转基因溶菌酶羊hLZ-J1品系特异性数字PCR方法及样本中痕量转基因成分检测

doi:10.16409/j.cnki.2095-039x.2022.09.007

中国生物防治学报 ›› 2022, Vol. 38 ›› Issue (5): 1109-1116.DOI: 10.16409/j.cnki.2095-039x.2022.09.007

转基因溶菌酶羊hLZ-J1品系特异性数字PCR方法及样本中痕量转基因成分检测

王艺洁¹, 徐文婷¹, 杨立桃¹, 梁晋刚²

1. 上海交通大学生命科学技术学院, 上海 200240;
2. 农业农村部科技发展中心, 北京 100176

收稿日期:2021-07-15 发布日期:2022-10-20
通讯作者: 梁晋刚,高级农艺师,E-mail:liangjingang@agri.gov.cn
作者简介:王艺洁,硕士研究生,E-mail:wangyijie000@sjtu.edu.cn
基金资助:
转基因重大专项(2016ZX08012-005);农业科研杰出人才培养计划(2021)

Development of an Event-Specific Droplet Digital PCR Assay for Quantification of the Transgene DNAs in Trace Samples of GM Goat hLZ-J1

WANG Yijie¹, XU Wenting¹, YANG Litao¹, LIANG Jingang²

1. School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China;
2. Development Center of Science and Technology, Ministry of Agriculture of People's Republic of China, Beijing 100176, China

Received:2021-07-15 Published:2022-10-20

摘要/Abstract

摘要： 人溶菌酶是一种天然抗生素，具有抗细菌、真菌，抗炎症，预防感染等功能。转基因溶菌酶山羊可以在乳腺中特异性表达，从而可以从羊乳中规模化制备溶菌酶。本研究旨在建立一种适用于痕量样本中转基因溶菌酶羊成分定量检测的品系特异性数字PCR方法，并利用该方法对山羊的血液、羊奶、粪便和环境土壤样本等进行检测，用于对转基因溶菌酶山羊成分的溯源。建立的数字PCR方法具有高特异性、灵敏度、准确性，检测限和定量限低至15个拷贝每个反应，可以对羊血、羊乳等制品进行准确的定量检测。研究结果还表明该方法可以准确测定粪便和环境土壤样本中的痕量转基因溶菌酶羊成分，可进一步用于转基因动物外源基因的水平转移检测及环境风险评估。

关键词: 数字PCR, 转基因溶菌酶羊, 品系特异性, 痕量样本

Abstract: Human lysozyme (hLZ) is a natural antibiotic,functioning to resist bacteria,fungi,inflammation and infection.Transgenic human lysozyme goat can express high levels of human lysozyme in milk for large scale production of lysozyme.Herein,we developed an event-specific droplet digital PCR (ddPCR) method for quantitative detection of transgenic hLZ goat and its derivates.This method presents high specificity,sensitivity and accuracy in goat blood and goat milk samples analysis.The limit of detection and limit of quantification were determined with 15 copies per reaction.Furthermore,this method was used to trace the transgenic hLZ goat content in goat feces and the living environmental soil samples,indicating that the developed ddPCR method can be used for monitoring horizontal gene transfer and environmental risk assessment of transgenic hLZ goat.

Key words: ddPCR, genetically modified human lysozyme goat, event-specific, trace samples

中图分类号:

S476

王艺洁, 徐文婷, 杨立桃, 梁晋刚. 转基因溶菌酶羊hLZ-J1品系特异性数字PCR方法及样本中痕量转基因成分检测[J]. 中国生物防治学报, 2022, 38(5): 1109-1116.

WANG Yijie, XU Wenting, YANG Litao, LIANG Jingang. Development of an Event-Specific Droplet Digital PCR Assay for Quantification of the Transgene DNAs in Trace Samples of GM Goat hLZ-J1[J]. Chinese Journal of Biological Control, 2022, 38(5): 1109-1116.

参考文献

[1] Hankiewicz J, Swierczek E. Lysozyme in human body fluids[J]. Clinica Chimica Acta, 1974, 57(3):205-209.
[2] Ibrahim H R, Imazato K, Ono H. Human lysozyme possesses novel antimicrobial peptides within its N-terminal domain that target bacterial respiration[J]. Journal of Agricultural and Food Chemistry, 2011, 59(18):10336-10345.
[3] Cooper C A, Maga E A, Murray J D. Production of human lactoferrin and lysozyme in the milk of transgenic dairy animals:past, present, and future[J]. Transgenic Research, 2015, 24(4):605-614.
[4] Ogundele M O. A novel anti-inflammatory activity of lysozyme:modulation of serum complement activation[J]. Mediators of Inflammation, 1998, 7(5):363-365.
[5] Samaranayake Y H, Samaranayake L P, Wu P C, et al. The antifungal effect of lactoferrin and lysozyme on Candida krusei and Candida albicans[J]. APMIS, 1997, 105(11):875-883.
[6] Lee-Huang S, Huang P L, Sun Y, et al. Lysozyme and RNases as anti-HIV components in β-core preparations of human chorionic gonadotropin[J]. Proceedings of the National Academy of Sciences of the United States of America, 1999, 96(6):2678-2681.
[7] Gill A O, Holley R A. Surface application of lysozyme, nisin, and EDTA to inhibit spoilage and pathogenic bacteria on ham and bologna[J]. Journal of Food Protection, 2000, 63(10):1338-1346.
[8] Proctor V A, Cunningham F E, Fung D Y C. The chemistry of lysozyme and its use as a food preservative and a pharmaceutical[J]. C R C Critical Reviews in Food Science and Nutrition, 1988, 26(4):359-395.
[9] Hughey V L, Wilger P A, Johnson E A. Antibacterial activity of hen egg white lysozyme against Listeria monocytogenes Scott A in foods[J]. Applied and Environmental Microbiology, 1989, 55(3):631-638.
[10] Chandan R C, Parry R M, Shahani K M. Lysozyme, lipase, and ribonuclease in milk of various species 1, 2[J]. Journal of Dairy Science, 1968, 51(4):606-607.
[11] Yu H, Chen J, Liu S, et al. Large-scale production of functional human lysozyme in transgenic cloned goats[J]. Journal of Biotechnology, 2013, 168(4):676-683.
[12] Maga E A, Shoemaker C F, Rowe J D, et al. Production and processing of milk from transgenic goats expressing human lysozyme in the mammary gland[J]. Journal of Dairy Science, 2006, 89(2):518-524.
[13] Gui T, Zhang M, Chen J, et al. In vitro evaluation of a mammary gland specific expression vector encoding recombinant human lysozyme for development of transgenic dairy goat embryos[J]. Biotechnology Letters, 2012, 34(8):1445-1452.
[14] Tong J, Wei H, Liu X, et al. Production of recombinant human lysozyme in the milk of transgenic pigs[J]. Transgenic Research, 2011, 20(2):417-419.
[15] Yang B, Wang J, Tang B, et al. Characterization of bioactive recombinant human lysozyme expressed in milk of cloned transgenic cattle[J]. PLoS ONE, 2011, 6(3):e17593.
[16] 卢军锋,李晓,杨公社,等.我国转基因动物及其安全性评价发展现状[J].中国兽医杂志, 2018, 54(2):62-64.
[17] 胡慧宇,李忠慧.转基因动物安全性评价[J].当代畜禽养殖业, 2020(11):35-36.
[18] Karalis D T, Karalis T, Karalis S, et al. Genetically modified products, perspectives and challenges[J]. Cureus, 2020, 12(3):e7306.
[19] Xu C C Y, Ramsay C, Cowan M, et al. Transgenes of genetically modified animals detected non-invasively via environmental DNA[J]. PLoS ONE, 2021, 16(8):e0249439.
[20] 李晓琳,吴绍强,刘晓飞,等.转基因动物及其产品的主要管理制度[J].畜牧与兽医, 2019, 51(2):130-134.
[21] Kamle S, Ali S. Genetically modified crops:detection strategies and biosafety issues[J]. Gene, 2013, 522(2):123-132.
[22] Pu T, Liu W, Wu Y, et al. ABIN1 alleviates inflammatory responses and colitis via facilitating A20 activity[J]. Therapeutic Advances in Chronic Disease, 2020, 11:2040622320944782.
[23] Nakagaki A, Hirano S, Urakawa A, et al. Transgenic mice with a tandem duplication of the Necdin gene overexpress Necdin[J]. Mamm Genome, 2018, 29(9/10):680-689.
[24] Salisu I B, Shahid A A, Yaqoob A, et al. Molecular approaches for high throughput detection and quantification of genetically modified crops:A review[J]. Frontiers in Plant Science, 2017, 8:1670.
[25] Wang Q, Cai Y, He Y, et al. Droplet digital PCR (ddPCR) method for the detection and quantification of goat and sheep derivatives in commercial meat products[J]. European Food Research and Technology, 2018, 244(4):767-774.
[26] Xu W, Cui J, Liu B, et al. An event-specific real-time PCR method for measuring transgenic lysozyme goat content in trace samples[J]. Foods, 2021, 10(5):925.
[27] Widmer F, Seidler R J, Watrud L S. Sensitive detection of transgenic plant marker gene persistence in soil microcosms[J]. Molecular Ecology, 1996, 5(5):603-613.
[28] Barnes M A, Turner C R. The ecology of environmental DNA and implications for conservation genetics[J]. Conservation Genetics, 2016, 17(1):1-17.

转基因溶菌酶羊hLZ-J1品系特异性数字PCR方法及样本中痕量转基因成分检测

Development of an Event-Specific Droplet Digital PCR Assay for Quantification of the Transgene DNAs in Trace Samples of GM Goat hLZ-J1

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 1

编辑推荐

Metrics

本文评价

联系我们