An Optimization on Identification of Irradiated Food by Fluorescence Quantitative PCR

doi:10.13560/j.cnki.biotech.bull.1985.2018-0481

Abstract

Abstract: Purpose of this study is to realize the identification ability of false positive samples and reduce template amount interference with real-time PCR results in optimizing irradiation food identification. Potato tuber was selected as material and treated with 60-Co in gradient dose（1-12 kGy）,irradiating,heating and freezing-thawing respectively. Analysis methods,including primer screening of transposon,detection limit analysis,comparison of DNA damage characteristics and dose curve regression,were used to explore the optimized effects of transposon primers in potato irradiation identification. As results,transposon primer combination of LTR2-5\18S-8 and LTR2-2\ACT-5 can be used for the identification of false positive samples,and the combination of LTR2-5\18S-5 can be used for irradiation identification. In the optimized method,the irradiation dose was linear with the exponential function of the initial cycle difference with the standard equation Y=-0.1581X+ 4.611 and determination coefficient R² 99.24%,which was superior to the original initial cycle difference-irradiation dose model. In conclusion,by the optimized method,the dose-effect relationship with real-time PCR in irradiation identification was improved with less possibility of false positive,and the result interference caused by template amount was eliminated.

Key words: irradiation identification, transposon, potato, DNA damage

LIU Mian-xue, FU Yi, ZHENG Yu, WANG Yan, PAN Lin, LI Hua, HUANG Min. An Optimization on Identification of Irradiated Food by Fluorescence Quantitative PCR[J]. Biotechnology Bulletin, 2018, 34(12): 77-83.

References

[1] Ahn J, Akram K, Kwak J, et al.Reliable screening of various foodstuffs with respect to their irradiation status:A comparative study of different analytical techniques[J]. Radiation Physics & Chemistry, 2013, 91(10):186-192.
[2] Stefanova R, Vasilev N, Spassov S.Irradiation of food, current legislation framework, and detection of irradiated foods[J]. Food Analytical Methods, 2010, 3(3):225-252.
[3] Akram K, Ahn J, Kwon J.Analytical methods for the identification of irradiated foods[J]. Current Research on Agriculture and Life Science, 2013, 3(1):1-10.
[4] Jo Y, Sanyal B, Ameer K, et al.Thermoluminescence analysis and DNA comet assay to identify grapes irradiated as a quarantine treatment[J]. European Food Research & Technology, 2017, 243(8):1397-1403.
[5] 刘宁, 高建民, 刘鹏, 等. 应用DNA彗星电泳法检测辐照食品[J]. 检验检疫学刊, 2014, 24(4):51-54.
[6] Zanardi E, Caligiani A, Novelli E.New insights to detect irradiated food:an overview[J]. Food Analytical Methods, 2017, 11(1):224-235.
[7] Löbrich M, Cooper P, Rydberg B.Non-random distribution of DNA double-strand breaks induced by particle irradiation[J]. Int J Radiat Biol, 1996, 70(5):493-503.
[8] 王锋, 王志东, 周洪杰, 等. 辐照食品检测技术方法与标准[J]. 食品与机械, 2009, 25(5):142-144.
[9] Sonntag C.Free-Radical-Induced DNA Damage and Its Repair[M]. Springer Berlin Heidelberg:2006.
[10] Sutherland B, Bennett P, et al.Clustered DNA damages induced in isolated DNA and in human cells by low doses of ionizing radiation[J]. Proc Natl Acad Sci USA, 2000, 97(1):103-108.
[11] Lee J, Levin R.New approach for discrimination of Vibrio vulnificus by real-time PCR before and after γ-irradiation[J]. Journal of Microbiological Methods, 2008, 73(1):1-6.
[12] Shehata M, Gomaa F, Helal Z.Effects of gamma and electron beam irradiation on viability and DNA elimination of Staphylococcus aureus[J]. Archives of Clinical Microbiology, 2011, 2(6):1-9.
[13] 刘正霞, 徐阳, 徐进梅, 等. 不同引物及数据分析方法对定量PCR结果的影响[J]. 南京医科大学学报:自然科学版, 2009, 8:1112-1117.
[14] Şakalar E, Arman K.A new RAPD-PCR based analytical assay for detection of sea bass and sea bream treated with ionizing radiation[J]. Food Science & Biotechnology, 2015, 24(4):1233-1237.
[15] 梁琳琳, 周明兵. 植物活性长末端重复序列反转录转座子研究进展[J]. 生物工程学报, 2016, 32(4):409-429.
[16] Yuan-fang X, Deng G, Peng L, et al. Effect of irradiation on sterili-zation and quality of edible areca nut[J]. Acta Agriculturae Nucleatae Sinica, 2014, 28(2):240-244.
[17] 胡广, 王乐, 等. 棉花miRNA表达量检测方法Stem-loop RT-PCR的建立[J]. 核农学报, 2017, 31(11):2121-2127.
[18] 中华人民共和国卫生部编. 食品卫生检验方法(理化部分)[M]. 北京:中国标准出版社, 1979.
[19] 冯燕纯, 陈杰荣, 郭允苗. 鼻咽癌患者组织样本中EB病毒DNA拷贝数与患者预后的相关性研究[J]. 热带医学杂志, 2017, 17(6):693-696.
[20] Şakalar E, Mol S.Determination of irradiation dose and distingui-shing between irradiated and non irradiated fish meat by real-time PCR[J]. Food Chemistry, 2015, 182:150-155.
[21] 李文建, 周光明, 卫增泉, 等. 重离子辐射哺乳动物细胞敏感性的分子机理[J]. 原子核物理评论, 2003, 20(1):42-47.
[22] Mehra M, Gangwar I, Shankar R.A deluge of complex repeats:the solanum genome[J]. PLoS One, 2015, 10(8):e0133962.
[23] Paz R, Kozaczek M, et al.Diversity, distribution and dynamics of full-length Copia and Gypsy LTR retroelements in Solanum lycopersicum[J]. Genetica, 2017, 145(4-5):417-430.
[24] Divashuk M, Khuat T, Kroupin P, et al.Variation in copy number of Ty3/Gypsy centromeric retrotransposons in the genomes of Thinopyrum intermedium and its diploid progenitors[J]. PLoS One, 2016, 11(4):e0154241.
[25] Wang K, Huang G, Zhu Y.Transposable elements play an important role during cotton genome evolution and fiber cell development[J]. Sci China Life Sci, 2016, 59(2):112-121.
[26] 刘春林, 官春云. 关于植物随机引物扩增多态性DNA标记的可靠性问题[J]. 植物生理学报, 2000, 36(1):56-59.
[27] 赵婧妤, 贺诗雨, 戴刘凤, 等. 60Co-γ射线辐照翠竹的生物学效应及RAPD分析[J]. 世界科技研究与发展, 2016, 6):1280-1286.
[28] 何丽娴, 王阳, 杨欢, 等. 电子束辐照金花葵幼苗的生物学效应及RAPD分析[J]. 北京师范大学学报:自然科学版, 2014, 1:52-57.
[29] 王金斌, 白蓝, 李文, 等. 同步检测动物源性成分的五重PCR的条件优化和检出限分析[J]. 核农学报, 2018, 32(3):506-514.