Establishment of real-time fluorescence loop-mediated isothermal amplification method for rapid detection of aflatoxin-producing fungi

CHEN Chen; TAO Ze; WANG Ke; LI Qingqing; AN Hongyu; LU Zenghui; LI Tuoping; LI Suhong

doi:10.13995/j.cnki.11-1802/ts.039164

PDF(4208 KB)

Food and Fermentation Industries ›› 2025, Vol. 51 ›› Issue (4) : 335-344. DOI: 10.13995/j.cnki.11-1802/ts.039164

Establishment of real-time fluorescence loop-mediated isothermal amplification method for rapid detection of aflatoxin-producing fungi

Author information +

History +

Abstract

Aflatoxins are microbial toxins that are highly hepatic-inducing, teratogenic and carcinogenic.The LAMP primers were designed according to the aflatoxin-producing regulatory gene aflR, the gothramoxin transmethylase gene omt-1, and the variegated aspergilloin A dehydrogenase gene ver-1, respectively, and a real-time loop-mediated isothermal amplification (LAMP) system was established and optimized.Results showed that the detection limits of Aspergillus flavus DNA for primers designed for aflR, omt-1, and ver-1 genes were 1.0×10^-2, 1.0×10^-3, and 1.0×10^-4 ng/μL, respectively.The system reacted positively with Aspergillus flavus and Aspergillus parasitica, but negatively with other bacteria, with the help of a thermostatic fluorescence reading device or according to the color change of the developer, it can quickly determine whether the strains to be tested are aflatoxins within 60 minutes.

Key words

aflatoxin-producing fungi / real-time fluorescent loop-mediated isothermal amplification / rapid detection / aflR / omt-1 / ver-1

Cite this article

EndNote

Ris (Procite)

Bibtex

Download Citations

CHEN Chen, TAO Ze, WANG Ke, LI Qingqing, AN Hongyu, LU Zenghui, LI Tuoping, LI Suhong. Establishment of real-time fluorescence loop-mediated isothermal amplification method for rapid detection of aflatoxin-producing fungi[J]. Food and Fermentation Industries, 2025, 51(4): 335-344 https://doi.org/10.13995/j.cnki.11-1802/ts.039164

References

[1] YU J J, CHANG P K, EHRLICH K C, et al. Clustered pathway genes in aflatoxin biosynthesis[J]. Applied and Environmental Microbiology, 2004, 70(3):1253-1262.
[2] 高晶. 饲料源寄生曲霉和纯绿青霉菌落生长及产毒的调控研究[D].兰州:兰州大学, 2023.
GAO J. Studies on the growth and toxin production of Aspergillus parasitica and Penicillium viridis in feed[D].Lanzhou:Lanzhou University, 2023.
[3] WANG C, SUN L L, ZHAO Q. A simple aptamer molecular beacon assay for rapid detection of aflatoxin B1[J]. Chinese Chemical Letters, 2019, 30(5):1017-1020.
[4] HE S, HUANG Q T, ZHANG Y, et al. Magnetic beads-based multicolor colorimetric immunoassay for ultrasensitive detection of aflatoxin B1[J]. Chinese Chemical Letters, 2021, 32(4):1462-1465.
[5] 刘楚君, 张莉蕴, 吴聪明, 等. 四极杆-线形离子阱液相色谱质谱联用法快速检测玉米油中黄曲霉毒素[J]. 食品安全导刊, 2023(20):84-88.
LIU C J, ZHANG L Y, WU C M, et al. Determination of aflatoxin in corn oil by liquid chromatography-quadrupole-linear ion trap mass spectrometry[J]. China Food Safety Magazine, 2023(20):84-88.
[6] 邹邵华. 粮食中黄曲霉毒素的产生机制和检测方法探究[J]. 粮食加工, 2023, 48(5):148-151.
ZOU S H. Exploration on the production mechanism and detection methods of aflatoxins in grain[J]. Grain Processing, 2023, 48(5):148-151.
[7] LIU Q J, LI X, WU R Q, et al. Development of an on-spot and rapid recombinase polymerase amplification assay for Aspergillus flavus detection in grains[J]. Food Control, 2021, 125:107957.
[8] 唐颂, 李岩松, 尚翠玲, 等. 玉米赤霉烯酮间接竞争ELISA方法的建立[J]. 食品工业科技, 2022, 43(4): 300-304.
TANG S, LI Y S, SHANG C L, et al. Establishment of an indirect competitive ELISA for zearalenone[J]. Science and Technology of Food Industry, 2022, 43(4): 300-304.
[9] 段春宇. 酶联免疫吸附法和免疫亲和柱-高效液相色谱法在检测饲料中黄曲霉毒素B1含量中的比对研究[J]. 中国动物保健, 2022, 24(5):131-132, 104.
DUAN C Y. Comparative study of enzyme-linked immunosorbent assay and immunoaffinity column high-performance liquid chromatography for detecting aflatoxin B1 content in feed [J]. China Animal Health, 2022, 24(5):131-132, 104.
[10] 刘颖, 金永鹏, 罗荪琳, 等. 高效液相色谱同步检测饲料中黄曲霉毒素B₁、玉米赤霉烯酮及呕吐毒素的方法[J]. 动物营养学报, 2021, 33(8):4684-4693.
LIU Y, JIN Y P, LUO S L, et al. A method for simultaneous determination of aflatoxin B₁, Zearalenone and deoxynivalenol in feed by high performance liquid chromatography[J]. Chinese Journal of Animal Nutrition, 2021, 33(8):4684-4693.
[11] 高志存, 石露莎, 余舒宁, 等. 酶联免疫吸附法与高效液相色谱法检测玉米中黄曲霉毒素B₁的比较[J]. 云南畜牧兽医, 2023(6):10-12.
GAO Z C, SHI L S, YU S N, et al. Comparison of enzyme-linked immunosorbent assay and high performance liquid chromatography in detecting aflatoxin B₁ in corn[J]. Yunnan Journal of Animal Science and Veterinary Medicine, 2023(6):10-12.
[12] 张欣悦. 中药材中黄曲霉菌及其毒素检测新技术研究[D]. 北京: 北京中医药大学, 2017.
ZHANG X Y. Research on new technologies for detection of Aspergillus flavus and its toxins in Chinese medicinal materials[D]. Beijing: Beijing University of Chinese Medicine, 2017.
[13] WONG Y P, OTHMAN S, LAU Y L, et al. Loop-mediated isothermal amplification (LAMP): A versatile technique for detection of micro-organisms[J]. Journal of Applied Microbiology, 2018, 124(3):626-643.
[14] 张照茹. 水稻纹枯病菌及稻曲病菌LAMP检测体系的建立与应用[D].沈阳:沈阳农业大学,2020.
ZHANG Z R. Establishment and application of LAMP detection system for rice sheath blight pathogen and rice smut pathogen[D].Shenyang: Shenyang Agricultural University, 2020.
[15] 于佳. 作物土传病害病原真菌和卵菌的三种分子检测方法研究[D]. 南京: 南京农业大学, 2019.
YU J. Research on three molecular detection methods for pathogenic Fungi and Egg Fungi of soil borne crop diseases[D]. Nanjing: Nanjing Agricultural University, 2019.
[16] 杜文芳. 环介导等温扩增及生物分析新方法研究[D]. 长沙: 湖南大学, 2018.
DU W F. A new method for loop mediated isothermal amplification and bioanalysis[D]. Changsha: Hunan University, 2018.
[17] 周思妍. 以LAMP-CRISPR/Cas12a为基础的儿童呼吸道病原检测方法的建立与应用[D]. 长春: 吉林大学, 2023.
ZHOU S Y. Establishment and application of a detection method for respiratory pathogens in children based on LAMP coupling CRISPR/Cas12a[D]. Changchun: Jilin University, 2023.
[18] 任显凤. 粮油黄曲霉与毒素同步检测及木霉阻控技术研究[D]. 北京: 中国农业科学院, 2020.
REN X F. Simultaneous detection and trichoderma's biocontrol on Aspergillus flavus and aflatoxins in grains and oil[D]. Beijing: Chinese Academy of Agricultural Sciences, 2020.
[19] 孙长坡, 常晓娇, 伍松陵, 等. 利用PCR-RFLP方法鉴别黄曲霉毒素产毒菌株[J]. 中国农业科学, 2014, 47(18):3675-3683.
SUN C P, CHANG X J, WU S L, et al. Identification of aflatoxin producing strains by using PCR-RFLP method[J]. Scientia Agricultura Sinica, 2014, 47(18):3675-3683.
[20] 李雨薇, 吕家硕, 于一凡, 等. 黄曲霉毒素降解技术研究及应用进展[J]. 生物技术进展, 2023, 13(6):853-862.
LI Y W, LYU J S, YU Y F, et al. Research and application progress in degradation of aflatoxin[J]. Current Biotechnology, 2023, 13(6):853-862.
[21] 马慧慧. 寄生曲霉菌在不同饲料原料中产生黄曲霉毒素B₁的差异性及其机制研究[D]. 武汉: 华中农业大学, 2015.
MA H H. The study on difference and mechanisim of different feedstuff on afatoxin B1 production by Aspergillus parasiticus[D].Wuhan: Huazhong Agricultural University, 2015.
[22] 凌莉, 叶正茂, 王溯源, 等. 成都市售辣椒中产黄曲霉毒素菌株的分离及其基因分析[J]. 四川大学学报(医学版), 2015, 46(5):684-687.
LING L, YE Z M, WANG S Y, et al. Isolation and study on the aflatoxin genes of aflatoxin-producing fungi in paprika samples in Chengdu[J]. Journal of Sichuan University (Medical Science Edition), 2015, 46(5):684-687.
[23] LIANG S H, SKORY C D, LINZ J E. Characterization of the function of the ver-1A and ver-1B genes, involved in aflatoxin biosynthesis in Aspergillus parasiticus[J]. Applied and Environmental Microbiology, 1996, 62(12):4568-4575.
[24] YU J, CARY J W, BHATNAGAR D, et al. Cloning and characterization of a cDNA from Aspergillus parasiticus encoding an O-methyltransferase involved in aflatoxin biosynthesis[J]. Applied and Environmental Microbiology, 1993, 59(11):3564-3571.
[25] 王欢, 薛健. 寄生曲霉快速分子检测方法的研究[J]. 农业与技术, 2022, 42(17):19-22.
WANG H, XUE J. Research on rapid molecular detection method for Parasitic aspergillus [J]. Agriculture and Technology, 2022, 42(17):19-22.
[26] 张桂枝,刘俊杰,潘梦洋,等. 复合植物精油和有机酸盐对发酵饲料的防霉效果及发酵品质的影响[J]. 黑龙江畜牧兽医, 2023(14): 91-95;101.
ZHANG G Z, LIU J J, PAN M Y, et al. The effect of compound plant essential oils and organic acid salts on the antifungal effect and fermentation quality of fermented feed[J]. Heilongjiang Animal Science and Veterinary Medicine, 2023(14): 91-95;101.
[27] 张宁慧, 王瑱, 毛垣元, 等. 植物精油及活性成分防控黄曲霉生长及其毒素产生的研究进展[J]. 中草药, 2020, 51(20):5348-5359.
ZHANG N H, WANG T, MAO Y Y, et al. Research progress on growth of Aspergillus flavus and its toxins production controlled by plant essential oils and its active ingredients[J]. Chinese Traditional and Herbal Drugs, 2020, 51(20):5348-5359.
[28] 徐丹, 郭芮, 贺竹梅, 等. 九种植物精油对黄曲霉抑制作用的比较[J]. 保鲜与加工, 2020, 20(2):79-84.
XU D, GUO R, HE Z M, et al. Comparison of the inhibitory effect of nine plant essential oils on Aspergillus flavus[J]. Storage and Process, 2020, 20(2):79-84.
[29] 王婷. 黄曲霉菌及其毒素合成关键酶抗体的研制[D]. 北京: 中国农业科学院, 2017.
WANG T. Preparation of antibodies against key enzymes of Aspergillus flavus and its toxin synthesis[D]. Beijing: Chinese Academy of Agricultural Sciences, 2017.
[30] 晏丽. 黄曲霉毒素产毒菌检测方法的研究及其应用[D]. 无锡: 江南大学, 2012.
YAN L. Development and application of detection methods for aflatoxins toxin-producing bacteria[D]. Wuxi: Jiangnan University, 2012.
[31] 张庆, 赵晓美, 王娉, 等. 食品中产毒真菌核酸检测方法的研究进展[J]. 中国农业科技导报, 2021, 23(3):91-98.
ZHANG Q, ZHAO X M, WANG P, et al. Advance on detection methods of nucleic acid for toxin-producing fungi in food[J]. Journal of Agricultural Science and Technology, 2021, 23(3):91-98.
[32] 陈冠果, 李可, 陆金虎,等.产黄曲霉毒素真菌双重荧光定量PCR检测技术的建立与应用[J]. 中国粮油学报, 2024, 39(2):188-195.
CHEN G G, LI K, LU J H, et al. Development and application of dual fluorescent quantitative PCR for detection of aflatoxins fungi[J]. Journal of the Chinese Cereals and Oils Association, 2024, 39(2):188-195.
[33] 冯优妍, 杨爱馥, 郑秋月, 等. 粮食中主要霉菌实时荧光PCR检测方法建立[J]. 粮食与饲料工业, 2020(4):1-3;9.
FENG Y Y, YANG A F, ZHENG Q Y, et al. Establishment of a real-time PCR method for detection of main fungus in grain[J]. Cereal & Feed Industry, 2020(4):1-3; 9.
[34] 刘启君. 食品中黄曲霉菌及黄曲霉毒素B1的快速检测方法研究[D]. 广州: 华南理工大学, 2021.
LIU Q J. Rapid detection of Aspergillus flavus and aflatoxins B1 in food[D].Guangzhou: South China University of Technology, 2021.