Rapid detection of wheat components in starch products using the Proofman-LMTIA method
Main Article Content
Keywords
wheat starch; Proofman probe; ladder-shape melting temperature isothermal amplification
Abstract
For rapid detection of wheat components in starch products, this study developed the analysis method based on the ladder-shape melting temperature isothermal amplification (LMTIA) coupled with proofreading enzyme-mediated probe cleavage (Proofman). With the internal transcribed spacer (ITS) gene of Triticum aestivum L. (wheat) as the target, specific primers and the Proofman probe were designed, the reaction temperature was opti-mized, and the sensitivity and the specificity of the Proofman-LMTIA method were determined. With the results indicating that the optimal reaction temperature of this Proofman-LMTIA method was 65°C, it can specifically detect wheat components rather than seven other species; the sensitivity was 10 fg (femtograms) of the genomic deoxyribonucleic acids (gDNAs) of Triticum aestivum L. and the detection limit was 0.1% of the artificially adul-terated wheat starch. The Proofman-LMTIA reaction could be completed in 20 minutes, and 1 of 12 commercial starch products was found to have adulterated wheat starch. In conclusion, the established Proofman-LMTIA method was suitable, or rapid and accurate, for the detection of wheat components in starch products.
References
Ayvaz H, Korkmaz F, Polat H, Ayvaz Z, Tuncel NB. Detection of einkorn flour adulteration in flour and bread samples using computer-based image analysis and near-infrared spectroscopy. Food Control 2021;127:108162. https://doi.org/10.1016/j.foodcont.2021.108162
Ankara U, Boyaci I, Yazar S, Koksel H. Rapid detection of common wheat flour addition to durum wheat flour and pasta using spectroscopic methods and chemometrics. J Cereal Sci 2023;109:103604. https://doi.org/10.1016/j.jcs.2022.103604
Büren V, Stadler M, Lüthy J. Detection of wheat adulteration of spelt flour and products by PCR. Eur Food Res Technol 2001;212:234–9. https://doi.org/10.1007/s002170000230
Caterina M, Bergami R, Scaramagli S, Delogu C, Andreani L, Carnevali P, et al. A digital PCR assay to quantify the percentages of hulled vs. hulless wheat in flours and flour-based products. Biology 2021;10:1138. https://doi.org/10.3390/biology10111138
Carloni E, Amagliani G, Omiccioli E, Ceppetelli V, Mastro MD, Rotundo L, et al. Validation and application of a quantitative real-time PCR assay to detect common wheat adulteration of durum wheat for pasta production. Food Chem 2017;224:86–91. https://doi.org/10.1016/j.foodchem.2016.12.053
Chen J, Zhang Y, Chen C, Zhang Y, Zhou W, Sang Y. Identification and quantification of cassava starch adulteration in different food starches by droplet digital PCR. PLoS ONE 2020;15:e0228624. https://doi.org/10.1371/journal.pone.0228624
Cai C, Hou X, Huang B, He J, Wu X, He Q. LAMP assay coupled CRISPR/LbCas12a system in a single-tube method for visual detection of meat adulteration. Food Control 2024;167:110809. https://doi.org/10.1016/j.foodcont.2024.110809
Cui P, Hu Z, Guo M, Wang Y, Xu D, Yao W, et al. Rapid and duplex detection of sweetpotato and maize components in starch products using Proofman-LMTIA method. Microchem J 2024;204:111082. https://doi.org/10.1016/j.microc.2024.111082
Caballero-Agosto ER, Sierra-Vega NO, Rolon-Ocasio Y, Samuel P, Infante-Castillo R, Fontalvo-Gomez M, et al. Detection and quantification of cornstarch and wheat flour as adulterants in milk powder by near- and mid-infrared spectroscopy coupled with chemometric routines. Food Chem Adv 2024;4:100582. https://doi.org/10.1016/j.focha.2023.100582
Cardoso VGK, Poppi RJ. Cleaner and faster method to detect adulteration in cassava starch using Raman spectroscopy and one-class support vector machine. Food Control 2021;125:107917. https://doi.org/10.1016/j.foodcont.2021.107917
Ding S, Chen GY, Wei YH, Dong J, Du F, Cui X, et al. Sequence-specific and multiplex detection of COVID-19 virus (SARS-CoV-2) using proofreading enzyme-mediated probe cleavage coupled with isothermal amplification. Biosens Bioelectron 2021;178:113041. https://doi.org/10.1016/j.bios.2021.113041
He H, Chen Y, Li G, Wang Y, Ou X, Guo J. Hyperspectral imaging combined with chemometrics for rapid detection of talcum powder adulterated in wheat flour. Food Control 2023;144, 109378. https://doi.org/10.1016/j.foodcont.2022.109378
Girolamo AD, Arroyo MC, Cervellieri S, Cortese M, Lippolis V. Detection of durum wheat pasta adulteration with common wheat by infrared spectroscopy and chemometrics: A case study. Lebenson Wiss Technol – Food Sci Technol 2020;127:109368. https://doi.org/10.1016/j.lwt.2020.109368
Grote U, Fasse A, Nguyen T, Erenstein O. Food security and the dynamics of wheat and maize value chains in Africa and Asia. Front Sustain Food Syst 2021;4:617009. https://doi.org/10.3389/fsufs.2020.617009
Glökler J, Lim TS, Ida J, Frohme M. Isothermal amplifications – A comprehensive review on current methods. Crit Rev BiochemMol Biol 2021;56:543–86. https://doi.org/10.1080/10409238.2021.1937927
Gu M, Xiao F, Wang B, Zhang Y, Ding C, Zhang G, et al. Study on detection of soybean components in edible oil with ladder-shape melting temperature isothermal amplification (LMTIA) assay. Anal Methods 2023;15:581–6. https://doi.org/10.1039/D2AY01719A
Guo X, Zhang P, Yue Y. Global wheat planting suitability under the 1.5°C and 2°C warming targets. Front Plant Sci 2024;15:1410388. https://doi.org/10.3389/fpls.2024.1410388
Hazard B, Trafford K, Lovegrove A, Uauy C, Shewry P. Strategies to improve wheat for human health. Nat Food 2020;1:475–80. https://doi.org/10.1038/s43016-020-0134-6
Kaldeli A, Zakidou P, Paraskevopoulou A. Volatilomics as a tool to ascertain food adulteration, authenticity, and origin. Compr Rev Food SciFood Saf 2024;23:13387. https://doi.org/10.1111/1541-4337.13387
Liu H, Wadood S, Xia Y, Liu Y, Gan RY. Wheat authentication: An overview on different techniques andchemometric methods. Crit Rev Food Sci Nutr 2023;63:33–56. https://doi.org/10.1080/10408398.2021.1942783
Luo X, Yang Y, LinX, Xiao J. Deciphering spike architecture formation towards yield improvement in wheat. J Genet Genom 2023;50:835–45. https://doi.org/10.1016/j.jgg.2023.02.015
Liu J, Wang Y, Li T，Huang K, Song C, Cui P, et al. Dual detection of Bupleurum scorzonerifolium Willd and Bupleurum chinense DC using proofman-LMTIA method. ChemBiol Technol Agric 2024;11:104. https://doi.org/10.1186/s40538-024-00637-2
Murakami T, Sumaoka J, Komiyama M. Sensitive isothermal detection of nucleic acid sequence by primer generation-rolling circle amplification. Nucleic Acids Res 2009;37:e19. https://doi.org/10.1093/nar/gkn1014
Marquez C, Lopez M, Ruisánchez I, Callao P. FTRaman and NIR spectroscopy data fusion strategy for multivariate qualitative analysis of food fraud. Talanta 2016;16180–6. https://doi.org/10.1016/j.talanta.2016.08.003
Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, et al. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res 2000;28:e63. https://doi.org/10.1093/nar/28.12.e63
Ndlovu PF, Magwaza LS, Tesfay SZ, Mphahlele RR. Vis-NIR spectroscopic and chemometric models for detecting contamination of premium green banana flour with wheat by quantifying resistant starch content. J Food Composit Anal 2021;102:104035. https://doi.org/10.1016/j.jfca.2021.104035
Pastor K, Acanski M, Vujic D, Kojic P. A rapid dicrimination of wheat, walnut and hazelnut flour samples using chemometric algorithms on GC/MS data. J Food Meas Charact 2019;13:2961–9. https://doi.org/10.1007/s11694-019-00216-2
Peter K, Richard B, Simon J. Wheat area expansion into northern higher latitudes and global food security. Agric Ecosyst Environ 2023;351:108499. https://doi.org/10.1016/j.agee.2023.108499
Schulze C, Geuthner AC, Mäde D. Development and validation of a method for quantification of common wheat, durum wheat, rye and barley by droplet digital PCR. Eur Food Res Technol 2021;247:2267–83. https://doi.org/10.1007/s00217-021-03786-y
Wang D G, Wang Y, Zhang M, Zhang Y, Sun J, Song C, et al. Ladder- shape melting temperature isothermal amplification of nucleic acids. Biotechniques 2021;71:358–69. https://doi.org/10.2144/btn-2020-0173
Wang Y, Wang BR, Wang DG. Detection of chicken adulteration in beef via ladder-shape melting temperature isothermal amplification (LMTIA) assay. Biotechnol Biotechnol Equip 2022;36:339345. https://doi.org/10.1080/13102818.2022.2081514
Wang YZ, Wang B, Wang D. Detection of pork adulteration in beef with ladder- shape melting temperature isothermal amplification (LMTIA) assay. CyTA – J Food 2022;20:244–50. https://doi.org/10.1080/19476337.2022.2129791
Wang YZ, Wang BR, Xu DD, Zhang M, Zhang XH, Wang DG. Development of a ladder-shape melting temperature isothermal amplification (LMTIA) assay for detection of African swine fever virus (ASFV). J Vet Sci 2022;23:e51. https://doi.org/10.4142/jvs.22001
Xu G, Hu L, Zhong H, Wang H, Yusa SI, WeissTC, et al. Cross priming amplification: Mechanism and optimization for isothermal DNA amplification. Sci Rep 2012;2:246. https://doi.org/10.1038/srep00246
Yao W, Xu D, Zhou D, Liu J, Zhang X, Wang Y, et al. Detection of the thaumatin-like protein gene from Brassica rapa var. oleifera in honey with the Proofman-LMTIA method. CyTA – J Food 2024;22. https://doi.org/10.1080/19476337.2024.2351912
Zhou J, Chen X, JIN M. Adulteration identification of wheat flour in chestnut flour based on differences in mycotoxin contamination by liquid chromatography-tandem mass spectrometry. Chin J Chromatogr 2022;40:303–12. https://doi.org/10.3724/SP.J.1123.2021.10021
Zhang Y, Wang Y, Ouyang X, Wang D, Xiao F, Sun J. Development of a ladder-shape melting temperature isothermal amplification (LMTIA) assay for the identification of cassava component in sweet potato starch noodles. Molecules 2022;27:3414. https://doi.org/10.3390/molecules27113414
Zhang X, Li Z, Zhang Y, Xu D, Zhang L, Xiao F, et al. Rapid discrimination of Panax quinquefolium and Panax ginseng using the Proofman-Duplex-LMTIA technique. Molecules 2023;28:6872. https://doi.org/10.3390/molecules28196872
Ankara U, Boyaci I, Yazar S, Koksel H. Rapid detection of common wheat flour addition to durum wheat flour and pasta using spectroscopic methods and chemometrics. J Cereal Sci 2023;109:103604. https://doi.org/10.1016/j.jcs.2022.103604
Büren V, Stadler M, Lüthy J. Detection of wheat adulteration of spelt flour and products by PCR. Eur Food Res Technol 2001;212:234–9. https://doi.org/10.1007/s002170000230
Caterina M, Bergami R, Scaramagli S, Delogu C, Andreani L, Carnevali P, et al. A digital PCR assay to quantify the percentages of hulled vs. hulless wheat in flours and flour-based products. Biology 2021;10:1138. https://doi.org/10.3390/biology10111138
Carloni E, Amagliani G, Omiccioli E, Ceppetelli V, Mastro MD, Rotundo L, et al. Validation and application of a quantitative real-time PCR assay to detect common wheat adulteration of durum wheat for pasta production. Food Chem 2017;224:86–91. https://doi.org/10.1016/j.foodchem.2016.12.053
Chen J, Zhang Y, Chen C, Zhang Y, Zhou W, Sang Y. Identification and quantification of cassava starch adulteration in different food starches by droplet digital PCR. PLoS ONE 2020;15:e0228624. https://doi.org/10.1371/journal.pone.0228624
Cai C, Hou X, Huang B, He J, Wu X, He Q. LAMP assay coupled CRISPR/LbCas12a system in a single-tube method for visual detection of meat adulteration. Food Control 2024;167:110809. https://doi.org/10.1016/j.foodcont.2024.110809
Cui P, Hu Z, Guo M, Wang Y, Xu D, Yao W, et al. Rapid and duplex detection of sweetpotato and maize components in starch products using Proofman-LMTIA method. Microchem J 2024;204:111082. https://doi.org/10.1016/j.microc.2024.111082
Caballero-Agosto ER, Sierra-Vega NO, Rolon-Ocasio Y, Samuel P, Infante-Castillo R, Fontalvo-Gomez M, et al. Detection and quantification of cornstarch and wheat flour as adulterants in milk powder by near- and mid-infrared spectroscopy coupled with chemometric routines. Food Chem Adv 2024;4:100582. https://doi.org/10.1016/j.focha.2023.100582
Cardoso VGK, Poppi RJ. Cleaner and faster method to detect adulteration in cassava starch using Raman spectroscopy and one-class support vector machine. Food Control 2021;125:107917. https://doi.org/10.1016/j.foodcont.2021.107917
Ding S, Chen GY, Wei YH, Dong J, Du F, Cui X, et al. Sequence-specific and multiplex detection of COVID-19 virus (SARS-CoV-2) using proofreading enzyme-mediated probe cleavage coupled with isothermal amplification. Biosens Bioelectron 2021;178:113041. https://doi.org/10.1016/j.bios.2021.113041
He H, Chen Y, Li G, Wang Y, Ou X, Guo J. Hyperspectral imaging combined with chemometrics for rapid detection of talcum powder adulterated in wheat flour. Food Control 2023;144, 109378. https://doi.org/10.1016/j.foodcont.2022.109378
Girolamo AD, Arroyo MC, Cervellieri S, Cortese M, Lippolis V. Detection of durum wheat pasta adulteration with common wheat by infrared spectroscopy and chemometrics: A case study. Lebenson Wiss Technol – Food Sci Technol 2020;127:109368. https://doi.org/10.1016/j.lwt.2020.109368
Grote U, Fasse A, Nguyen T, Erenstein O. Food security and the dynamics of wheat and maize value chains in Africa and Asia. Front Sustain Food Syst 2021;4:617009. https://doi.org/10.3389/fsufs.2020.617009
Glökler J, Lim TS, Ida J, Frohme M. Isothermal amplifications – A comprehensive review on current methods. Crit Rev BiochemMol Biol 2021;56:543–86. https://doi.org/10.1080/10409238.2021.1937927
Gu M, Xiao F, Wang B, Zhang Y, Ding C, Zhang G, et al. Study on detection of soybean components in edible oil with ladder-shape melting temperature isothermal amplification (LMTIA) assay. Anal Methods 2023;15:581–6. https://doi.org/10.1039/D2AY01719A
Guo X, Zhang P, Yue Y. Global wheat planting suitability under the 1.5°C and 2°C warming targets. Front Plant Sci 2024;15:1410388. https://doi.org/10.3389/fpls.2024.1410388
Hazard B, Trafford K, Lovegrove A, Uauy C, Shewry P. Strategies to improve wheat for human health. Nat Food 2020;1:475–80. https://doi.org/10.1038/s43016-020-0134-6
Kaldeli A, Zakidou P, Paraskevopoulou A. Volatilomics as a tool to ascertain food adulteration, authenticity, and origin. Compr Rev Food SciFood Saf 2024;23:13387. https://doi.org/10.1111/1541-4337.13387
Liu H, Wadood S, Xia Y, Liu Y, Gan RY. Wheat authentication: An overview on different techniques andchemometric methods. Crit Rev Food Sci Nutr 2023;63:33–56. https://doi.org/10.1080/10408398.2021.1942783
Luo X, Yang Y, LinX, Xiao J. Deciphering spike architecture formation towards yield improvement in wheat. J Genet Genom 2023;50:835–45. https://doi.org/10.1016/j.jgg.2023.02.015
Liu J, Wang Y, Li T，Huang K, Song C, Cui P, et al. Dual detection of Bupleurum scorzonerifolium Willd and Bupleurum chinense DC using proofman-LMTIA method. ChemBiol Technol Agric 2024;11:104. https://doi.org/10.1186/s40538-024-00637-2
Murakami T, Sumaoka J, Komiyama M. Sensitive isothermal detection of nucleic acid sequence by primer generation-rolling circle amplification. Nucleic Acids Res 2009;37:e19. https://doi.org/10.1093/nar/gkn1014
Marquez C, Lopez M, Ruisánchez I, Callao P. FTRaman and NIR spectroscopy data fusion strategy for multivariate qualitative analysis of food fraud. Talanta 2016;16180–6. https://doi.org/10.1016/j.talanta.2016.08.003
Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, et al. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res 2000;28:e63. https://doi.org/10.1093/nar/28.12.e63
Ndlovu PF, Magwaza LS, Tesfay SZ, Mphahlele RR. Vis-NIR spectroscopic and chemometric models for detecting contamination of premium green banana flour with wheat by quantifying resistant starch content. J Food Composit Anal 2021;102:104035. https://doi.org/10.1016/j.jfca.2021.104035
Pastor K, Acanski M, Vujic D, Kojic P. A rapid dicrimination of wheat, walnut and hazelnut flour samples using chemometric algorithms on GC/MS data. J Food Meas Charact 2019;13:2961–9. https://doi.org/10.1007/s11694-019-00216-2
Peter K, Richard B, Simon J. Wheat area expansion into northern higher latitudes and global food security. Agric Ecosyst Environ 2023;351:108499. https://doi.org/10.1016/j.agee.2023.108499
Schulze C, Geuthner AC, Mäde D. Development and validation of a method for quantification of common wheat, durum wheat, rye and barley by droplet digital PCR. Eur Food Res Technol 2021;247:2267–83. https://doi.org/10.1007/s00217-021-03786-y
Wang D G, Wang Y, Zhang M, Zhang Y, Sun J, Song C, et al. Ladder- shape melting temperature isothermal amplification of nucleic acids. Biotechniques 2021;71:358–69. https://doi.org/10.2144/btn-2020-0173
Wang Y, Wang BR, Wang DG. Detection of chicken adulteration in beef via ladder-shape melting temperature isothermal amplification (LMTIA) assay. Biotechnol Biotechnol Equip 2022;36:339345. https://doi.org/10.1080/13102818.2022.2081514
Wang YZ, Wang B, Wang D. Detection of pork adulteration in beef with ladder- shape melting temperature isothermal amplification (LMTIA) assay. CyTA – J Food 2022;20:244–50. https://doi.org/10.1080/19476337.2022.2129791
Wang YZ, Wang BR, Xu DD, Zhang M, Zhang XH, Wang DG. Development of a ladder-shape melting temperature isothermal amplification (LMTIA) assay for detection of African swine fever virus (ASFV). J Vet Sci 2022;23:e51. https://doi.org/10.4142/jvs.22001
Xu G, Hu L, Zhong H, Wang H, Yusa SI, WeissTC, et al. Cross priming amplification: Mechanism and optimization for isothermal DNA amplification. Sci Rep 2012;2:246. https://doi.org/10.1038/srep00246
Yao W, Xu D, Zhou D, Liu J, Zhang X, Wang Y, et al. Detection of the thaumatin-like protein gene from Brassica rapa var. oleifera in honey with the Proofman-LMTIA method. CyTA – J Food 2024;22. https://doi.org/10.1080/19476337.2024.2351912
Zhou J, Chen X, JIN M. Adulteration identification of wheat flour in chestnut flour based on differences in mycotoxin contamination by liquid chromatography-tandem mass spectrometry. Chin J Chromatogr 2022;40:303–12. https://doi.org/10.3724/SP.J.1123.2021.10021
Zhang Y, Wang Y, Ouyang X, Wang D, Xiao F, Sun J. Development of a ladder-shape melting temperature isothermal amplification (LMTIA) assay for the identification of cassava component in sweet potato starch noodles. Molecules 2022;27:3414. https://doi.org/10.3390/molecules27113414
Zhang X, Li Z, Zhang Y, Xu D, Zhang L, Xiao F, et al. Rapid discrimination of Panax quinquefolium and Panax ginseng using the Proofman-Duplex-LMTIA technique. Molecules 2023;28:6872. https://doi.org/10.3390/molecules28196872
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Apr 11, 2025
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