Abstract
As common toxigenic fungi genera, Aspergillus, Penicillium, and Fusarium, have attracted the attention of governments and people all over the world due to their human carcinogenicity, teratogenicity, and hepatotoxicity. Accordingly, effective and quantitative detection method for toxigenic fungi before mycotoxins produced should be established to ensure food safety. In this paper, two duplex-droplet digital PCR (ddPCR) were developed and optimized for toxigenic fungi. The detection limits of the target genes, including the AflR, Och, Pen, and Fus of toxigenic fungi, were 26 copies/reaction, 15 copies/reaction, 161 copies/reaction, and 29 copies/reaction, respectively. Notably, the detection limit of duplex ddPCR was three orders of magnitude higher than that of tradition real-time fluorescence PCR reaction. Moreover the efficiency and sensitivity of the established method were also higher than those of real-time fluorescence PCR. The linear quantitative range of copy number of AflR and Och genes in AflR/Och duplex system and Pen and Fus genes in Pen/Fus duplex system were both 2 × 10−7–2 × 10−3 ng/μL. From DNA extraction to target gene detection, time consume of 96 samples was within 6 h, achieving the purpose of high-throughput and rapid detection.
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Data Availability
The datasets supporting the conclusions of this article are included within the article and its additional files.
References
Agriopoulou S, Stamatelopoulou E, and Varzakas T (2020) Advances in occurrence, importance, and mycotoxin control strategies: prevention and detoxification in foods. Foods 9(2) 137 https://www.mdpi.com/2304-8158/9/2/137
Alshannaq A, and Yu JH (2017) Occurrence, toxicity, and analysis of major mycotoxins in food. Int J Environ Res Public Health 14(6) 632 https://www.mdpi.com/1660-4601/14/6/632
Chen J-Q, Healey S, Regan P, Laksanalamai P, Hu Z (2017) PCR-based methodologies for detection and characterization of Listeria monocytogenes and Listeria ivanovii in foods and environmental sources. Food Sci Human Wellness 6(2):39–59. https://doi.org/10.1016/j.fshw.2017.03.001
Commission CA (2010) CAC/GL 74-2010. Guidelines on performance criteria and validation of methods for detection, identification and quantification of specific DNA sequences and specific proteins in foods. Rome: Codex Alimentarius Commission
Eskola M, Kos G, Elliott CT, Hajšlová J, and Mayar S (2019) Worldwide contamination of food-crops with mycotoxins: validity of the widely cited 'FAO estimate' of 25%. Critical Rev Food Sci Nutr 16(60):2773–2789. https://doi.org/10.1080/10408398.2019.1658570
Gallo A, Giuberti G, Frisvad JC, Bertuzzi T, Nielsen KF (2015) Review on mycotoxin issues in ruminants: occurrence in forages, effects of mycotoxin ingestion on health status and animal performance and practical strategies to counteract their negative effects. Toxins 7:3057–3111. https://doi.org/10.3390/toxins7083057
Gil-Serna J, Vázquez C, Sardiñas N, González-Jaén MT, Patiño B (2009) Discrimination of the main ochratoxin A-producing species in Aspergillus section Circumdati by specific PCR assays. Int J Food Microbiol 136(1):83–87. https://doi.org/10.1016/j.ijfoodmicro.2009.09.018
Haas D, Pfeifer B, Reiterich C, Partenheimer R, Reck B, Buzina W (2013) Identification and quantification of fungi and mycotoxins from Pu-erh tea. Int J Food Microbiol 166(2):316–322. https://doi.org/10.1016/j.ijfoodmicro.2013.07.024
Hassan YI, and Zhou T (2018) Promising detoxification strategies to mitigate mycotoxins in food and feed. Toxins, 10(3), 116 https://www.mdpi.com/2072-6651/10/3/116
Hayat A, Paniel N, Rhouati A, Marty J-L, Barthelmebs L (2012) Recent advances in ochratoxin A-producing fungi detection based on PCR methods and ochratoxin A analysis in food matrices. Food Control 26(2):401–415. https://doi.org/10.1016/j.foodcont.2012.01.060
He L, Simpson DJ, Gänzle MG (2020) Detection of enterohaemorrhagic Escherichia coli in food by droplet digital PCR to detect simultaneous virulence factors in a single genome. Food Microbiol 90:103466. https://doi.org/10.1016/j.fm.2020.103466
von Hertwig AM, Sant'Ana AS, Sartori D, da Silva JJ, Nascimento MS, Iamanaka BT, . . . Taniwaki MH (2018) Real-time PCR-based method for rapid detection of Aspergillus niger and Aspergillus welwitschiae isolated from coffee. J Microbiol Methods 148 87-92 https://doi.org/10.1016/j.mimet.2018.03.010
Hindson BJ, Ness KD, Masquelier DA, Belgrader P, Heredia NJ, Makarewicz AJ, . . . Legler TC (2011) High-throughput droplet digital PCR system for absolute quantitation of DNA copy number. Anal chem 83 (22), 8604-8610. https://doi.org/10.1021/ac202028g
Lei S, Gu X, Zhong Q, Duan L, Zhou A (2020) Absolute quantification of Vibrio parahaemolyticus by multiplex droplet digital PCR for simultaneous detection of tlh, tdh and ureR based on single intact cell. Food Control 114:107207. https://doi.org/10.1016/j.foodcont.2020.107207
Li H, Fu T, Yun Z, Lv T, Yuan L, Xu B (2017) Effect comparison of five methods to extract fungal genomic DNA as PCR templates. Chin Agric Sci Bull. https://doi.org/10.1016/j.fct.2013.07.047
Marin S, Ramos AJ, Cano-Sancho G, Sanchis V (2013) Mycotoxins: occurrence, toxicology, and exposure assessment. Food Chem Toxicol 60:218–237. https://doi.org/10.1016/j.fct.2013.07.047
Mitchell NJ, Bowers E, Hurburgh C, Wu F (2016) Potential economic losses to the US corn industry from aflatoxin contamination. Food Addit Contam Part A Chem Anal Control Expo Risk Assess 33(3):540–550. https://doi.org/10.1080/19440049.2016.1138545
Niessen L (2007) PCR-based diagnosis and quantification of mycotoxin producing fungi. Int J Food Microbiol 119(1):38–46. https://doi.org/10.1016/j.ijfoodmicro.2007.07.023
Patiño B, González-Salgado A, González-Jaén MT, Vázquez C (2005) PCR detection assays for the ochratoxin-producing Aspergillus carbonarius and Aspergillus ochraceus species. Int J Food Microbiol 104(2):207–214. https://doi.org/10.1016/j.ijfoodmicro.2005.02.011
Pereira VL, Fernandes JO, Cunha SC (2014) Mycotoxins in cereals and related foodstuffs: a review on occurrence and recent methods of analysis. Trends Food Sci Technol 36(2):96–136. https://doi.org/10.1016/j.tifs.2014.01.005
Raguseo C, Gerin D, Pollastro S, Rotolo C, Rotondo PR, Faretra F, De MiccolisAngelini RM (2021) A duplex-droplet digital PCR assay for simultaneous quantitative detection of Monilinia fructicola and Monilinia laxa on Stone Fruits. Front Microbiol 12:747560–747560. https://doi.org/10.3389/fmicb.2021.747560
Ren J, Deng T, Huang W, Chen Y, Ge Y (2017) A digital PCR method for identifying and quantifying adulteration of meat species in raw and processed food. PLoS ONE 12(3):e0173567–e0173567. https://doi.org/10.1371/journal.pone.0173567
Rodrigues P, Venâncio A, Lima N (2012) Mycobiota and mycotoxins of almonds and chestnuts with special reference to aflatoxins. Food Res Int 48(1):76–90. https://doi.org/10.1016/j.foodres.2012.02.007
Rodríguez A, Rodríguez M, Andrade MJ, Córdoba JJ (2012) Development of a multiplex real-time PCR to quantify aflatoxin, ochratoxin A and patulin producing molds in foods. Int J Food Microbiol 155(1):10–18. https://doi.org/10.1016/j.ijfoodmicro.2012.01.007
Santos Pereira C, Cunha SC, Fernandes JO (2019) Prevalent mycotoxins in animal feed: occurrence and analytical methods. Toxins 11(5):290. https://doi.org/10.3390/toxins11050290
Sardiñas N, Gil-Serna J, Santos L, Ramos AJ, González-Jaén MT, Patiño B, Vázquez C (2011) Detection of potentially mycotoxigenic Aspergillus species in Capsicum powder by a highly sensitive PCR-based detection method. Food Control 22(8):1363–1366. https://doi.org/10.1016/j.foodcont.2011.02.013
Sim JH, Tian F, Jung SY, Auh J-H, Chun HS (2018) Multiplex polymerase chain reaction assays for the detection of the zearalenone chemotype of Fusarium species in white and brown rice. Int J Food Microbiol 269:120–127. https://doi.org/10.1016/j.ijfoodmicro.2018.02.003
Stakheev AA, Ryazantsev DY, Gagkaeva TY, Zavriev SK (2011) PCR detection of Fusarium fungi with similar profiles of the produced mycotoxins. Food Control 22(3):462–468. https://doi.org/10.1016/j.foodcont.2010.09.028
Sudharsan S, Malka B, Varda Z, Moshe K, Anatoly T, Elazar Q, Edward S (2017) Rapid detection and identification of mycotoxigenic fungi and mycotoxins in stored wheat grain. Toxins 9(10):302. https://doi.org/10.3390/toxins9100302
Suman M (2021) Last decade studies on mycotoxins’ fate during food processing: an overview. Curr Opin Food Sci 41:70–80. https://doi.org/10.1016/j.cofs.2021.02.015
Ülger TG, Uçar A, Çakıroğlu FP, Yilmaz S (2020) Genotoxic effects of mycotoxins. Toxicon 185:104–113. https://doi.org/10.1016/j.toxicon.2020.07.004
Varzakas T (2016) Quality and safety aspects of cereals (wheat) and their products. Crit Rev Food Sci Nutr 56(15):2495–2510. https://doi.org/10.1080/10408398.2013.866070
Welke JE (2019) Fungal and mycotoxin problems in grape juice and wine industries. Curr Opin Food Sci 29:7–13. https://doi.org/10.1016/j.cofs.2019.06.009
Yu N, Ren J, Huang W, Xing R, Chen Y (2021) An effective analytical droplet digital PCR approach for identification and quantification of fur-bearing animal meat in raw and processed food. Food Chem 10:129525. https://doi.org/10.1016/j.foodchem.2021.129525
Yu N, Han J, Deng T, Chen L, Zhang J, Xing R, . . . Chen Y (2020) A novel analytical droplet digital PCR method for identification and quantification of raw health food material powder from Panax notoginseng. Food Analytical Methods 1-9 https://doi.org/10.1007/s12161-020-01887-0
Zain ME (2011) Impact of mycotoxins on humans and animals. J Saudi Chem Soc 15(2):129–144. https://doi.org/10.1016/j.jscs.2010.06.006
Zhang Q, Zhao X, Chen Y, Liu B, Wang P (2020) [Establishment of rapid detection method for multiple real-time fluorescent PCR of toxin-producing fungi]. Wei Sheng Yan Jiu = J Hyg Res 49(6):881–888. https://doi.org/10.19813/j.cnki.weishengyanjiu.2020.06.002
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This work was supported by the National Key Research and Development Program of China (Grant Number 2017YFC1601200).
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Ping Wang: formal analysis, writing—review and editing, investigation, and validation. Tianming Qu: methodology, formal analysis, writing—original draft. Xiaomei Zhao: data curation, methodology, supervision. Yiqiang Ge: methodology and formal analysis. Ying Chen: conceptualization, writing—review and editing, supervision, project administration, and funding acquisition.
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Ping Wang declares that she has no conflict of interest. Tianming Qu declares that he has no conflict of interest. Xiaomei Zhao declares that she has no conflict of interest. Yiqiang Ge declares that he has no conflict of interest. Ying Chen declares that she has no conflict of interest.
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Wang, P., Qu, T., Zhao, X. et al. An Efficient Droplet Digital PCR Approach for Detection DNA at Low Concentrations of Toxigenic Fungi in Food Products. Food Anal. Methods 15, 1695–1706 (2022). https://doi.org/10.1007/s12161-022-02236-z
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DOI: https://doi.org/10.1007/s12161-022-02236-z