RRS1-F/RRS1-R and 0,5 UAmpliTaq Gold polymerase activated with hot-start. The concentration of MgCl2 was optimized by changing it in the range from 0.5 mM up to 4.5 mM. Optimization was done also for the annealing temperature, in order to fi nd an appropriate value for annealing the two pairs of primers, one for the transgene covering part of the CMV 35S promoter and the part of the CTP4 EPSPS inserted gene, and the second pair for the “housekeeping” gene. concentration for these reactions (Fig. 4A). The optimization of the temperature was done with 12 different values in the range from 55 °C to 65 °C. The analysis on a2.5% agarose gel shows that the strength of the signal started to be weaker at temperatures above 62.2 °C (Fig. 4B, #11and #12). The rest of the samples that were amplifi ed at values between 55.8 °C and 62.2 °C did not show any differences in the bands; that is an indicator for the possibility for primers annealing at the temperatures with awider range. (Fig. 4B). Due to the similar conditions in both reactions atrial was con- Fig. 3: The DNA isolation was performed using the slightlymodified CTAB method. What is the result? The results from the MgCl2 optimization were visualized on 2.5% agarose gel. The samples with 0.5 mM and 1.0mMMgCl2 did not show any fragments, while in the samples with 1.5mMand 2.0 mM MgCl2 aweak signal was registered. The samples that had concentration of MgCl2 from 2.5 mM up to 4.5 mM have shown visible and clear fragments. Due to the fact that the higher MgCl2 concentration usually results in unspecifi camplifi - cation, the 2.5 mM MgCl2 was considered to be the most suitable
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