ABSTRACT applicability of a PCR based GMO testing

ABSTRACTConsidering the increase of the total cultivated land areadedicated to genetically modified organisms (GMO), Growing number of GMOsintroduced to the market, Intending to ensure consumers’ freedom of choice andtheir perception toward GMOs and the need to comply with various local GMOlegislations, efficient and accurate analytical methods are needed for theirdetection and identification.This paper describes the development and applicability of a PCRbased GMO testing of the most frequent constructs used in Transgenic Soy, Maizeand Rice varieties. To comply with GMO analytical quality requirements, anegative and a positive control were analyzed in parallel. In addition, aninternal positive control was also included in each reaction as well for thedetection of potential PCR inhibition.

Tested on non-GM materials,on different sample inputs and on proficiency test samples, the method offeredhigh specificity and sensitivity with an absolute limit of detection of 0.25%GM construct in the samples. Convenient  and cost efficient, this approach fits thepurpose of GMO testing laboratories. Keywords: Genetically modified organisms (GMOs), DNA Extraction, Polymerase Chain Reaction (PCR),Element-specific, Detection, ACaMV”Cauliflower Mosaic Virus (HYPERLINK”http://gmo-crl.

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jrc.ec.europa.eu/gmomethods/search?db=gmometh=id%3AQL-ELE*%26ft%3ACaMV”CaMVHYPERLINK”http://gmo-crl.

jrc.ec.europa.eu/gmomethods/search?db=gmometh=id%3AQL-ELE*%26ft%3ACaMV”)35S promoter (p-35S), Anopaline%20synthase%20terminator”nopalineHYPERLINK “http://gmo-crl.jrc.ec.

europa.eu/gmomethods/search?db=gmometh&q=id%3AQL-ELE*%26ft%3Anopaline%20synthase%20terminator”HYPERLINK”http://gmo-crl.jrc.ec.europa.eu/gmomethods/search?db=gmometh&q=id%3AQL-ELE*%26ft%3Anopaline%20synthase%20terminator”synthaseHYPERLINK”http://gmo-crl.

jrc.ec.europa.eu/gmomethods/search?db=gmometh&q=id%3AQL-ELE*%26ft%3Anopaline%20synthase%20terminator”terminator (t-nos), Soy, Maize, Rice INTRODUCTIONSince the first GMO approval in 1996, adoption of GM technologyhas been growing exponentially and the number of GM crops introduced to themarket has been increasing (Rosa et al.,2016).

In parallel, During the last decade, an intensive development of GMOdetection methods, additionally triggered by the establishment of legislationframes and requests for the traceability and labeling in many countries,worldwide have been occurred (Žel et al,2012). Moreover, these new biotech organisms will harbor novel and sometimesunique characteristics (Stein and Rodriguez-Cerezo 2009a). On the other hand, aprolonged effort exists to develop and optimize more efficient detectionmethodologies to face with all the new challenges for the analysis of thediverse and growing number of GM events (Žel et al, 2012).Thisintroduced recombinant deoxyribonucleic acid (rDNA) sequence, also called atransgenic sequence, which differentiates the GM plant from its non-GMcounterpart at the DNA level, is used as a target for the DNA-based detectionof the GM plants (Žel et al, 2012).In response to constantly increasing number of GM eventscommercialized in various parts of the world and to guarantee the traceabilityof GMO in food/feed chain and the consumer’s freedom of choice, regulatoryframeworks were established in many countries around the world (Marie-Alice Fraiture, et al., 2016)such as in Iran.

Their implementations, needs more efficient methods for thedetection of genetically modified organisms (GMOs). Sincethe introduction of mandatory labeling requirements in Iran in 1392, differentanalytical approaches have been developed for GMO identification and the mostdirect and widely used detection methods target the genetic modificationitself, i.e.

, the modified DNA, and are based on the amplification of thespecific DNA targets using the PCR technique (Miraglia et al., 2004; Rodríguez-Lázaro etal., 2007).Commonlyused transgenic elements in the usual GM events are Cauliflower Mosaic Virus 35S promoter(p35S) and nopalinesynthase terminator (t-nos)(Ruttnik et al., 2010). MATERIALS AND METHODS SamplesCertifiedreference materials (CRMs) of GM ERM-BF410a (Roundup Ready Soybean),ERM-BF417d MON863 x MON 810 Maize, ERM-BF420c Maize, ERM-BF419b Sugar beet and0306-I8  LLRice62 were purchased from theInstitute for Reference Materials and Measurements (IRMM, Geel, Belgium) andAmerican Oil Chemists’ Society (AOCS, Urbana, IL, USA). Fifty-sevensoybean-containing food samples consist of Soy protein isolate, Chocolate,Soybean, Lecithin, Soy milk, Soy concentrate, Oil, Fiber, Two-thousand-six-hundred-and-oneRice samples, Sixty-four Maize containing food and feed samples (Corn seeds,powder, Corn oil, Frozen and dried samples) were received from Food and drugadministration, different companies, or taken from domestic markets duringthree years (2015 – 2017). DNA IsolationSampleswere processed using the QIAamp DNeasy Mini Kit based on silica gel membranetechnology which allowed an efficient extraction of complete DNA from planttissues and processed food samples.

 Target selection: To allow an efficient detection and identification of GMmaterial, two different categories of targets were selected. 1)Plant endogenous sequences were chosen to specifically detect soya, maize, ricewhich provides information on the host species of the transgenic material andalso indicates potential plant cross-contamination in raw material. 2)Considered as key indicators of GM material, the frequently used CauliflowerMosaic Virus 35S promoter (p-35S), Nopaline synthase terminator fromAgrobacterium tumefaciens (t-NOS), were selected.  Oligonucleotideprimers Oligonucleotide primers were designed using the Oligo7 Primeranalysis Software (Molecular Biology Insights, Inc.

Colorado Springs, CO, USA).All oligonucleotides were ordered as customised assays at Metabions, Germany.  PCR AmplificationPCR runs were performed using an ABI Simpliamp System (LifeTechnologies, USA). Each amplification reaction contained 1X  reaction buffer; 0.2-0.4M of each primer;0.5-1 U Taq DNA Polymerase (Sinaclon), 1.5mM MgCl2, 0.

8 mM each dATP, dCTP,dGTP, and dTTP (Sinaclon, Iran) for soybean-specific amplifications, 0.4 mMdNTPs for maize-specific amplifications or 0.2 mM dNTPs for Rice-specific amplifications. Amplification profileswere as follows: denaturation for 3 min at 95ºC; 35 cycles (Rice 30 cycles) of30s denaturation step at 95ºC, 30s an annealing/elongation step at 55ºC, and60-120s (Rice 30 s) at 72ºC; and a final extension of 3-5 min at 72ºC. Gel ElectrophoresisAmplified DNA fragments were electrophoresed on 2% agarose gelsin 1X TAE buffer, and bands were visualized by safe staining and UVtransillumination.  RESULTS SpecificityTo determine the specificity of the method, plant materials andGM events with a high GM content (?1%) were tested in duplicate. To assess thereliability of the PCR runs, a negative control, a non template control(Distilled water) and a positive control were analyzed in each run. All theassays successfully amplified on the positive control, while no amplificationcurves were observed with NTC.

The generic plant assay successfully amplifiedon all the plant species tested (Table 1) and did not lead to any signals on animal DNA(beef, pig, horse, sheep, goat and chicken).Sensitivity To determine the sensitivity of the different assays, plantmaterials and GM events with GM content ranged from 5 percent to 0.0625% wereanalyzed. Since the limit of detection of an assay is the amount of analyte whichcan be detected by the analytical method at least 95 % of the time, a minimumof 20 replicates were tested for each target in at least three different runs.The generic plant assay allowed a very sensitive detection of plant material(LOD of 0.25 %), known to be suitable for the detection of very low levels ofplant genetic material (Laube et al., 2010) which could be very usefulfor the detection of residual plant DNA in highly processed products, such asstarch or lecithin.

 Figure 1- Sample serial dilution forlimit of detection determination. Lanes 1 to 9: 35S Amplicons; L1:5%, L2: 2%, L3: 1%, L4: 0.5%, L5: 0.25%, L6: 0.125%,  L7: 0.0625%, L8: NTC, L9: Positive control (10%),L10: 100 bp DNA Size marker, Lanes 11 to 20: NOS Amplicons; L11: 5%, L12: 2%,L13: 1%, L14: 0.5%, L15: 0.25%, L16: 0.

125%, L17: 0.0625%, L18: NTC, L19: Positive control (10%).GM Target andIndigenous gene DetectionIn this study, Fifty-sevensoybean-containing food samples, Two-thousand-six-hundred-and-one Rice samples,Sixty-four Maize containing food and feed samples were received from Food anddrug administration, different companies, or taken from domestic markets duringthree years (1394 – 1396). DNA isolation from both CRMs and food products wasperformed using the QIAamp DNeasy Mini Kit which allowed an efficientextraction of complete DNA from plant tissues and processed food samples withthe majority of samples showing A260/280 ratio between 1.8 and 2. The extractedDNA samples with good quality and purity were subjected to PCR amplification toidentify Plant endogenous sequences as well as Mosaic Virus 35S promoter (pCaMV35S) and Agrobacterium tumefaciens Nopaline synthase terminator (tNOS) specifictargets. The agarose gel electrophoresis of the PCR products showed theexpected bands of 189 bp and 199 bp for the introduced genes of CaMV 35Spromoter and NOS terminator, respectively. Besides,fragments of SPS, Lectin, and Zein geneswere also amplified to confirm the presence of Rice, Soya, Maize in thesamples.

Product sizes of each of these indigenous genes are 277 bp, 216 bp,329 bp respectively (Figure 2). The results of gel electrophoresis for DNAtemplates isolated from the samples tested with CaMV35s and NOS primer pairsare shown in figure 1. The results obtained from qualitative PCR test onTwo-thousand-six-hundred-and-one Rice samples, Fifty-seven soybean-containingfood samples and Sixty-four Maize containing food and feed samples containingSPS, Lectin, Zein genes, respectively showed that in total, 1.75% of Maizesamples and 6.25% of Soya samples were GM positive and no GM positive Ricesample was detected.DiscussionThe method used in this study is a fast,inexpensive and easy to perform procedure regarding the hands-on steps and itcan be implemented in every lab with a Thermal cycler. Only minute amounts ofprimers are used when setting up a PCR master mix.

Therefore, these primers canbe used for a large number of samples once synthesized and stored properly(?20?C). By combining an automated DNA extraction method with PCR, a largenumber of samples can be screened for the presence of GM events at the sametime. The applicability of this method was alsoproved by the whole workflow (DNA extraction in combination with a PCR assay)can be carried out in a single day.Larger number of rice samples for GMevaluation compared to soya and Maize samples was due to this crop’s strategicplace in Iranian people food chain and its economic value for the country. Lowpercentage of GM soy bean and Maize samples can be related to restricted numberof samples sent for our lab or precision of choice of government for choosingwhich country or provider to choose to import Non-GM crops or it can be due to highfrequency of GM Maize and GM Soy in the market. Since 33 percent of all Maize and78 percent of soy cultivated worldwide is GM, so finding a provider which sellsnon-GM products can be extremely rare specially in the case of soybean. So theimporters simply won’t send the samples to laboratories  because there is a high probability that thetests results come positive.

The tested samples for GMO, targeted bythe developed PCR module, were chosen according to their importance. Theobtained detection limits of our developed PCR assay is 0.25% of GM element inthe sample which is fulfilling to meet the legal requirements of labelingthreshold in Iran.

It should be mentioned that even when usinga very complex matrix like processed food, Lecithin and oil as startingmaterials, the developed DNA extraction-PCR system revealed reliable results. Thesensitivity of the developed method is high enough to detect even traces of GM elementsin food and feed samples.For routine GM crop surveillance, thismodule can be applied as a useful screening tool for the detection of commonlyused GM events in different samples. However, the developed assay is aqualitative assay. Once the samples are screened for the presence of GM, aquantitative method needs to be performed, if quantification is required. ConclusionAs a summary, the utmost optimization of the PCR developed inthis method offers a broad, simple and cost efficient strategy in GMO analysis.The described Method allows the detection of three routine types of samples toobtain their content. The assay would theoretically enable the screening ofcommon worldwide known GMOs described in public databases.

Easy to perform, sensitive,specific and cost-effective, this method fits for purpose of GMO testinglaboratories, complying with the legal and analytical requirements described bydifferent authorities.  Acknowledgments:Wewould like to thank Mrs. Mahdizadeh, Mr.

Mousavi, Food and drug administrationReference laboratory, Tehran Iran for their valuable contribution, as well asParsian Biotech laboratory personnel for their collaboration.