New multiplex PCR methods for rapid screening of genetically modified organisms in foods

Abstract

We present novel multiplex PCR methods for rapid and reliable screening of genetically modified organisms (GMOs). New designed PCR primers targeting four frequently used GMO specific sequences permitted identification of new DNA markers, in particular 141 bp fragment of cauliflower mosaic virus (CaMV) 35S promoter, 224 bp fragment of Agrobacterium tumefaciens nopaline synthase (NOS) terminator, 256 bp fragment of 5-enolppyruvylshikimate-phosphate synthase (epsps) gene and 258 bp fragment of Cry1Ab delta-endotoxin (cry1Ab) gene for GMO screening. The certified reference materials containing Roundup Ready soybean (RRS) and maize MON 810 were applied for the development and optimization of uniplex and multiplex PCR systems. Evaluation of amplification products by agarose gel electrophoresis using negative and positive controls confirmed high specificity and sensitivity at 0.1% GMO for both RRS and MON 810. The fourplex PCR was developed and optimized that allows simultaneous detection of three common transgenic elements, such as: CaMV 35S promoter, NOS terminator, epsps gene together with soybean-specific lectin gene. The triplex PCR developed enables simultaneous identification of transgenic elements, such as: 35S promoter and cry1Ab gene together with maize zein gene. The analysis of different processed foods demonstrated that multiplex PCR methods developed in this study are useful for accurate and fast screening of GM food products.

Keywords: DNA marker; food; genetically modified organism; multiplex PCR; screening.

Figure 1

Uniplex PCR amplification of GMO-specific regions using primer pairs: P35S for/P35S rev (A), TNOS for/TNOS rev (B), EPSPS for/EPSPS rev (C), Cry1 for/Cry1 rev (D). Samples (A): lane 1, water, lanes 2–5. Roundup Ready soybean (RRS set): 0, 0.1, 0.5, and 10%; (B) lane 1–4. RRS set: 0, 0.1, 0.5, and 10%; lane 5. soybean seeds, lane 6. water; (C) lane 1, soybean seeds, lanes 2–5. RRS set: 0, 0.1, 0.5, and 10%; lane 6. water; (D). lanes 1–6. Maize MON 810 set: 0, 0.1, 0.5, 1, 2, and 5%, lane 7. water. M, Molecular weight marker (Qiagen GelPilot 100 bp ladder): 100, 200, 300, 400, 500, and 600 bp.

Figure 2

Fourplex PCR for detection of GM Soybean (Roundup Ready soya) using primer pairs: LECT for/LECT rev, P35S for/ P35S rev, TNOS for/ TNOS rev, and EPSPS for/ EPSPS rev. Lanes 1–4. Certified reference material of Roundup Ready soybean (RRS) set: 0%, 0.1% RRS, 0.5% RRS, 10% RRS, lane 5. water. M, Molecular weight markers (Qiagen GelPilot 100 bp ladder): 100, 200, 300, 400, 500, and 600 bp.

Figure 3

Triplex PCR for detection of MON 810 maize using primer pairs: ZEIN for/ZEIN rev, P35S for/P35S rev, and Cry1 for/ Cry1 rev. Lanes 1–6. Certified reference material of MON810 maize set: 0% (blank), 0.1, 0.5, 1, 2, and 5%, lane 7. water. M, Molecular weight markers (Qiagen GelPilot 100 bp ladder): 100, 200, 300, 400, 500, and 600 bp.

Figure 4

Screening of different soybean products to evaluate the presence of soybean and GMOs using fourplex PCR with primer pairs: LECT for/LECT rev, P35S for/P35S rev, TNOS for/TNOS rev, and EPSPS for/EPSPS rev. Lanes 1, 2. Certified reference material of Roundup Ready soybean (RRS): 0.1% RRS and 0.5% RRS, lane 3. soybean seeds, lane 4. Soya flakes, lane 5. Soybean sauce, M, Molecular weight markers (Qiagen GelPilot 100 bp ladder): 100, 200, 300, 400, 500, and 600 bp.

Figure 5

Screening of different wheat products to evaluate the presence of soybean ingredients and GMOs using fourplex PCR with primer pairs: LECT for/LECT rev, P35S for/P35S rev, TNOS for/TNOS rev, and EPSPS for/EPSPS rev. Lane 1. bread 1, lane 2. Bread 2, lane 3. 0.5% RRS, lane 4. crispbread, lane 5. 0% RRS, lane 6. dried crust, 7. soybean seeds. M, Molecular weight markers (Qiagen GelPilot 50 bp ladder): 50, 100, 150, 200, 250, 300, 350, 400, and 500 bp.

Figure 6

Screening of different maize products to evaluate the presence of maize and GMOs using threeplex PCR with primer pairs: ZEIN for/ZEIN rev, P35S for/P35S rev and Cry1 for/Cry1 rev. Lane 1. flour, lane 2. chips, lane 3. Flakes 1, lane 4. Flakes 2, lane 5. 2% maize Bt176, lane 6. 2% maize MON810, lane 7. Water. M, Molecular weight markers (Qiagen GelPilot 100 bp ladder): 100, 200, 300, 400, 500, and 600 bp.