荧光定量PCR辐照食品鉴定技术的优化研究

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

生物技术通报 ›› 2018, Vol. 34 ›› Issue (12): 77-83.doi: 10.13560/j.cnki.biotech.bull.1985.2018-0481

荧光定量PCR辐照食品鉴定技术的优化研究

刘绵学^{1, 2}, 伏毅^{1, 2}, 郑宇¹, 王艳^{1, 2}, 潘琳¹, 李华¹, 黄敏^{1, 2}

1. 四川省原子能研究院辐照保藏四川省重点实验室,成都 610101;
2. 四川省原子能研究院生物技术研究所,成都 610101

收稿日期:2018-05-24 出版日期:2018-12-26 发布日期:2018-12-24
作者简介:刘绵学,男,博士,研究方向：分子生物学及辐照食品鉴定;E-mail：35837681@qq.com
基金资助:
四川省科技支撑计划项目（2016SZ0075,2016NZ0117）,四川省应用基础项目（2017JY0242,2016JY0030,2018JY0531,18YYJC0284）,四川省科技计划重点研发项目（2018GZ0003）

An Optimization on Identification of Irradiated Food by Fluorescence Quantitative PCR

LIU Mian-xue^{1, 2}, FU Yi^{1, 2}, ZHENG Yu¹, WANG Yan^{1, 2}, PAN Lin¹, LI Hua¹, HUANG Min^{1, 2}

1. Irradiation Preservation Key Laboratory of Sichuan Province,Sichuan Institute of Atomic Energy,Chengdu 610101;
2. Department of Biotechnology,Sichuan Institute of Atomic Energy,Chengdu 610101

Received:2018-05-24 Published:2018-12-26 Online:2018-12-24

摘要/Abstract

摘要： 为优化荧光定量PCR辐照食品鉴定技术方法,实现对假阳性样本的鉴别能力,降低模板量对鉴定结果的干扰效应。选择马铃薯作为研究材料,分别进行1-12 kGy的⁶⁰Co辐照、加热和冻融处理,通过转座子引物筛选、检出限分析、DNA损伤特征比较和剂量曲线回归等方法,探究转座子引物的应用对马铃薯辐照鉴定结果的优化效果。结果显示,转座子引物组合LTR2-5\18S-8与LTR2-2\ACT-5可用于假阳性样本的鉴别,引物组合LTR2-5\18S-5可用于辐照鉴定。优化后方法中“初始循环数差值的指数函数-辐照剂量”标准曲线方程为Y=-0.1581X+ 4.611,线性关系R²为99.24%,优于“初始循环数差值-辐照剂量”的原有模式。通过转座子引物的应用与方法优化,增强了假阳性鉴别能力,消除了模板差异,增强了荧光定量PCR辐照鉴定技术的“剂量-效应关系”。

关键词: 辐照鉴定, 转座子, 马铃薯, DNA损伤

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

刘绵学, 伏毅, 郑宇, 王艳, 潘琳, 李华, 黄敏. 荧光定量PCR辐照食品鉴定技术的优化研究[J]. 生物技术通报, 2018, 34(12): 77-83.

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.

参考文献

[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.

荧光定量PCR辐照食品鉴定技术的优化研究

An Optimization on Identification of Irradiated Food by Fluorescence Quantitative PCR

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

编辑推荐

Metrics

本文评价

[1]	刘雯锦, 马瑞, 刘升燕, 杨江伟, 张宁, 司怀军. 马铃薯StCIPK11的克隆及响应干旱胁迫分析[J]. 生物技术通报, 2023, 39(9): 147-155.
[2]	于国红, 刘朋程, 李磊, 李明哲, 崔海英, 郝洪波, 郭安强. 不同基因型马铃薯对干旱胁迫的生理响应[J]. 生物技术通报, 2022, 38(5): 56-63.
[3]	高蒙, 李富婷, 魏湛林, 张赛行, 白如仟, 尚轶, 马玲. 二倍体马铃薯中SCF^SLF复合体的组分分析[J]. 生物技术通报, 2022, 38(4): 117-125.
[4]	雷春霞, 李灿辉, 陈永坤, 龚明. 马铃薯块茎形成的生理生化基础和分子机制[J]. 生物技术通报, 2022, 38(4): 44-57.
[5]	冯建英, 李立芹, 鲁黎明. 马铃薯bHLH转录因子家族全基因组鉴定与表达分析[J]. 生物技术通报, 2022, 38(2): 21-33.
[6]	高波, 马娟, 李秀花, 李焦生, 王容燕, 陈书龙. 马铃薯腐烂茎线虫Dd-mel-26基因的克隆与功能分析[J]. 生物技术通报, 2021, 37(7): 107-117.
[7]	彭洁, 邓孟胜, 张杰, 刘石锋, 罗李飞, 王西瑶. 马铃薯StSN2的克隆、定位及表达分析[J]. 生物技术通报, 2021, 37(5): 11-18.
[8]	郑英转, 吕燕, 杨东旭, 李国威, 王洪洋, 李灿辉. 基于液氮研磨法的流式细胞术检测马铃薯倍性的研究[J]. 生物技术通报, 2021, 37(1): 282-288.
[9]	李培根, 要雅倩, 宋吉祥, 王天琪, 周波, 王冰, 林榕姗. 马铃薯根际产IAA芽孢杆菌的分离鉴定及促生效果研究[J]. 生物技术通报, 2020, 36(9): 109-116.
[10]	李静舒, 赵佳. 生防细菌ML-3抗菌蛋白发酵条件的优化及其防治应用[J]. 生物技术通报, 2020, 36(6): 83-92.
[11]	黄凤君, 李佳皓, 刘瑞林, 余丽萍, 王西瑶, 李立芹. 马铃薯StKUP12的表达及参与钾营养的功能研究[J]. 生物技术通报, 2020, 36(4): 54-60.
[12]	王宇, 邓孟胜, 徐驰, 余丽萍, 谢良帅, 李立芹, 王西瑶. 马铃薯StSSR2的克隆与表达分析[J]. 生物技术通报, 2020, 36(4): 61-69.
[13]	叶明旺, 李灿辉, 龚明. 基因组编辑技术在马铃薯精准分子育种中的应用及研究展望[J]. 生物技术通报, 2020, 36(3): 9-17.
[14]	吴志胜, 傅高惠, 罗文骏, 刘俊杰, 陈慧芳, 白银山. 精准高效外源DNA整合技术研究进展[J]. 生物技术通报, 2020, 36(3): 29-37.
[15]	庞鹏湘, 常燕楠, 尉瑞敏, 郜刚. 马铃薯StSRP1的克隆、表达及生物信息学分析[J]. 生物技术通报, 2019, 35(7): 10-16.