Application of Real-time Quantitative PCR Technology in Food Rapid Detection
-
摘要: 荧光定量聚合酶链式反应（real-time quantitative polymerase chain reaction，qPCR）是一种将聚合酶链式反应（polymerase chain reaction，PCR）扩增与实时检测相结合的技术，也是近年来食品快速检测技术的研究热点之一。与传统PCR技术相比，它具有耗时短、操作简单、灵敏度高、特异性强等优势。本文简要概述了荧光定量PCR技术的基本原理、发展历程、分类及特点，综述了荧光定量PCR技术在食源性致病菌、掺杂掺假、转基因食品、过敏原、食源性寄生虫、可食用昆虫等食品检测中的应用，并对荧光定量PCR技术及其在食品快速检测中应用的前景进行了展望。
-
关键词:
- 荧光定量聚合酶链式反应(qPCR) /
- 快速检测 /
- 食源性病原菌 /
- 掺杂掺假 /
- 过敏原
Abstract: Real-time quantitative polymerase chain reaction (qPCR) is a technology that combines polymerase chain reaction (PCR) amplification with real-time detection, and also one of the research hotspots of food rapid detection technology in recent years. It has the advantages of short time-consuming, simple operation, high sensitivity, and strong specificity, compared with traditional PCR technology. This article briefly outlines the basic principles, development process, classification, and characteristics of real-time qPCR technology. The application in the detection of food-borne pathogens, adulteration, genetically modified foods, allergens, food-borne parasites, and edible insects is reviewed. The prospects of real-time qPCR technology and its application in food rapid detection are predicted. -
表 1 荧光定量PCR在食源性致病菌检测中的应用情况
Table 1. Application of real-time qPCR in the detection of food-borne pathogens
致病菌种类 被检食品 荧光定量PCR技术 参考
文献单增李斯特菌 肉制品 TaqMan探针 [21] 鲜切甜瓜 [22] 鲜切蔬菜、牛奶、奶酪 [23] 冷冻蔬菜 [24] 生禽肉、动物性海产品、鲜鸡蛋、熟肉制品、
中式凉拌菜、米面制品、坚果类、蔬菜荧光探针 [25] 大肠杆菌 猪肉、牛奶 TaqMan探针 [26] 牡蛎 [27] 生菜、卷心菜 荧光探针 [28] 志贺氏菌 牛奶 SYBR Green染料 [29] 乳品 TaqMan探针 [30] 冷冻蔬菜 [24] 鲜猪肉 − [31] 生禽肉、动物性海产品、鲜鸡蛋、熟肉制品、
中式凉拌菜、米面制品、坚果类、蔬菜荧光探针 [25] 沙门氏菌 乳品 TaqMan探针 [30] 奶粉 [32] 肉制品 [33] 冷冻蔬菜 [24] 牛乳 荧光染料 [34] 冷冻肉制品 荧光探针 [35] 生禽肉、动物性海产品、鲜鸡蛋、熟肉制品、
中式凉拌菜、米面制品、坚果类、蔬菜[25] 金黄色葡萄球菌 肉类、乳制品 SYBR Green II染料 [36] 奶粉 TaqMan探针 [32] 冷冻蔬菜 [24] 生禽肉、动物性海产品、鲜鸡蛋、熟肉制品、
中式凉拌菜、米面制品、坚果类、蔬菜荧光探针 [25] 蜡样芽胞杆菌 肉制品 TaqMan探针 [37] 鲜牛奶 荧光探针 [38] 生禽肉、动物性海产品、鲜鸡蛋、熟肉制品、
中式凉拌菜、米面制品、坚果类、蔬菜[25] 副溶血性弧菌 水产品 SYBR Green I染料 [39] 生禽肉、动物性海产品、鲜鸡蛋、熟肉制品、
中式凉拌菜、米面制品、坚果类、蔬菜荧光探针 [25] 酿脓链球菌 新鲜猪肉 TaqMan探针 [40] 克罗诺杆菌 奶粉 TaqMan探针 [32] 
下载: 导出CSV
表 2 标准培养方法、常规PCR与荧光定量PCR比较
Table 2. Comparison of standard culture method, conventional PCR and real-time qPCR
下载: 导出CSV
表 3 荧光定量PCR在过敏原检测中的应用
Table 3. Application of real-time qPCR in the detection of allergens
食品类型 过敏原 检测限 检测方法 目的基因 参考文献 商业食品 核桃 2.5 pg SYBR Green Jug r 1, 3, 4 [65] 坚果糊 榛子 2 mg/kg TaqMan hsp 1 [66] 板栗粉与小麦粉混合物 板栗 100 mg/kg TaqMan Cas s 9 [67] 商业食品 花生、榛子、开心果、杏仁、
腰果、澳洲坚果、核桃、山核桃0.1 mg/kg TaqMan ITS [68] 商业食品 小麦、荞麦、花生 10 mg/kg TaqMan 18S r RNA [69] 实验室样品 大豆、芹菜 0.01% TaqMan atp A, mtd [70] 饼干，婴儿食品，酸奶 猕猴桃 25 mg/kg SYBR Green Act c2 [71] 桃 20 mg/kg Pru p2.01B 苹果 50 mg/kg Mal d1 功能性食品 油菜籽蜂花粉 1 pg/μL~1 ng/μL TaqMan-QSY PEP [72] 茶花蜂花粉 mat K 莲花蜂花粉 ITS 松花粉 mat K 玫瑰蜂花粉 ITS 杨柳蜂花粉 ITS 虞美人蜂花粉 ITS 猕猴桃蜂花粉 ITS 调味酱 鱼 0.01 pg TaqMan,Eva Green 16S rRNA [73] 调味酱 虾 0.1 pg TaqMan 16S rRNA [74] 猪肉饺子、通心粉沙拉、蛤蜊汁 螃蟹 0.1～105 mg/kg − 12S rRNA [75] 食用油、蛤蜊汤、炸玉米饼混合物 大西洋鳕鱼、太平洋鳕鱼、白眼狭鳕鱼 1～10 mg/kg − COI [76] 牛奶 α-乳清蛋白 0.05 ng TaqMan-MGB α-LA [64] 火腿、香肠 牛奶蛋白 6 fg TaqMan 12S rRNA [77]
下载: 导出CSV
-
[1] 李双, 韩殿鹏, 彭媛, 等. 食品安全快速检测技术研究进展[J]. 食品安全质量检测学报,2019,10(17):5575−5581. [2] 杜清春. 布鲁氏菌实时荧光定量PCR快速检测技术的建立及初步应用[D]. 大理: 大理大学, 2019. [3] 索原杰. 多重实时荧光PCR致病菌检测方法的构建及其在牛奶中的应用[D]. 杭州: 浙江大学, 2018. [4] 黄小玲, 张登, 廖嘉明, 等. 荧光定量PCR技术的原理及其在植物研究中的应用[J]. 安徽农业科学,2018,46(25):36−40. doi: 10.3969/j.issn.0517-6611.2018.25.011 [5] 刘永嘉, 单非. 浅谈分子生物学在食品微生物检验中的应用和前景[J]. 食品安全导刊,2018(33):73, 75. [6] Higuchi R, Dollinger G, Walsh P S, et al. Simultaneous amplification and detection of specific DNA sequences[J]. Technology,1992,10(4):413−417. [7] Vaïtilingom M, Pijnenburg H, Gendre F, et al. Real-time quantitative PCR detection of genetically modified maximizer maize and roundup ready soybean in some representative foods[J]. Journal of Agricultural and Food Chemistry,1999,47(12):5261−5266. doi: 10.1021/jf981208v [8] Fu Z, Rogelj S, Kieft T L. Rapid detection of Escherichia coli O157: H7 by immunomagnetic separation and real-time PCR[J]. International Journal of Food Microbiology,2005,99(1):47−57. doi: 10.1016/j.ijfoodmicro.2004.07.013 [9] Liang N J, Dong J, Luo L X, et al. Detection of viable Salmonella in lettuce by propidium monoazide real-time PCR[J]. Journal of Food Science,2011,76(4):M234−M237. doi: 10.1111/j.1750-3841.2011.02123.x [10] 王金凤, 王建昌, 张伟, 等. GNM C7-8实时荧光定量PCR同时快速检测8种食源性致病菌[J]. 食品安全质量检测学报,2018,9(9):2090−2095. doi: 10.3969/j.issn.2095-0381.2018.09.018 [11] 李婷婷, 张桂兰, 王之莹, 等. 羊肉掺假鉴别快速荧光定量PCR芯片制备及应用研究[J]. 生物技术进展,2018,8(6):522−529. [12] 刘宽, 王芬, 陈晓平, 等. 一种基于荧光定量PCR的自动化检测装备初探[J]. 食品工业,2018,39(9):185−189. [13] 金鹭, 陈传君, 林华, 等. 基于实时荧光PCR对肉制品中羊肉的精确定量[J]. 食品与发酵工业,2020,46(8):246−253. [14] 王斌. 一种复用型一步法荧光定量PCR试剂盒: CN209854118U[P]. 2019-12-27. [15] 杨艳, 王桂姬, 周广运, 等. 多种绿色DNA嵌入染料实时荧光PCR检测猪DNA效果比较[J]. 中国食品学报,2018,18(6):283−289. [16] 杨艳, 王桂姬, 周广运, 等. PCR技术在肉类成分定量分析中的应用研究进展[J]. 食品工业科技,2016,37(17):360−365. [17] 纪冬, 辛绍杰. 实时荧光定量PCR的发展和数据分析[J]. 生物技术通讯,2009,20(4):598−600. doi: 10.3969/j.issn.1009-0002.2009.04.041 [18] 常志远. 转基因食品分子生物学检测方法的建立及应用[D]. 长春: 长春理工大学, 2019. [19] 彭媛媛, 武煊, 陶晓奇. 实时荧光PCR技术定量检测肉类掺假的研究进展[J]. 食品与发酵工业,2019,45(15):279−287. [20] 陈晨. 微滴式数字PCR技术用于牛羊肉掺假定量检测方法的研究[D]. 保定: 河北农业大学, 2018. [21] Alía A, Andrade M J, Córdoba J J, et al. Development of a multiplex real-time PCR to differentiate the four major Listeria monocytogenes serotypes in isolates from meat processing plants[J]. Food Microbiology,2020,87:103367. doi: 10.1016/j.fm.2019.103367 [22] 关棣锴, 胡文忠, 朴永哲, 等. TaqMan探针法荧光定量PCR检测鲜切甜瓜中单核细胞增多性李斯特菌的研究[J]. 食品工业科技,2014,35(19):297−300. [23] Kim D H, Chon J W, Kim H, et al. Comparison of culture, conventional and real-time PCR methods for Listeria monocytogenes in foods[J]. Korean Journal for Food Science of Animal Resources,2014,34(5):665−673. doi: 10.5851/kosfa.2014.34.5.665 [24] 王晓. 多重实时荧光定量PCR同时检测冷冻蔬菜中4种食源性致病菌[J]. 食品安全质量检测学报,2018,9(22):5917−5921. doi: 10.3969/j.issn.2095-0381.2018.22.021 [25] 章沙沙. 实时荧光定量PCR检测食品中常见食源性致病菌[J]. 食品与发酵科技,2016,52(4):87−89. [26] 王芳妹, 钟文涛, 王淑好, 等. 5种致泻大肠埃希氏菌实时荧光定量PCR快速检测技术[J]. 食品与机械,2019,35(5):88−95. [27] Miotto M, Barretta C, Ossai S O, et al. Optimization of a propidium monoazide-qPCR method for Escherichia coli quantification in raw seafood[J]. International Journal of Food Microbiology,2020,318:108467. doi: 10.1016/j.ijfoodmicro.2019.108467 [28] Kim J H, Oh S W. Optimization of bacterial concentration by filtration for rapid detection of foodborne Escherichia coli O157: H7 using real-time PCR without microbial culture enrichment[J]. Journal of Food Science,2019,84(11):3241−3245. doi: 10.1111/1750-3841.14836 [29] 戴陈伟, 童琳, 武昌俊, 等. 实时荧光定量PCR技术快速检测志贺氏菌[J]. 食品安全质量检测学报,2019,10(23):8037−8041. [30] Demirci M, Yigin A, Altun S K, et al. Salmonella spp. and Shigella spp. detection via multiplex real-time PCR and discrimination via MALDI-TOF MS in different animal raw milk samples[J]. Nigerian Journal of Clinical Practice,2019,22(8):1083−1090. [31] 杨旭. 免疫磁分离-两重荧光定量PCR法快速检测鲜猪肉中金葡菌和志贺氏菌[J]. 食品安全导刊,2017(36):108. doi: 10.3969/j.issn.1674-0270.2017.36.084 [32] 林碧莲, 柯振华, 陈筱婷, 等. 多重实时荧光定量PCR快速检测婴幼儿奶粉中沙门氏菌、克罗诺杆菌和金黄色葡萄球菌[J]. 食品安全质量检测学报,2017,8(11):4375−4381. doi: 10.3969/j.issn.2095-0381.2017.11.049 [33] 於颖, 顾其芳, 陈敏, 等. 肉制品中沙门菌实时荧光定量聚合酶链反应标准质粒的构建[J]. 上海预防医学,2017,29(4):273−276, 280. [34] 姚笛, 徐磊, 佐兆杭, 等. 牛乳中沙门氏菌的荧光定量PCR检测方法的建立[J]. 中国乳品工业,2019,47(7):42−45. doi: 10.3969/j.issn.1001-2230.2019.07.009 [35] 庄平, 余以刚, 周冬根, 等. SD-PMA-qPCR快速检测冷冻肉制品中活性沙门氏菌的研究[J]. 现代食品科技,2017,33(7):289−294. [36] 郭梦冉, 董兵, 李聪, 等. 荧光定量PCR检测金黄色葡萄球菌方法的建立及应用[J]. 河北农业大学学报,2018,41(3):72−76, 83. [37] 杨滴, 赵宇明, 秦鹏钧, 等. 肉制品中蜡样芽胞杆菌实时荧光PCR检测方法的研究[J]. 肉类工业,2017(6):49−52. doi: 10.3969/j.issn.1008-5467.2017.06.012 [38] 刘新梅, 程逸宇, 吴海晶, 等. 食品中蜡样芽孢杆菌芽孢EMA-qPCR检测[J]. 食品安全导刊,2019(15):175−177. [39] 陈琳, 周青青, 顾青, 等. 实时定量PCR法快速检测水产品中的副溶血性弧菌[J]. 浙江农业学报,2019,31(5):823−828. doi: 10.3969/j.issn.1004-1524.2019.05.19 [40] 王远洋, 刘钟栋, 兰全学. 应用实时荧光PCR法检测食品中的酿脓链球菌[J]. 中国食品添加剂,2018(5):161−166. [41] 吴孟娟. 基于纳米抗体的免疫磁珠捕获-qPCR技术建立河弧菌的快速检测方法[D]. 南昌: 南昌大学, 2019. [42] 李涛, 王艳, 王粮子, 等. 乳制品中四种病原菌多重荧光PCR检测方法的建立与应用[J]. 基因组学与应用生物学,2017,36(9):3807−3813. [43] 尤祯丹, 韩国全, 陈传君, 等. 即食食品中单增李斯特氏菌快速检测技术的研究进展[J]. 食品工业科技,2020,41(10):358−362. [44] 刘易伟, 胡文忠, 刘程惠, 等. 鲜切果蔬中常见致病菌及其PCR快速检测方法[J]. 食品工业科技,2013,34(17):360−364. [45] 叶欣. 餐饮业即食食品微生物污染调查及金黄色葡萄球菌快速检测方法建立[D]. 杭州: 浙江农林大学, 2019. [46] 郭蔷, 冮洁, 武晓松. 采用荧光定量PCR方法快速检测蔬菜中沙门氏菌[J]. 食品研究与开发,2016,37(7):123−127. doi: 10.3969/j.issn.1005-6521.2016.07.030 [47] Osopale B A, Adewumi G A, Witthuhn R C, et al. A review of innovative techniques for rapid detection and enrichment of Alicyclobacillus during industrial processing of fruit juices and concentrates[J]. Food Control,2019,99:146−157. doi: 10.1016/j.foodcont.2018.12.032 [48] Zhao Y Y, Xia D D, Ma P P, et al. Advances in the detection of virulence genes of Staphylococcus aureus originate from food[J]. Food Science and Human Wellness,2020,9(1):40−44. doi: 10.1016/j.fshw.2019.12.004 [49] Ibrahim W A, Abd El-Ghany W A, Nasef S A, et al. A comparative study on the use of real time polymerase chain reaction (RT-PCR) and standard isolation techniques for the detection of Salmonellae in broiler chicks[J]. International Journal of Veterinary Science and Medicine,2014,2(1):67−71. doi: 10.1016/j.ijvsm.2013.11.001 [50] Chen X Y, Lu L X, Xiong X H, et al. Development of a real-time PCR assay for the identification and quantification of bovine ingredient in processed meat products[J]. Scientific Reports,2020,10(1):2052. doi: 10.1038/s41598-020-59010-6 [51] Tan L L, Ahmed S A, Ng S K, et al. Rapid detection of porcine DNA in processed food samples using a streamlined DNA extraction method combined with the SYBR Green real-time PCR assay[J]. Food Chemistry,2020,309:125654. doi: 10.1016/j.foodchem.2019.125654 [52] Kim M J, Suh S M, Kim S Y, et al. Development of a real-time PCR assay for the detection of donkey (Equus asinus) meat in meat mixtures treated under different processing conditions[J]. Foods,2020,9(2):130. doi: 10.3390/foods9020130 [53] Li T T, Jalbani Y M, Zhang G L, et al. Detection of goat meat adulteration by real-time PCR based on a reference primer[J]. Food Chemistry,2019,277:554−557. doi: 10.1016/j.foodchem.2018.11.009 [54] Guo L, Qian J P, Guo Y S, et al. Simultaneous identification of bovine and equine DNA in milks and dairy products inferred from triplex TaqMan real-time PCR technique[J]. Journal of Dairy Science,2018,101(8):6776−6786. doi: 10.3168/jds.2018-14408 [55] Di Domenico M, Di Giuseppe M, Wicochea Rodríguez J D, et al. Validation of a fast real-time PCR method to detect fraud and mislabeling in milk and dairy products[J]. Journal of Dairy Science,2017,100(1):106−112. doi: 10.3168/jds.2016-11695 [56] Yang Y G, Liu M C, Niu N, et al. Identification of small berry species in food and juice using TaqMan-based real-time PCR[J]. Journal of AOAC International,2019,102(5):1552−1566. doi: 10.1093/jaoac/102.5.1552 [57] Ferreira T, Farah A, Oliveira T C, et al. Using real-time PCR as a tool for monitoring the authenticity of commercial coffees[J]. Food Chemistry,2016,199:433−438. doi: 10.1016/j.foodchem.2015.12.045 [58] Villa C, Costa J, Oliveira M B P P, et al. Novel quantitative real-time PCR approach to determine safflower (Carthamus tinctorius) adulteration in saffron (Crocus sativus)[J]. Food Chemistry,2017,229:680−687. doi: 10.1016/j.foodchem.2017.02.136 [59] Moon B, Kim W, Park I, et al. Establishment of a PCR assay for the detection and discrimination of authentic Cordyceps and adulterant species in food and herbal medicines[J]. Molecules,2018,23(8):1932. doi: 10.3390/molecules23081932 [60] 王凤军, 叶素丹, 包永华, 等. 多重实时荧光PCR快速检测转基因大豆及其加工产品[J]. 中国粮油学报,2018,33(9):135−141. doi: 10.3969/j.issn.1003-0174.2018.09.021 [61] 权永兵, 黄永辉, 徐淼锋, 等. 改良十六烷基三甲基溴化铵-磁珠核酸提取方法及在植物转基因检测中的应用[J]. 食品安全质量检测学报,2019,10(23):8042−8047. [62] 王洪健, 许银叶, 曾伟婷. 蛋白粉中转基因成分实时荧光定量PCR检测方法的建立[J]. 食品与发酵科技,2018,54(1):123−126. [63] 邵彪, 周小兰, 王琳琳, 等. 肉制品中植源性转基因成分多重荧光定量PCR检测方法的建立[J]. 肉类研究,2018,32(1):41−45. [64] Guan X, Cai Q, Zhang W J, et al. Development of a real-time quantitative PCR assay using a TaqMan minor groove binder probe for the detection of α-lactalbumin in food[J]. Journal of Dairy Science,2016,99(3):1716−1724. doi: 10.3168/jds.2015-10255 [65] Linacero R, Ballesteros I, Sanchiz A, et al. Detection by real time PCR of walnut allergen coding sequences in processed foods[J]. Food Chemistry,2016,202:334−340. doi: 10.1016/j.foodchem.2016.01.132 [66] Piknová Ľ, Janská V, Kuchta T, et al. Comparison of real-time polymerase chain reaction and enzyme-linked immunosorbent assay for sensitive and quantitative detection of hazelnuts in nut pastes[J]. Journal of AOAC International,2018,101(6):1864−1867. doi: 10.5740/jaoacint.18-0017 [67] Sanchiz Á, Ballesteros I, López-García A, et al. Chestnut allergen detection in complex food products: Development and validation of a real-time PCR method[J]. LWT,2020,123:109067. doi: 10.1016/j.lwt.2020.109067 [68] García A, Madrid R, García T, et al. Detection of food allergens by taqman real-time PCR methodology[M]//Methods in Molecular Biology. New York, NY: Springer New York, 2017: 95-108. [69] Miyazaki A, Watanabe S, Ogata K, et al. Real-time PCR detection methods for food allergens (wheat, buckwheat, and peanuts) using reference plasmids[J]. Journal of Agricultural and Food Chemistry,2019,67(19):5680−5686. doi: 10.1021/acs.jafc.9b01234 [70] 汪永信, 程潇, 安虹, 等. 实时荧光PCR法同时检测食物中大豆和芹菜致敏原成分[J]. 生物技术通报,2016,32(1):69−73. [71] Graziano S, Gullì M, Marmiroli N. Detection of allergen coding sequences of kiwi, peach, and apple in processed food by qPCR[J]. Journal of the Science of Food and Agriculture,2018,98(8):3129−3139. [72] Yang Y G, Wang H Y, Liu M C, et al. A multiplex real-time PCR method applied to detect eight pollen species in food for the prevention of allergies[J]. European Food Research and Technology,2019,245(10):2195−2207. doi: 10.1007/s00217-019-03327-8 [73] Fernandes T J R, Costa J, Oliveira M B P P, et al. Exploiting 16S rRNA gene for the detection and quantification of fish as a potential allergenic food: A comparison of two real-time PCR approaches[J]. Food Chemistry,2018,245:1034−1041. doi: 10.1016/j.foodchem.2017.11.068 [74] Fernandes T J R, Costa J, Oliveira M B P P, et al. A new real-time PCR quantitative approach for the detection of shrimp crustaceans as potential allergens[J]. Journal of Food Composition and Analysis,2018,72:7−14. doi: 10.1016/j.jfca.2018.05.012 [75] Eischeid A C, Stadig S R. A group-specific, quantitative real-time PCR assay for detection of crab, a crustacean shellfish allergen, in complex food matrices[J]. Food Chemistry,2018,244:224−231. doi: 10.1016/j.foodchem.2017.10.045 [76] Eischeid A C. A method to detect allergenic fish, specifically cod and pollock, using quantitative real-time PCR and COI DNA barcoding sequences[J]. Journal of the Science of Food and Agriculture,2019,99(5):2641−2645. doi: 10.1002/jsfa.9466 [77] Villa C, Costa J, Mafra I. Detection and quantification of milk ingredients as hidden allergens in meat products by a novel specific real-time PCR method[J]. Biomolecules,2019,9(12):E804. doi: 10.3390/biom9120804 [78] Murphy H R, Lee S, Da Silva A J. Evaluation of an improved US food and drug administration method for the detection of Cyclospora cayetanensis in produce using real-time PCR[J]. Journal of Food Protection,2017,80(7):1133−1144. doi: 10.4315/0362-028X.JFP-16-492 [79] Temesgen T T, Robertson L J, Tysnes K R. A novel multiplex real-time PCR for the detection of Echinococcus multilocularis, Toxoplasma gondii, and Cyclospora cayetanensis on berries[J]. Food Research International,2019,125:108636. doi: 10.1016/j.foodres.2019.108636 [80] Frey C F, Oakley J R, Lobanov V A, et al. A novel protocol to isolate, detect and differentiate taeniid eggs in leafy greens and berries using real-time PCR with melting curve analysis[J]. Parasites & Vectors,2019,12:590. [81] Kim S Y, Kim M J, Jung S K, et al. Development of a fast real-time PCR assay based on TaqMan probe for identification of edible rice grasshopper (Oxya chinensis) in processed food products[J]. Food Research International,2019,116:441−446. doi: 10.1016/j.foodres.2018.08.059 [82] Kim M J, Jung S K, Kim S Y, et al. Development of detection method for edible silkworm (Bombyx mori) using real-time PCR[J]. Food Control,2018,94:295−299. doi: 10.1016/j.foodcont.2018.07.021 [83] Vandeweyer D, Milanović V, Garofalo C, et al. Real-time PCR detection and quantification of selected transferable antibiotic resistance genes in fresh edible insects from Belgium and the Netherlands[J]. International Journal of Food Microbiology,2019,290:288−295. doi: 10.1016/j.ijfoodmicro.2018.10.027 [84] Milanović V, Osimani A, Roncolini A, et al. Investigation of the dominant microbiota in ready-to-eat grasshoppers and mealworms and quantification of carbapenem resistance genes by qPCR[J]. Frontiers in Microbiology,2018,9:3036. doi: 10.3389/fmicb.2018.03036 -
