Development of methods for qualitative and quantitative analysis and monitoring of GMO in soy and corn based processed foods

Transcription

1 Development of methods for qualitative and quantitative analysis and monitoring of GMO in soy and corn based processed foods Cheol-soo, Lee. Department of food industry Korea Health Development Institute

2 Abstract In response to the implementation of labeling regulations for genetically modified(gm) foods, the effective analytical methods such as Polymerase Chain Reaction(PCR) to identify and quality modified genes in raw food ingredients have been successfully developed. However these are not directly applicable to test processed foods. Therefore, this study was conducted to develop the effective analytical protocols to identify modified genes derived from GM soy and corn in the processed foods. We extracted DNA from 8 different soy-based, 10 different corn-based food and 4 different DNA extraction methods such as Wizard, DNeasy, CTAB and phenolchloroform extraction, respectively, and then modified gene derived from soy or corn were identified using PCR method for which the analytical kit containing three primer(35s promotor, NOS terminator, reference gene) and taq DNA polymerase was used. The aim of study was to develop qualitative PCR detection methods as well as sampling and DNA preparation methods to genetic alteration in different processed foods derived from genetically modified soybean and maize, respectively

3 Introduction Approximately forty-different kinds of genetically modified foods(gmo) including soy and corn are distributed throughout the world. Because we have a very low degree of self-supply for soy and corn, tremendous amounts of domestically consumed soy and corn are dependent on imports which are produced using biotechnology. Along with criticism on the safety of GMO, there is an international consensus for GMO labeling on the food label with regard to precautional principle and consumer's right. Responding to this, the regulation of GMO labeling in Korea is going to be in effect on March, Based on the developed methods for GMO testing, methods of sampling and preparation for soy and corn-based processed foods distributed in our market will be further established. In addition, the consumption levels of GMO containing foods among korean will be examined, and these results will provide an valuable scientific information for establishing any standards relating to regulations on GMO and negotiation for international trade.

4 Methodology Material and methods Material Corn and soy based food samples were obtained from mainly developing companies and Korean traditional food samples of approximately 18 samples( Soy-based food(8), and corn-based food(10)) Method Wizard TM method 100mg of sample were mixed with 860ul extraction buffer(10mm Tris-HCl) ph 7.5, 150mM NaCl,3mM EDTA,1%SDS), 100ul 5M Guanidine-HCl, 40ul Proteinase K(20mg/ml) and 3ul RNase(100mg/ml). The sample incubated for at least 3h 65C(waterbath). After centrifugation( at 14,000 rpm, 10 min.) Transfer 500 ul of the supernatant was combined with Wizard TM mini column (Promega No A7211)in 1.5ml eppendorf tube, The minicolumn were washed with 700ul of 80% isopropanol, centrifugate at 14,000rpm 2min and dried at 65 C for 5min at dry oven. The DNA eluted with 60ul of preheated (65C) elution buffer let for 2 min at room temperature and centrifugated at 13,000rpm 1min. At this point we should have enough DNA to run PCR reactions. Optional we checked 5ul of the elute on a agrose gel

5 DNeasy TM method 100mg of sample were mixed with 600ul AP1 extraction buffer). The sample Mixed and incubated for at least 5min 65C(waterbath) four time. After incubation added 130 ul of AP2 and keep on ice for i 30 min.spin down the precipitations with shedder TM mini column at 14,000 rpm, 5 min After centrifugation( at 14,000 rpm, 5 min.) Transfer 450 ul of the supernatant to a new eppendorf tube. Add AP3 225 ul and ethanol 450 ul to the supernatant, (total volume of 1025 ul 450 ul of the supernatant was combined with DNeasy TM mini column (Qiagen No 69104)in 1.5ml eppendorf tube. The minicolumn were washed with 500ul of AP4, centrifugate at 14,000rpm 2min and dried at 65 C for 5min at dry oven. The DNA eluted with 100ul of preheated (65C) elution buffer let for 2 min at room temperature and centrifugated at 13,000rpm 1min. At this point we should have enough DNA to run PCR reactions. Optional we checked 5ul of the elute on a agrose gel Phenol/Cholroform precipitation method 100mg of sample were mixed with2.5-3 volumes of 95% ethanol/0.12 M sodium acetate to the DNA sample contained in a 1.5 ml microcentrifuge tube, invert to mix, and incubate in an ice-water bath for at least 10 minutes.centrifuge at 12,000 rpm in a microcentrifuge for 15 minutes at 4 C, decant the supernatant, and drain inverted on a paper towel. Add 70% ethanol (corresponding to about two volume of the original sample), incubate at room temperature for 5-10 minutes and centrifuge again for 5 minutes, and decant and drain the tube, as above. Place the tube in a dry-oven and dry the DNA pellet for about 5-10 minutes, or until dry. Always dissolve dried DNA in 10 mm Tris-HCl, ph , 0.1 mm EDTA (termed 10:0.1 TE buffer).

6 CTAB method 100mg of sample were mixed with preheated (65C) 750ul CTAB isolation buffer(2%(w/v) CTAB(sigma), 1.4M NaCl,0.2%(v/v)2-mercaptoethanol, 20mM EDTA,100mM Tris-HCl(pH8.0)) incubate at 60C with occuational gentle swirling. Extract once with chloroform-isoamylalcohol(24:1; v/v) and mixed gently but roughly. DNA solution centrifuge at 14,000rpm for 10 minutes at room temperature. Use table top centrifuge.transfer the upper aqueous phase to a 2nd tube; add 2/3 volume isopropanol or EtOH and mix by inverting quickly 2 or 3 times to precipitate the DNA. Hook the precipitate out with a hooked pasteur pipet. Transfer the precipitate to a 50 ml polypropylene screwcap centrifuge tube containing about 20ml 76% EtOH/10mm NH4Ac; set for about 20 minute.transfer precipitate to 1.5 ml microtube containing 1 ml of TE buffer. Polymerase Chain Reaction(PCR ) We used four oligonucleotides such as 35S promotor primer(205bp), NOS teminator primer(200bp), B-actin primer(495bp), zein primer(250bp). Those were purchased Cogenbiotech co.ltd and used as a primer for PCR. For confirmation of the optimised protocol, All PCR reaction were performed in final volume of 25μl in 0.5ml tube containing 5 μl of 5 X reaction buffer, 2 μl of Primermixture,1 μl of Taq DNA polymerase (1U/ μl ), and X μl of DW. X μl template DNA solution. PCR program was as follow ; denaturation at 94 for 5 min followed 40 cycles of PCR cosisted of annealing at 60 for 40 sec, polymerization 72 for 30sec, denaturation at 94 for 2min..

7 Result 1 Detection of soybean based food with B-actin gene Wizard DNA purification TM B-actin Dneasy plant mini prep TM Phenol/chloroform extraction and precipitation CTAB M Material : M: ladder Bean paste(1-4 lane), Mixed seasoning( lane 5) Tofu(lane 6), Bean sprout(lane 7), Soy beverage(lane 8) Nuddle(lane 9), GMO soybean( lane 10) Fig.!

8 Result 1-1 Detection of Maize based foood with zein gene Wizard DNA purification zein Dneasy plant mini prep TM Phenol/chloroform extraction and precipitation CTAB M Material : M : ladder Corn chip(1-4 lane), Frying corn powder(lane 5-6) Mixed seasoning( lane 7), Corn soup(lane 8-10) Popcorn(lane 11-12), GMO maize( lane 13) Fig.2

9 Result 2-1 Processed Soybean with 35S promotor Wizard DNA purification TM 35S Promotor Dneasy plant mini prep TM Phenol/chloroform extraction and precipitation CTAB M N Material : M : Ladder, N: negative control Bean paste(1-4 lane), Mixed seasoning( lane 5) Tofu(lane 6), Bean sprout(lane 7) Soy beverage(lane 8), Nuddle(lane 9), GMO soybean( lane 10) Fig.3

10 Result 2-2 Processed Soybean with NOS terminator Wizard DNA purification TM Nos terminator Dneasy plant mini prep TM Phenol/chloroform extraction and precipitation CTAB M N Material : M : Ladder, N: negative control, Bean paste(1-4 lane), Mixed seasoning( lane 5) Tofu(lane 6), Bean sprout(lane 7), Soy beverage(lane 8) Nuddle(lane 9), GMO soybean( lane 10) Fig.4

11 Result 3-1 Detection of Maize based foood with 35S promotor Wizard DNA purification TM 35S promotor Dneasy plant mini prep TM Phenol/chloroform extraction and precipitation CTAB M N Material : M : ladder, N: Negative control Corn chip(1-4 lane), Frying corn powder(lane 5-6) Mixed seasoning( lane 7), Corn soup(lane 8-10) Popcorn(lane 11-12), GMO maize( lane 13) Fig.5

12 Result 3-2 Detection of Maize based foood with NOS terminator Wizard DNA purification TM NOS terminator DNeasy plant mini prep TM Phenol/chloroform extraction and precipitation CTAB M N Material : M : ladder, N: Negative control Corn chip(1-4 lane), Frying corn powder(lane 5-6) Mixed seasoning( lane 7), Corn soup(lane 8-10) Popcorn(lane 11-12), GMO maize( lane 13) Fig.6

13 Conclusion The DNA extraction using column such as DNeasy method or Wizard method resulted in 100% or 62.5% detection rate(proporation of identified sample per total number of analyzed sample) for soy-based processed foods, respectively, and 91.7% or 100% detection rate for corn-based processed foods, respectively. However, the extraction methods using solvent such as CTAB or phenol-chloroform did not lead to good resolution (less than 50% detection rate) in identifying modified genes. Therefore, the data in this study indicate that the DNA extraction from the processed foods using column such as DNeasy or Wizard are superior to the methods using solvent with regard to identification or modified gene in the processed foods by PCR.

14 Result The DNA extraction using column such as DNeasy method or Wizard method were effective DNA extration method for soy or corn based food (Fig.1,2). But Phenol/chloroform extraction and CTAB method were poor resolution(fig. 1,2). Korean traditional soybean based food, bean paste,which had variable resolution(fig. 1 lane 1-4). It was assumed that bean paste manufacture step was somewhat different each factory and destroyed its DNA as fallow fermentation. Therefore It must further study as shelf life time. The transgenic plant used gene which is under the control of a 35S promoter and a NOS-terminator. The detection describe, which was designed to detect the GMO( such as Roudup ready, Starlink, MaizeGard) is based on a PCR amplifying parts of 35S promoter and NOS- terminator. We detected identifying modified genes. DNeasy method or Wizard method had well detected all inserted gene( Fig, 3-6). In several cases of soy-based food, Phenol-chloroform detected only one parts of 35S promoter. CTAB more poor resolution about inserted gene Therefore, the data will showed that phenol/chloroform and CTAB is not fit of DNA extraction method. It is different result between that of EU method.