草莓中诺如病毒和甲肝病毒的多重实时荧光逆转录PCR检测方法的建立
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摘要
目的建立草莓中诺如病毒GI、诺如病毒GII和甲肝病毒等3种食源性病毒的多重实时荧光RT-PCR检测方法,并应用于实际样品检测。方法对草莓样品进行前处理、病毒富集、病毒RNA提取和纯化后,先采用单重实时荧光RT-PCR进行检测,随后进行多重实时荧光RT-PCR反应条件优化,建立多重实时荧光RT-PCR检测方法并分析其特异性和灵敏度。结果所采用的病毒富集和核酸提取方法可以实现病毒的有效富集和抑制剂的去除,建立的多重实时荧光RT-PCR方法特异性强(100%),对草莓样品中诺如病毒GI、诺如病毒GII和甲肝病毒的检测灵敏度分别为56.2 RT-PCR50/20 g、31.6 RT-PCR50/20 g和31.4 CCID50/20 g。同时对50份样品进行检测,结果均为阴性。结论所建立的检测方法快速、灵敏、特异性强,适用于草莓产品中诺如病毒GI、诺如病毒GII和甲肝病毒的同时检测。
Objective To establish a multiplex real-time fluorescence RT-PCR detection method of norovirus GI,GII and hepatitis A virus in strawberry,and apply to actual sample. Methods After pretreatment of strawberry samples,viruses were concentrated,extracted and purified for virus RNA. The samples were determined by real-time fluorescence RT-PCR and multiplex real-time fluorescence RT-PCR. The specificity,sensitivity,and practical applications of the methods were also analyzed. Results The nucleic acid extraction method could effectively remove the inhibitory factors. The methods showed high specificity with no cross amplifications of other virus. The detection limit of multiplex real-time fluorescence PCR for norovirus GI,GII and hepatitis in strawberry was 56. 2 RT-PCR50/20 g,31. 6 RT-PCR50/20 g and 31. 4 CCID50/20 g,respectively. According to the test results of 50 inspection samples,the results were all negative. Conclusion The established system of nucleic acid extraction and real-time fluorescence RT-PCR detection method is suitable for the simultaneous detection of norovirus GI,GII and hepatitis A virus in strawberry.
Objective To establish a multiplex real-time fluorescence RT-PCR detection method of norovirus GI,GII and hepatitis A virus in strawberry,and apply to actual sample. Methods After pretreatment of strawberry samples,viruses were concentrated,extracted and purified for virus RNA. The samples were determined by real-time fluorescence RT-PCR and multiplex real-time fluorescence RT-PCR. The specificity,sensitivity,and practical applications of the methods were also analyzed. Results The nucleic acid extraction method could effectively remove the inhibitory factors. The methods showed high specificity with no cross amplifications of other virus. The detection limit of multiplex real-time fluorescence PCR for norovirus GI,GII and hepatitis in strawberry was 56. 2 RT-PCR50/20 g,31. 6 RT-PCR50/20 g and 31. 4 CCID50/20 g,respectively. According to the test results of 50 inspection samples,the results were all negative. Conclusion The established system of nucleic acid extraction and real-time fluorescence RT-PCR detection method is suitable for the simultaneous detection of norovirus GI,GII and hepatitis A virus in strawberry.
引文
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[15]张其刚,潘文良,李想,等.猪胃黏蛋白偶联磁珠和聚乙二醇富集检测青葱和葡萄中诺如病毒的比较研究[J].食品科学,2012,33(16):241-245.
[16]周阳,栾军,蒋鲁岩,等.实时荧光RT-PCR检测冷冻草莓中诺如病毒[J].食品安全质量检测学报,2013,4(2):515-520.
[17]莫雪梅,高东微.运用SYBR Green I荧光实时RT-PCR法检测草莓中甲肝病毒[J].食品科学,2010,31(14):153-157.
[18]曾军荣,李荣娇,王瑜玲,等.常见腹泻病毒的多重荧光RTPCR检测方法的建立及临床应用[J].广东医学,2011,32(13):1738-1740.
[19]Hoehne M,Schreier E.Detection of norovirus genogroup I and IIby multiplex real-time RT-PCR using a 3'-minor groove binderDNA probe[J].BMC Infectious Diseases,2005,6(9):1-6.
[20]纪蕾,韩健康,吴晓芳,等.多重荧光RT-PCR同时检测GI型和GII型诺如病毒方法的建立[J].中国人兽共患病学报,2011,27(4):311-315.
[21]ISO/TS 15216-2.Microbiology of food and animal feed-Horizontal method for determination of hepatitis A virus and norovirus in food using real-time RT-PCR-Part 2:method for qualitative detection[S].2013.
[22]张颖,吴风亮.RT-PCR方法检测贝类中诺沃克样病毒的研究[J].食品科学,2008,29(5):347-351.
[23]陈广全,饶红,张慧媛,等.用RT-PCR法检测食品中诺沃克样病毒[J].食品与发酵工业,2004,30(12):106-111.
[24]Marshall J A,Bruggink L D.Laboratory diagnosis of Norwalk viruse[J].Clin Lab,2006,52(11):571-581.
[25]卓菲,赵洁玲,文凤兰,等.实时荧光PCR检测诺瓦克病毒的应用研究[J].实用预防医学,2008,15(3):889-890.
[2]Myrmel M,Rimstad E,Wasteson Y.Immunomagnetic separation of a Norwalk-like virus(genogroup I)in artificially contaminated environmental water samples[J].Int J Food Microbiol,2001,62(1),17-26.
[3]Mead P S,Slutsker L,Diets V,et al.Food-related illness and death in the United States[J].Emerg Infect Dis,1999,5(5):607-625.
[4]Cotterelle B,Drougard C,Rolland J,et al.Outbreak of norovirus infection associated with the consumption of frozen raspberries,France,March 2005[J].Euro Surveill,2005,10(4):E050428.1.
[5]Falkenhorst G,Krussell L,Lisby M,et al.Imported frozen raspberries cause a series of norovirus outbreaks in Denmark,2005[J].Euro Surveill,2005,10(38):E050922.2.
[6]Le Guyader F S,Mittelholzer C,Haugarreau L,et al.Detection of noroviruses in raspberries associated with a gastroenteritis outbreak[J].Int J Food Microbiol,2004,97(2):179-186.
[7]Hernández F,Monge R,Jiménez C,et al.Rotavirus and hepatitis A virus on market lettuce(Latuca sativa)in Costa Rica[J].Int J Food Microbiol,1997,37(2/3):221-223.
[8]Rutjes S A,Lodder-Verschoor F,van der Poel W H M,et al.Detection of noroviruses in foods:a study on virus extraction procedures in foods implicated in outbreaks of human gastroenteritis[J].J Food Prot,2006,69(8),1949-1956.
[9]Lopman B A,Adak G K,Reacher M H,et al.Two epidemiologic patterns of norovirus outbreaks:surveillance in England and Wales,1992-2000[J].Emerg Infect Dis,2003,9(1):71-77.
[10]陈广全,饶红,傅浦博,等.食品中诺沃克样病毒和甲肝病毒检测方法研究进展[J].检验检疫科学,2005,15(6):51-54.
[11]袁巧,王晨,李晖,等.贝类中诺如病毒检测方法的研究进展[J].华南预防医学,2012,38(50):30-33.
[12]饶红,陈广全,冯骞,等.蔬菜及水果中NLVs和甲肝检测的研究[J].中国食品卫生杂志,2007,19(2):131-134.
[13]Hee-Yeon K,In-Shin K,In-Gyun H,et al.Optimization of methods for detecting norovirus on various fruit[J].Journal of Virological Methods,2008,153(2),104-110.
[14]Stals A,Baert L,Van Coillie E,et al.Evaluation of a norovirus detection methodology for soft red fruits[J].Food Microbiology,2011,28(1):52-58.
[15]张其刚,潘文良,李想,等.猪胃黏蛋白偶联磁珠和聚乙二醇富集检测青葱和葡萄中诺如病毒的比较研究[J].食品科学,2012,33(16):241-245.
[16]周阳,栾军,蒋鲁岩,等.实时荧光RT-PCR检测冷冻草莓中诺如病毒[J].食品安全质量检测学报,2013,4(2):515-520.
[17]莫雪梅,高东微.运用SYBR Green I荧光实时RT-PCR法检测草莓中甲肝病毒[J].食品科学,2010,31(14):153-157.
[18]曾军荣,李荣娇,王瑜玲,等.常见腹泻病毒的多重荧光RTPCR检测方法的建立及临床应用[J].广东医学,2011,32(13):1738-1740.
[19]Hoehne M,Schreier E.Detection of norovirus genogroup I and IIby multiplex real-time RT-PCR using a 3'-minor groove binderDNA probe[J].BMC Infectious Diseases,2005,6(9):1-6.
[20]纪蕾,韩健康,吴晓芳,等.多重荧光RT-PCR同时检测GI型和GII型诺如病毒方法的建立[J].中国人兽共患病学报,2011,27(4):311-315.
[21]ISO/TS 15216-2.Microbiology of food and animal feed-Horizontal method for determination of hepatitis A virus and norovirus in food using real-time RT-PCR-Part 2:method for qualitative detection[S].2013.
[22]张颖,吴风亮.RT-PCR方法检测贝类中诺沃克样病毒的研究[J].食品科学,2008,29(5):347-351.
[23]陈广全,饶红,张慧媛,等.用RT-PCR法检测食品中诺沃克样病毒[J].食品与发酵工业,2004,30(12):106-111.
[24]Marshall J A,Bruggink L D.Laboratory diagnosis of Norwalk viruse[J].Clin Lab,2006,52(11):571-581.
[25]卓菲,赵洁玲,文凤兰,等.实时荧光PCR检测诺瓦克病毒的应用研究[J].实用预防医学,2008,15(3):889-890.