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食品与发酵工业  2020, Vol. 46 Issue (8): 219-225    DOI: 10.13995/j.cnki.11-1802/ts.022950
  分析与检测 本期目录 | 过刊浏览 | 高级检索 |
基于实时荧光定量PCR检测的沙门氏菌标准物质验证
王菲1, 张玲2, 杨佳怡2, 叶子弘1, 俞晓平1*
1 (中国计量大学,浙江省生物计量及检验检疫技术重点实验室,浙江 杭州,310018)
2 (中国计量科学研究院,北京, 100013)
Verification of reference material for detecting Salmonella based on the real-time fluorescence quantitative PCR method
WANG Fei1, ZHANG Ling2, YANG Jiayi2, YE Zihong1, YU Xiaoping1*
1 (College of Life Sciences, China Jiliang University, Hangzhou 310018, China)
2 (National Institute of Metrology, Beijing 100013, China)
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摘要 该研究拟采用沙门氏菌核酸标准物质验证实时荧光定量PCR检测沙门氏菌,并对沙门氏菌核酸标准物质的适用性、均匀性、稳定性进行考察。使用不同的PCR仪进行检测比较,建立不同的PCR酶体系,并在沙门氏菌核酸标准物质中添加保护剂,以此来验证沙门氏菌核酸标准物质的适用性;同时根据线性图、参数表对沙门氏菌核酸标准物质进行均匀性、短期稳定性和反复冻融稳定性分析。结果表明研究设计的荧光定量PCR方法辅以标准物质具有良好的种属特异性,适用于常见的PCR仪和不同的酶体系,保护剂不影响沙门氏菌核酸的荧光扩增。研制的沙门氏菌标准物质样品均匀,未稀释的沙门氏菌核酸检测样在实验中6 d内量值保持稳定,反复冻融10次后的量值也相对稳定,溶解于保护剂的低浓度沙门氏菌核酸检测样稳定性加强,但低浓度沙门氏菌核酸检测样短期稳定性及反复冻融实验量值不稳定。辅以标准物质的实时荧光定量PCR技术检测准确度高且稳定,可用来验证各实验室的检测能力,实现微生物核酸检测技术的快速、准确、可溯源。
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王菲
张玲
杨佳怡
叶子弘
俞晓平
关键词:  荧光定量PCR  沙门氏菌  标准物质  稳定性  验证    
Abstract: Salmonella is a kind of food-borne pathogen which belongs to Enterobacteriaceae. However, the content of Salmonella in food is usually low, the routine test is time-consuming, so the detection with high sensitivity, repeatability and accuracy is needed. The nucleic acid reference material of Salmonella was used to verify the real-time fluorescence quantitative PCR (qRT-PCR) method for detecting Salmonella, the applicability, uniformity and stability of nucleic acid reference material of Salmonella were investigated in this study. To optimize the nucleic acid reference materials for verifying qRT-PCR method in detection of Salmonella, various enzyme systems for PCR and protective agents for nucleic acid reference materials were studied. The homogeneity, short-term stability and repeated freeze-thaw stability of nucleic acid reference materials were analyzed based on the linear graph and parameter table. The results showed that qRT-PCR method with relevant nucleic acid reference materials in this study was specific to Salmonella amplification, it was applicable to commonly used PCR instruments and various enzyme systems. The protective agent in reference materials did not inhibit the fluorescence amplification of Salmonella. The quality evaluation of reference material indicated that Salmonella nucleic acid samples were uniform, it remained stable within 6 days in the stability test without dilution. The quantity was stable after repeated freezing and thawing for 10 times. And the stability of diluted Salmonella nucleic acid sample was enhanced after adding protective agent. However, the short-term stability and quantity of diluted Salmonella nucleic acid samples were unstable in repeated freezing and thawing tests. The RT-PCR with reference material has high accuracy and stability in detecting Salmonella. It can be used to verify the detection ability of laboratories in rapidly and accurately detecting microbial nucleic acid with traceability.
Key words:  qRT-PCR    Salmonella    reference materials    stability    verification
收稿日期:  2019-12-03                出版日期:  2020-04-25      发布日期:  2020-05-20      期的出版日期:  2020-04-25
基金资助: 国家重点研发计划项目(2018YFC1200502;2018YFE0201602;2017YFF0204600;2017YFF004601)
作者简介:  硕士研究生(俞晓平研究员为通讯作者,E-mail:yxp@cjlu.edu.cn)
引用本文:    
王菲,张玲,杨佳怡,等. 基于实时荧光定量PCR检测的沙门氏菌标准物质验证[J]. 食品与发酵工业, 2020, 46(8): 219-225.
WANG Fei,ZHANG Ling,YANG Jiayi,et al. Verification of reference material for detecting Salmonella based on the real-time fluorescence quantitative PCR method[J]. Food and Fermentation Industries, 2020, 46(8): 219-225.
链接本文:  
http://sf1970.cnif.cn/CN/10.13995/j.cnki.11-1802/ts.022950  或          http://sf1970.cnif.cn/CN/Y2020/V46/I8/219
[1] 佘容,罗丹,刘耀敏,等.TaqMan荧光定量PCR在饲料沙门氏菌检测中的应用评估[J]. 中国预防兽医学报, 2015, 37(12): 947-951.
[2] 庄春红,吴小凤,郑迎翔,等.福建省泉州地区2013-2016年沙门氏菌的PFGE分子分型及耐药性研究[J]. 医学动物防制, 2019, 35(6):540-543.
[3] XIONG Dan, SONG Li, PAN Zhiming, et al. Identification and Discrimination of Salmonella enterica serovar gallinarum biovars pullorum and gallinarum based on a one-step multiplex PCR[J]. Frontiers in Microbiology, 2018, 9:1 718.
[4] 段小丽,董立伟,朱国强,等.双重PCR检测肠炎沙门氏菌方法的建立[J]. 中国家禽, 2012, 34(17): 20-22.
[5] CARLONJ E,ROTUDOL,BRAND I, et al. Rapid and simultaneous detection of Salmonella spp., Escherichia coli O157, and Listeria monocytogenes by magnetic capture hybridization and multiplex real-time PCR[J]. Folia Microbiologica, 2018, 63(6):735-742.
[6] ALFAMAE R G,HESSEL C T,DEOLIVEIRA E S, et al. Assessment of temperature distribution of cold and hot meals in food services and the prediction growth of Salmonella spp. and Listeria monocytogenes[J]. Food Control, 2019,91:501-970.
[7] 杨云斌,许月琴.不同选择性培养基中沙门氏菌和干扰菌的分离鉴定[J]. 食品安全质量检测学报, 2019,10(4): 977-982.
[8] 刘骆强,姚艳玲,管佳丽,等.5种食源性致病菌PCR检测方法的建立[J]. 食品安全质量检测学报, 2019,10(5): 1 330-1 335.
[9] NADIN-DAVIS S, POPE L, OGUNREMI D, et al. A real-time PCR regimen for testing environmental samples for Salmonella enterica subsp. enterica serovars of concern to the poultry industry, with special focus on Salmonella Enteritidis[J].Canadian Journal of Microbiology, 2019, 65(2): 162-173.
[10] 汪华,李俊岐,常怡虹.运用实时荧光PCR法辅助鉴定能力验证样品中的沙门氏菌[J]. 食品安全质量检测学报, 2019, 10(3): 598-602.
[11] 陈旭,毛瑞,乔宇,等.基于竞争性互补介导核酸恒温扩增技术快速检测沙门氏菌[J]. 沈阳农业大学学报, 2019,50(2): 179-184.
[12] 张红莉,李勇,许均华,等.4种检测方法对巧克力中亚利桑那沙门氏菌的检测结果比较[J]. 食品研究与开发, 2019,40(9):172-176.
[13] 黄宝莹,余之蕴,林耀文,等. 四种方法检测食品中沙门氏菌的比较[J]. 食品工业科技, 2014,35(15):185-187;192.
[14] 国家卫生和计划生育委员会, 国家食品药品监督管理总局. 食品安全国家标准食品微生物学检测沙门氏菌检验GB4789.4——2016[S]. 北京:中国标准出版社, 2017.
[15] LIU Yuejiao, SINGH P, MUSTAPHA A. Multiplex high resolution melt-curve real-time PCR assay for reliable detection of Salmonella[J].Food Control, 2018, 91(9): 225-230.
[16] 赵莹彤,浑婷婷,詹悦维,等.基于微流控的真菌单细胞捕获和培养[J]. 微生物学通报, 2019, 46(3): 522-530.
[17] 刘祥琴,刘小琦.荧光定量PCR仪检测HBV DNA结果比对分析[J]. 检验医学与临床, 2016, 13(6): 791-792.
[18] 李小龙,吴群,徐岩.乙醇与酸度协同作用推动芝麻香型白酒固态发酵过程的微生物群落演替[J]. 微生物学通报, 2019, 46(1): 1-10.
[19] 全国标准物质管理委员会. 标准物质定值原则和统计学原理[M]. 北京:中国质检出版社, 2011:72-73.
[20] LUGERT R, SCHETTLER C, GROSS U. Comparison of different protocols for DNA preparation and PCR for the detection of fungal pathogens in vitro[J]. Mycoses, 2006, 49(4):298-304.
[21] FEY H, PFISTER H, RÜEGG O. Comparative evaluation of different enzyme-linked immunosorbent assay systems for the detection of staphylococcal enterotoxins A, B, C, and D[J]. Clin Microbiol. 1984, 19(1):34-38.
[22] VECCIO P D. Effect of osmoregulatory solutes on the thermal stability of calf-thymus DNA[J]. Journal of the Chemical Society, Faraday Transactions, 1996, 92(8):1 361-1 367.
[23] 汤妍雯,曹帅英,黄世英,等.溴化丁基橡胶门尼黏度标准物质均匀性与稳定性评价[J]. 合成材料老化与应用, 2018,47(5):83-87.
[24] YANG Mengrui, LIU Fang, WANG Min, et al. Development of a whole liquid egg certified reference material for accurate measurement of enrofloxacin residue[J].Food Chemistry, 2019, 309:125 253.
[25] DJEBBL-SIMMONS D, XU W Q, JANES M, et al. Survival and inactivation of Salmonella enterica serovar Typhimurium on food contact surfaces during log, stationary and long-term stationary phases[J]. Food Microbiology, 2019, 84:103 272.
[26] HAIM-VILMOVSKY L. High-throughput single-cell real-time quantitativePCR analysis[J]. Methods in Molecular Biology, 2019,1979:177-183.
[27] NIU D L, ZHAO Y E, GONG X J, et al. Screening of reference genes and quantitative real-time PCR detection and verification in Dermatophagoides farinae under temperature stress[J]. Experimental Parasitology, 2019,206:107 754.
[28] SAKURIA A,NOMURAN,NANBAR, et al. Rapid typing of influenza viruses using super high-speed quantitative real-time PCR[J]. Journal of Virological Methods, 2011,178(1-2):75-81.
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