食品中甲型肝炎病毒RNA提取方法的比较及应用
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摘要
目的:对食品中甲型肝炎病毒的3种RNA提取方法进行综合比较,以优选出最佳的核酸提取方法,为各级实验室开展食源性甲型肝炎病毒核酸检验提供技术参考。方法:分别采用ABI AM1836、QIAGEN 74104和ROCHE11858882001三种商业化核酸提取试剂盒对人工污染甲型肝炎病毒的食品样品进行核酸提取,根据3种核酸提取方法的提取效率、抑制剂去除效率、检测灵敏度、综合应用指标进行病毒RNA的优化,且采用优化后的提取方法进行实际样品的检测。结果:综合病毒各项评价指标显示,ROCHE 11858882001在食品中提取甲型肝炎病毒RNA效果最优,其次为QIAGEN 74104和ABI AM1836。101份样品的检测结果显示,一份蓝靛果样品为甲型肝炎病毒核酸阳性,其余样品均为阴性。结论:ROCHE 11858882001是一种快捷有效的病毒RNA提取方法,适用于食品中甲型肝炎病毒的日常检测工作。
Objective: Three RNA extraction kits were compared to select the best one for RNA extraction from hepatitis A virus(HAV) in foods, aiming to provide a technical basis for foodborne HAV RNA detection in laboratories. Methods:After artificial contamination of known negative samples, three commercial nucleic acid extraction kits, ABI AM1836,QIAGEN 74104 and ROCHE 11858882001, were used to extract HAV RNA and their performance was evaluated in terms of extraction ef?ciency, inhibitor removal ef?ciency and sensitivity, and the optimized extraction kit was applied to real samples. Results: ROCHE 11858882001 was the best extraction kit, followed by QIAGEN 74104 and ABI AM1836. Of 101 samples, one sample of blue honeysuckle was positive while the rest were negative. Conclusion: ROCHE 11858882001 is a rapid and effective method for extracting HAV RNA, and thus it is suitable for the routine detection of foodborne HAV.
Objective: Three RNA extraction kits were compared to select the best one for RNA extraction from hepatitis A virus(HAV) in foods, aiming to provide a technical basis for foodborne HAV RNA detection in laboratories. Methods:After artificial contamination of known negative samples, three commercial nucleic acid extraction kits, ABI AM1836,QIAGEN 74104 and ROCHE 11858882001, were used to extract HAV RNA and their performance was evaluated in terms of extraction ef?ciency, inhibitor removal ef?ciency and sensitivity, and the optimized extraction kit was applied to real samples. Results: ROCHE 11858882001 was the best extraction kit, followed by QIAGEN 74104 and ABI AM1836. Of 101 samples, one sample of blue honeysuckle was positive while the rest were negative. Conclusion: ROCHE 11858882001 is a rapid and effective method for extracting HAV RNA, and thus it is suitable for the routine detection of foodborne HAV.
引文
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[8]房保海,岳志芹,赵玉然,等.果蔬生产用水中甲肝病毒检测方法和质控体系的研究[J].卫生研究,2017,46(1):99-102.DOI:1000-8020(2017)01-0099-05.
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[22]雷永良,王晓光,梅少林,等.食品中食源性病毒快速检测方法的建立研究[J].中国卫生检验杂志,2014(6):855-857.DOI:1004-8685(2014)06-0855-03.
[23]徐蕾蕊,魏海燕,马丹,等.MS2噬菌体在贝类食源性病毒检测中过程质控应用[J].中国公共卫生,2016,32(11):1584-1590.DOI:10.11847/zgggws2016-32-11-37.
[24]TERZI G,ALBAYRAK H,SIRIKEN B,et al.Detection of enteroviruses and hepatitis A virus RNA in cow milk by RT-PCR[J].Acta Veterinaria,2010,60(2/3):197-204.DOI:10.2298/AVB1003197T.
[25]吕艳,王静静,罗瑶瑶,等.五种核酸提取试剂盒对新城疫病毒RNA提取效率的比较[J].中国动物检疫,2017,34(6):98-101.DOI:10.3969/j.issn.1005-944X.2017.06.028.
[26]王赤华,曾勇.甲型H1N1流感病毒采用不同核酸提取方法的比较研究[J].临床和实验医学杂志,2017,16(3):230-232.DOI:10.3969/j.issn.1671-4695.2017.03.007.
[27]赵怡,陈苏红,刘志红,等.6种核酸提取试剂盒对甲型H1N1流感病毒核酸提取效果的比较[J].军事医学,2010,34(2):111-114.DOI:10.3969/j.issn.1674-9960.2010.02.003.
[28]饶红,陈广全,曾静,等.RT-PCR法检测贝类中的甲肝病毒的研究[J].食品与发酵工业,2006,32(9):122-125.DOI:10.3321/j.issn:0253-990X.2006.09.030.
[29]饶红,陈广全,冯骞,等.蔬菜及水果中NLVs和HAV检测的研究[J].中国食品卫生杂志,2007,19(2):131-134.DOI:10.3969/j.issn.1004-8456.2007.02.010.
[30]DUBOIS E,AGIER C,TRAORéO,et al.Modified concentration method for the detection of enteric viruses on fruits and vegetables by reverse transcriptase-polymerase chain reaction or cell culture[J].Journal of Food Protection,2002,65(12):1962-1969.DOI:10.4315/0362-028X-65.12.1962.
[31]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.
[32]LI D,BUTOT S,ZUBER S,et al.Monitoring of foodborne viruses in berries and considerations on the use of RT-PCR methods in surveillance[J].Food Control,2018,89:235-240.DOI:10.1016/j.foodcont.2018.02.024.
[2]訾梅,蔡皓东.甲肝疫苗的不良反应综合报道[J].药物流行病学杂志,2006,15(4):218-222.DOI:10.3969/j.issn.1005-0698.2006.04.011.
[3]施建平,杨锦玲.某农村幼儿园52例病毒性甲型肝炎暴发的流行病学分析[J].宁夏医学杂志,2010,32(9):856-857.DOI:10.3969/j.issn.1001-5949.2010.09.062.
[4]张艺,许大庆,张雪峰,等.2005-2015年安陆市甲型病毒性肝炎流行病学分析[J].应用预防医学,2016,22(6):509-510.DOI:10.3969/j.issn.1673-758X.2016.06.012.
[5]焦永真,韩剑秋,王宪明,等.1988年上海甲型肝炎暴发流行中从毛蚶分离到甲型肝炎病毒[J].病毒学报,1990(4):312-315.DOI:10.13242/j.cnki.bingduxuebao.000621.
[6]俞顺章.甲型肝炎流行促进了“大卫生”的诞生[J].上海预防医学,2017,29(1):1-3.DOI:10.19428/j.cnki.sjpm.2017.01.001.
[7]李楠,王佳慧,李凤琴,等.检测鲜草中G II型诺如病毒的两种富集方法比较[J].中国食品卫生杂志,2015,27(3):242-246.DOI:10.13590/j.cjfh.2015.03.005.
[8]房保海,岳志芹,赵玉然,等.果蔬生产用水中甲肝病毒检测方法和质控体系的研究[J].卫生研究,2017,46(1):99-102.DOI:1000-8020(2017)01-0099-05.
[9]中国质量新闻网.山东检验检疫局:冷冻草莓别样甜[R/O L].(2015-02-27)[2018-03-19].http://www.cqn.com.cn/news/zggmsb/diyi/1008917.html.
[10]新华网.冷冻草莓澳洲陷甲肝病毒“乌龙”中国企业损失惨重[R/OL].(2015-12-9)[2015-12-9].http://www.xinhuanet.com/overseas/2015-12/15/c_128531031_3.htm.
[11]国际果蔬报道.山东草莓雪上加霜:被新西兰初级产业-部召回[R/OL].(2015-12-9)[2018-03-19].http://www.guojiguoshu.com/article/1759?qt-popular_content=3.
[12]SINCERO T C,LEVIN D B,SIM?ES C M,et al.Detection of hepatitis A virus(HAV)in oysters(Crassostrea gigas)[J].Water Research,2006,40(5):895-902.DOI:10.1016/j.watres.2005.12.005.
[13]DE PAULA V S,VILLAR L M,COIMBRA GASPAR A M.Comparison of four extraction methods to detect hepatitis A virus RNAin serum and stool samples[J].Brazilian Journal of Infectious Diseases an Official Publication of the Brazilian Society of Infectious Diseases,2003,7(2):135-141.DOI:10.1590/S1413-86702003000200007.
[14]CHIRGWIN J M,PRZYBYLA A E,MACDONALD R J,et al.Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease[J].Biochemistry,1979,18(24):5294-5299.DOI:10.1021/bi00591a005.
[15]张丽,杨莲茹,吴绍强.核酸提取方法的研究进展[J].中国动物检疫,2011,28(12):75-78.DOI:10.3969/j.issn.1005-944X.2011.12.032.
[16]吴星,黄维金,周诚,等.五种乙型肝炎病毒核酸荧光定量检测试剂盒的比较[J].中华传染病杂志,2009,27(3):142-146.DOI:10.3760/cma.j.issn.1000-6680.2009.03.004.
[17]王聪,陈之遥,武海萍,等.三种核酸共提取试剂盒对血液病毒提取效能的比较研究[J].临床误诊误治,2011,24(8):6-9.DOI:10.3969/j.issn.1002-3429.2011.08.003.
[18]XU R,SHIEH Y C,STEWART D S.Comparison of RNA extraction kits for the purification and detection of an enteric virus surrogate on green onions via RT-PCR[J].Journal of Virological Methods,2017,239:61-68.DOI:10.1016/j.jviromet.2016.10.016.
[19]International Organization for Standards.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:ISO/TS 15216-2[S].Geneva:International Organization for Standardization,2013:1-27.
[20]高世光,王海燕,郭慧,等.草莓中甲型肝炎病毒检测[J].食品科学,2014,35(24):213-218.DOI:10.7506/spkx1002-6630-201424041.
[21]房保海,岳志芹,孙涛,等.果蔬产品中甲型肝炎病毒风险评估和基因分型研究[J].病毒学报,2016,32(4):484-489.DOI:10.13242/j.cnki.bingduxuebao.002990.
[22]雷永良,王晓光,梅少林,等.食品中食源性病毒快速检测方法的建立研究[J].中国卫生检验杂志,2014(6):855-857.DOI:1004-8685(2014)06-0855-03.
[23]徐蕾蕊,魏海燕,马丹,等.MS2噬菌体在贝类食源性病毒检测中过程质控应用[J].中国公共卫生,2016,32(11):1584-1590.DOI:10.11847/zgggws2016-32-11-37.
[24]TERZI G,ALBAYRAK H,SIRIKEN B,et al.Detection of enteroviruses and hepatitis A virus RNA in cow milk by RT-PCR[J].Acta Veterinaria,2010,60(2/3):197-204.DOI:10.2298/AVB1003197T.
[25]吕艳,王静静,罗瑶瑶,等.五种核酸提取试剂盒对新城疫病毒RNA提取效率的比较[J].中国动物检疫,2017,34(6):98-101.DOI:10.3969/j.issn.1005-944X.2017.06.028.
[26]王赤华,曾勇.甲型H1N1流感病毒采用不同核酸提取方法的比较研究[J].临床和实验医学杂志,2017,16(3):230-232.DOI:10.3969/j.issn.1671-4695.2017.03.007.
[27]赵怡,陈苏红,刘志红,等.6种核酸提取试剂盒对甲型H1N1流感病毒核酸提取效果的比较[J].军事医学,2010,34(2):111-114.DOI:10.3969/j.issn.1674-9960.2010.02.003.
[28]饶红,陈广全,曾静,等.RT-PCR法检测贝类中的甲肝病毒的研究[J].食品与发酵工业,2006,32(9):122-125.DOI:10.3321/j.issn:0253-990X.2006.09.030.
[29]饶红,陈广全,冯骞,等.蔬菜及水果中NLVs和HAV检测的研究[J].中国食品卫生杂志,2007,19(2):131-134.DOI:10.3969/j.issn.1004-8456.2007.02.010.
[30]DUBOIS E,AGIER C,TRAORéO,et al.Modified concentration method for the detection of enteric viruses on fruits and vegetables by reverse transcriptase-polymerase chain reaction or cell culture[J].Journal of Food Protection,2002,65(12):1962-1969.DOI:10.4315/0362-028X-65.12.1962.
[31]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.
[32]LI D,BUTOT S,ZUBER S,et al.Monitoring of foodborne viruses in berries and considerations on the use of RT-PCR methods in surveillance[J].Food Control,2018,89:235-240.DOI:10.1016/j.foodcont.2018.02.024.