Qualitative and Quantitative Real-Time PCR Methods for Assessing False-Positive Rates in Genetically Modified Organisms Based on the Microbial-Infection-Linked HPT Gene
Abstract
:1. Introduction
2. Results
2.1. Routine Detection of GMO in Soybean Sprouts
2.2. Isolation and Characterization of the HPT Gene Sequence
2.3. Design of Primers and Probes
2.4. Specificity and Sensitivity Test of Singleplex Qualitative
2.5. Determination of the Applicability of Qualitative PCR
2.6. Verification of qPCR Specificity
2.7. Standard Curve and Repeatability of qPCR
2.8. Limits of Detection and Quantification with qPCR
2.9. Analysis of HPT-Positive Seed Material Using qPCR
2.10. Development of a Qualitative and Quantitative Duplex PCR
3. Discussion
4. Materials and Methods
4.1. Materials
4.2. DNA Extraction and Purification
4.3. Genome Walking PCR
4.4. Construction of a Reference Plasmid
4.5. Primer and Probe Design
4.6. Qualitative PCR Conditions
4.7. qPCR Conditions
4.8. Specificity Tests
4.9. Sensitivity Tests
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
- Matsuoka, T.; Kuribara, H.; Takubo, K.; Akiyama, H.; Miura, H.; Goda, Y.; Kusakabe, Y.; Isshiki, K.; Toyoda, M.; Hino, A. Detection of recombinant DNA segments introduced to genetically modified maize (Zea mays). J. Agric. Food Chem. 2002, 50, 2100–2109. [Google Scholar] [PubMed]
- Dörries, H.H.; Remus, I.; Grönewald, A.; Grönewald, C.; Berghöf-Jager, K. Development of a qualitative, multiplex real-time PCR kit for screening of genetically modified organisms (GMOs). Anal. Bioanal. Chem. 2010, 396, 2043–2054. [Google Scholar] [PubMed]
- Randhawa, G.J.; Chhabra, R.; Singh, M. Multiplex PCR-Based Simultaneous Amplification of Selectable Marker and Reporter Genes for the Screening of Genetically Modified Crops. J. Agric. Food Chem. 2009, 57, 5167–5172. [Google Scholar] [PubMed]
- Wolf, C.; Scherzinger, M.; Wurz, A.; Pauli, U.; Hübner, P.; Lüthy, J. Detection of cauliflower mosaic virus by the polymerase chain reaction: Testing of food components for false-positive 35S-promoter screening results. Eur. Food Res. Technol. 2000, 210, 367–372. [Google Scholar]
- Cankar, K.; Ravnikar, M.; Zel, J.; Gruden, K.; Toplak, N. Real time polymerase chain reaction detection of cauliflower mosaic virus to complement the 35S screening assay for genetically modified organisms. J. AOAC Int. 2005, 88, 814–822. [Google Scholar]
- Chaouachi, M.; Fortabat, M.N.; Kebdani, N.; Audeon, C.; Romaniuk, M.; Bertheau, Y. An accurate real-time PCR test for the detection and quantification of cauliflower mosaıc virus (CaMV): Applicable in GMO screening. Eur. Food Res. Technol. 2008, 227, 789–798. [Google Scholar]
- Becker, R.; Ulrich, A. Improved detection and quantification of cauliflower mosaic virus in food crops: Assessing false positives in GMO screening based on the 35S promoter. Eur. Food Res. Technol. 2018, 244, 1861–1871. [Google Scholar]
- Bak, A.; Emerson, J.B. Multiplex quantitative PCR for single reaction genetically modified (GM) plant detection and identification of false positive GM plants linked to Cauliflower mosaic virus (CaMV) infection. BMC Biotechnol. 2019, 19, 73. [Google Scholar]
- Moor, D.; Liniger, M.; Grohmann, L.; Felleisen, R. Real-time PCR method for the detection of figwort mosaic virus (FMV) to complement the FMV 34S promoter-specific PCR assay used for screening of genetically modified plants. Eur. Food Res. Technol. 2012, 235, 835–842. [Google Scholar]
- Sundar, I.K.; Sakthive, N. Advances in selectable marker genes for plant transformation. J. Plant Physiol. 2008, 165, 1698–1716. [Google Scholar]
- Wakasa, Y.; Ozawa, K.; Takaiwa, F. Higher-level accumulation of foreign gene products in transgenic rice seeds by the callus-specific selection system. J. Biosci. Bioeng. 2009, 107, 78–83. [Google Scholar] [PubMed]
- Van den Elzen, P.J.; Townsend, J.; Lee, K.Y.; Bedbrook, J.R. A chimaeric hygromycin resistance gene as a selectable marker in plant cells. Plant Mol. Biol. 1985, 5, 299–302. [Google Scholar] [CrossRef]
- Ortiz, J.P.A.; Reggiardo, M.I.; Ravizzini, R.A.; Altabe, S.G.; Cervigni, G.D.L.; Spitteler, M.A.; Morata, M.M.; Elias, F.E.; Vallejos, R.H. Hygromycin resistance as an efficient selectable marker for wheat stable transformation. Plant Cell Rep. 1996, 15, 877–881. [Google Scholar] [PubMed]
- Yadav, D.; Shavrukov, Y.; Bazanova, N.; Chirkova, L.; Borisjuk, N.; Kovalchuk, N.; Ismagul, A.; Parent, B.; Langridge, P.; Hrmova, M.; et al. Constitutive overexpression of the TaNF-YB4 gene in transgenic wheat significantly improves grain yield. J. Exp. Bot. 2015, 66, 6635–6650. [Google Scholar] [CrossRef] [PubMed]
- Bartlett, J.G.; Alves, S.C.; Smedley, M.; Snape, J.W.; Harwood, W.A. High-throughput Agrobacterium-mediated barley transformation. Plant Methods 2008, 4, 22. [Google Scholar] [CrossRef]
- Lu, C.; Song, G.; Wu, G.; Wu, Y.; Cao, Y.; Li, J.; Luo, J. Detection of Genetically Modified Plants and Derived Products—Qualitative PCR Method for the Marker Genes. NPTII HPT and PMI. Announcement by the Ministry of Agriculture. No.1782-2-2012. 2012, pp. 1–9. Available online: http://www.bjstandard.com/standard/271346.html (accessed on 5 February 2013).
- Huang, X.; Gao, H.; Li, X.; Ling, X.; Zhu, S.; Chen, H.; Pan, L.; Cao, J.; Zhang, G. Detection of Genetically Modified Organisms and Derived Products—Qualitative Real-Time Polymerase Chain Reaction (PCR) Methods. GB/T 19495.4-2018. 2018. Available online: https://www.sdtdata.com/fx/foodcodex?p=tsLibCard&s=fcv1&act=doDownload (accessed on 17 September 2018).
- European Network of GMO Laboratories (ENGL). Definition of Minimum Performance Requirements for Analytical Methods of GMO Testing. 2015. Available online: http://gmo-crl.jrc.ec.europa.eu/guidancedocs.htm (accessed on 20 October 2015).
- Yue, Y.; Wu, G.; Wu, Y.; Lu, C. Development of qualitative PCR method targeting marker genes in transgenic plants. Chin. J. Oil Crop. Sci. 2011, 33, 280–289. [Google Scholar]
- Nabi, N.; Chaouachi, M.; Zellama, M.S.; Ben Hafsa, A.; Mrabet, B.; Saïd, K.; Fathia, H.S. A new QRT-PCR assay designed for the differentiation between elements provided from Agrobacterium sp. in GMOs plant events and natural Agrobacterium sp. bacteria. Food Chem. 2016, 196, 58–65. [Google Scholar]
- Holst-Jensen, A.; Rønning, S.B.; Løvseth, A.; Berdal, K.G. PCR technology for screening and quantification of genetically modified organisms (GMOs). Anal. Bioanal. Chem. 2003, 375, 985–993. [Google Scholar]
- Li, J.; Wu, Y.; Li, X.; Wang, Y.; Zhang, L.; Li, Y.; Wu, G. Developing a matrix reference material for screening of transgenic rice. Anal. Bioanal. Chem. 2015, 407, 9153–9163. [Google Scholar]
- Porebski, S.; Bailey, L.G.; Baum, B.R. Modification of a CTAB DNA extraction protocol for plants containing high polysaccharide and polyphenol components. Plant Mol. Biol. Rep. 1997, 15, 8–15. [Google Scholar] [CrossRef]
- Stephen, F.A.; Warren, G.; Webb, M.; Eugene, W.M.; David, J.L. Basic local alignment search tool. J. Mol. Biol. 1990, 215, 403–410. [Google Scholar]
Target Species Plant | 18S rRNA | Qualitative PCR | qPCR | ||
---|---|---|---|---|---|
Ghpt-F3/R3 | HPT Gene | Ghpt-QF3/QR3/QP | HPT Gene | ||
Arabidopsis thaliana | + | − | − | − | − |
Soybean (Glycine max) | + | − | − | − | − |
Maize (Zea mays) | + | − | − | − | − |
Peanuts (Arachis hypogaea) | + | − | − | − | − |
Rice (Oryza sativa) | + | − | − | − | − |
Rapeseed (Brassica napus) | + | − | − | − | − |
Cotton (Gossypium hirsutum) | + | − | − | − | − |
Wheat (Triticum aestivum) | + | − | − | − | − |
Sugar beet (Beta vulgaris) | + | − | − | − | − |
Tomato (Lycopersicon esculentum Mill) | + | − | − | − | − |
GM soybean mixed sample | + | − | − | − | − |
GM maize mixed sample | + | − | − | − | − |
GM rapeseed mixed sample | + | − | − | − | − |
GM cotton mixed sample | + | − | − | − | − |
TT51-1 | + | − | − | − | − |
G6H1 | + | − | − | − | − |
Kefeng6 | + | − | + | − | + |
Kemingdao | + | − | + | − | + |
SD rice | + | − | + | − | + |
Repeat | Slope | Mean | SD | RSD (%) | R2 | Mean | SD | RSD (%) | Efficiency | Mean | SD | RSD (%) |
---|---|---|---|---|---|---|---|---|---|---|---|---|
1 | −3.327 | −3.341 | 0.024 | 0.717 | 0.999 | 0.999 | 0.001 | 0.058 | 99.70% | 99.17% | 0.009 | 0.932 |
2 | −3.328 | 0.999 | 99.70% | |||||||||
3 | −3.369 | 1.000 | 98.10% |
Copy Number | Repeat | Ct Value | Mean of Ct Values | SD | RSD (%) | Mean of All Ct Values | SDr | RSDr (%) | ||
---|---|---|---|---|---|---|---|---|---|---|
1 | 2 | 3 | ||||||||
500,000 | 1 | 21.28 | 21.59 | 21.40 | 21.42 | 0.16 | 0.73 | 21.41 | 0.03 | 0.15 |
2 | 21.34 | 21.52 | 21.42 | 21.43 | 0.09 | 0.42 | ||||
3 | 21.30 | 21.44 | 21.37 | 21.37 | 0.07 | 0.33 | ||||
50,000 | 1 | 24.58 | 24.82 | 24.77 | 24.72 | 0.13 | 0.51 | 24.73 | 0.01 | 0.03 |
2 | 24.68 | 24.80 | 24.73 | 24.74 | 0.06 | 0.24 | ||||
3 | 24.66 | 24.81 | 24.73 | 24.73 | 0.08 | 0.30 | ||||
5000 | 1 | 27.95 | 28.20 | 28.14 | 28.10 | 0.13 | 0.46 | 28.06 | 0.03 | 0.11 |
2 | 27.94 | 28.13 | 28.04 | 28.04 | 0.10 | 0.34 | ||||
3 | 27.94 | 28.17 | 28.06 | 28.06 | 0.12 | 0.41 | ||||
500 | 1 | 31.24 | 31.64 | 31.58 | 31.49 | 0.22 | 0.69 | 31.42 | 0.06 | 0.18 |
2 | 31.37 | 31.52 | 31.31 | 31.40 | 0.11 | 0.34 | ||||
3 | 31.35 | 31.52 | 31.26 | 31.38 | 0.13 | 0.42 | ||||
50 | 1 | 34.64 | 34.38 | 35.06 | 34.69 | 0.34 | 0.99 | 34.77 | 0.11 | 0.30 |
2 | 34.62 | 35.04 | 34.54 | 34.73 | 0.27 | 0.77 | ||||
3 | 35.03 | 34.82 | 34.83 | 34.89 | 0.12 | 0.34 |
Plasmid Copy Number | Repeat | Ct Value | Mean of Ct Values | SD | RSD (%) | Mean of All Ct Values | SDr | RSDr (%) | ||
---|---|---|---|---|---|---|---|---|---|---|
1 | 2 | 3 | ||||||||
50 | 1 | 34.79 | 34.45 | 34.32 | 34.52 | 0.24 | 0.70 | 34.59 | 0.06 | 0.17 |
2 | 34.87 | 34.68 | 34.32 | 34.62 | 0.28 | 0.81 | ||||
3 | 34.46 | 35.14 | 34.25 | 34.62 | 0.47 | 1.34 | ||||
20 | 1 | 35.75 | 35.62 | 35.32 | 35.56 | 0.22 | 0.62 | 36.18 | 0.55 | 1.51 |
2 | 36.81 | 36.11 | 36.92 | 36.61 | 0.44 | 1.20 | ||||
3 | 36.05 | 36.25 | 36.76 | 36.35 | 0.37 | 1.01 | ||||
10 | 1 | 37.09 | 37.07 | 36.65 | 36.94 | 0.25 | 0.67 | 36.75 | 0.34 | 0.94 |
2 | 35.76 | 36.97 | 36.34 | 36.36 | 0.61 | 1.66 | ||||
3 | 38.77 | 36.34 | 35.79 | 36.97 | 1.59 | 4.29 | ||||
5 | 1 | 37.32 | 37.32 | 40.58 | 38.41 | 1.88 | 4.90 | 38.36 | 0.23 | 0.60 |
2 | 38.1 | 38.57 | 37.68 | 38.12 | 0.45 | 1.17 | ||||
3 | 37.83 | 40.41 | 37.47 | 38.57 | 1.60 | 4.16 | ||||
1 | 1 | 38.34 | 38.46 | 40.31 | 39.04 | 1.10 | 2.83 | 39.06 | / | / |
2 | NA | 38.71 | NA | 38.71 | / | / | ||||
3 | NA | 39.44 | NA | 39.44 | / | / |
Samples | Ct Values of the HPT Gene | Ct Values of Microorganisms Assay | ||||
---|---|---|---|---|---|---|
1 | 2 | 3 | 1 | 2 | 3 | |
no template control | NA | NA | NA | NA | NA | NA |
non-GM rice | NA | NA | NA | NA | NA | NA |
plasmid of pFP-hpt | NA | NA | NA | 26.83 | 26.77 | 27.03 |
Kemingdao | 23.88 | 23.74 | 23.86 | NA | NA | NA |
Routine testing rice 2519 | 28.62 | 28.54 | 28.61 | 26.91 | 26.91 | 27.03 |
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. |
© 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
Share and Cite
Li, Y.; Xiao, F.; Zhai, C.; Li, X.; Wu, Y.; Gao, H.; Li, J.; Zhai, S.; Liu, B.; Wu, G. Qualitative and Quantitative Real-Time PCR Methods for Assessing False-Positive Rates in Genetically Modified Organisms Based on the Microbial-Infection-Linked HPT Gene. Int. J. Mol. Sci. 2022, 23, 10000. https://doi.org/10.3390/ijms231710000
Li Y, Xiao F, Zhai C, Li X, Wu Y, Gao H, Li J, Zhai S, Liu B, Wu G. Qualitative and Quantitative Real-Time PCR Methods for Assessing False-Positive Rates in Genetically Modified Organisms Based on the Microbial-Infection-Linked HPT Gene. International Journal of Molecular Sciences. 2022; 23(17):10000. https://doi.org/10.3390/ijms231710000
Chicago/Turabian StyleLi, Yunjing, Fang Xiao, Chao Zhai, Xiaofei Li, Yuhua Wu, Hongfei Gao, Jun Li, Shanshan Zhai, Biao Liu, and Gang Wu. 2022. "Qualitative and Quantitative Real-Time PCR Methods for Assessing False-Positive Rates in Genetically Modified Organisms Based on the Microbial-Infection-Linked HPT Gene" International Journal of Molecular Sciences 23, no. 17: 10000. https://doi.org/10.3390/ijms231710000