贝类中诺如病毒实时荧光定量PCR检测方法的建立及应用
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摘要
目的建立实时荧光定量PCR法检测GⅠ、GⅡ型诺如病毒的方法。对东海区域市场内零售贝类海产品中诺如病毒携带情况进行监测,为海产品食品安全和风险预警提供科学依据。方法采集浙江沿海地区市场上常见贝类,利用实时荧光定量PCR检测技术对贝类进行检测。结果 2 955份样品中有63份样品携带诺如病毒,其中杂色蛤、太平洋牡蛎、僧帽牡蛎、毛蚶中均检出GⅡ型诺如病毒。贝类样品中太平洋牡蛎的检出率最高,为15.64%。贝类中诺如病毒在冬季(11月-次年2月)的检出率明显高于夏季(6月-9月),其季节分布差异有统计学意义(χ~2=31.3,P<0.05)。结论东海区域贝类中,特别是生食或半熟食贝类中诺如病毒的携带水平较高、分布较广,应持续做好贝类中诺如病毒携带监测,同时做好风险预警。
Objective To establish the method of detecting GI and GII norovirus through real-time RT-PCR,so as to provide scientific basis for seafood food safety and early risk warning through the detection of the norovirus in retail shellfish seafood in the area of East China Sea.Methods Common shellfish on the market of Zhejiang coastal areas was collected and then detected through RT-PCR detection technology.Results Among the 2 955 samples,63 samples carried norovirus.To be specific,GII norovirus was detected in venerupis variegata,crassostrea gigas,ostrea cucullata and scapharca subcrenata.Among the shellfish samples,crassostrea gigas had the highest detection rate of norovirus(15.64%).Besides,the detection rate of norovirus in shellfish in winter(November to February) was significantly higher than that in summer(June to September),with the season distribution difference statistically significant(χ~2= 31.3,P<0.05).Conclusion Shellfish in the East China sea area,especially raw or half cooked shellfish,had high incidence and wide distribution of norovirus.Thus,it is advisable to constantly monitor norovirus carrying in shellfish and do early risk warning measure.
Objective To establish the method of detecting GI and GII norovirus through real-time RT-PCR,so as to provide scientific basis for seafood food safety and early risk warning through the detection of the norovirus in retail shellfish seafood in the area of East China Sea.Methods Common shellfish on the market of Zhejiang coastal areas was collected and then detected through RT-PCR detection technology.Results Among the 2 955 samples,63 samples carried norovirus.To be specific,GII norovirus was detected in venerupis variegata,crassostrea gigas,ostrea cucullata and scapharca subcrenata.Among the shellfish samples,crassostrea gigas had the highest detection rate of norovirus(15.64%).Besides,the detection rate of norovirus in shellfish in winter(November to February) was significantly higher than that in summer(June to September),with the season distribution difference statistically significant(χ~2= 31.3,P<0.05).Conclusion Shellfish in the East China sea area,especially raw or half cooked shellfish,had high incidence and wide distribution of norovirus.Thus,it is advisable to constantly monitor norovirus carrying in shellfish and do early risk warning measure.
引文
[1]U.S.Department of Health&Human Services.Norovirus Worldwide[EB/OL].[2015-09-30].http://www.cdc.gov/norovirus/worldwide.html.
[2]Ahmed SM,Hall AJ,Robinson AE,et al.Global prevalence of norovirus in cases of gastroenteritis:a systematic review and metaanalysis[J].Lancet Infect Dis,2014,14(8):725-730.
[3]Ahmed SM,Lopman BA,Levy K.A systematic review and metaanalysis of the global seasonality of norovirus[J].PLo S ONE,2013,8(10):e75922.
[4]Siebenga JJ,Vennema H,Zheng DP,et al.Norovirus illness is a global problem:emergence and spread of norovirus GII.4 variants,2001–2007[J].J Infect Dis,2009,200(5):802-812.
[5]陈传德,周晓红,莫艳玲,等.实时荧光定量PCR定量检测诺如病毒方法的建立及其评价[J].中国卫生检验杂志,2010,20(3):467-469,558.
[6]纪蕾,韩建康,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.
[7]钱明明,许海燕,宗晴,等.常规RT-PCR与荧光定量PCR检测GⅠ、GⅡ型诺如病毒方法的建立及应用[J].中国人兽共患病学报,2015,31(7):602-611.
[8]高世光,冯华炜,林天闻,等.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[J].中国食品卫生杂志,2015,27(5):512-517.
[9]柳淑芳,李振,周德庆.青岛地区贝类产品中诺如病毒的感染和流行初探[J].渔业科学进展,2009,30(1):61-66.
[10]Westrell T,Dusch V,Ethelberg S,et al.Norovirus out-breaks linked to oyster consumotion in the United Kingdom,Norway,France,Sweden and Denmark[EB/OL].[2010-05-25].http://eurosurveillance.org/images/dynamic/EE/V15N12/art195-24.
[11]李晖,黄吉成,方苓,等.贝类海产品中诺如病毒快速检测方法研究[J].中国卫生检验杂志,2006,16(11):1300-1302.
[12]李海波,邵亚萍,张宏萍,等.南通地区沿海贝类诺瓦克病毒携带情况调查[J].中国卫生检验杂志,2011,21(9):2310-2311.
[13]苏来金,马丽萍,徐仰丽,等.山东半岛地区贝类中诺如病毒污染状况与病毒鉴定初报[J].中国人兽共患病学报,2010,26(5):451-454.
[14]周德庆,苏来金,赵峰,等.诺如病毒在贝类中的富集特性与机制研究进展[J].病毒学报,2015,31(3):313-317.
[15]郭志刚,杨作升,范德江,等.长江口泥质区的季节性沉积效应[J].地理学报,2003,58(4):591-597.
[2]Ahmed SM,Hall AJ,Robinson AE,et al.Global prevalence of norovirus in cases of gastroenteritis:a systematic review and metaanalysis[J].Lancet Infect Dis,2014,14(8):725-730.
[3]Ahmed SM,Lopman BA,Levy K.A systematic review and metaanalysis of the global seasonality of norovirus[J].PLo S ONE,2013,8(10):e75922.
[4]Siebenga JJ,Vennema H,Zheng DP,et al.Norovirus illness is a global problem:emergence and spread of norovirus GII.4 variants,2001–2007[J].J Infect Dis,2009,200(5):802-812.
[5]陈传德,周晓红,莫艳玲,等.实时荧光定量PCR定量检测诺如病毒方法的建立及其评价[J].中国卫生检验杂志,2010,20(3):467-469,558.
[6]纪蕾,韩建康,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.
[7]钱明明,许海燕,宗晴,等.常规RT-PCR与荧光定量PCR检测GⅠ、GⅡ型诺如病毒方法的建立及应用[J].中国人兽共患病学报,2015,31(7):602-611.
[8]高世光,冯华炜,林天闻,等.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[J].中国食品卫生杂志,2015,27(5):512-517.
[9]柳淑芳,李振,周德庆.青岛地区贝类产品中诺如病毒的感染和流行初探[J].渔业科学进展,2009,30(1):61-66.
[10]Westrell T,Dusch V,Ethelberg S,et al.Norovirus out-breaks linked to oyster consumotion in the United Kingdom,Norway,France,Sweden and Denmark[EB/OL].[2010-05-25].http://eurosurveillance.org/images/dynamic/EE/V15N12/art195-24.
[11]李晖,黄吉成,方苓,等.贝类海产品中诺如病毒快速检测方法研究[J].中国卫生检验杂志,2006,16(11):1300-1302.
[12]李海波,邵亚萍,张宏萍,等.南通地区沿海贝类诺瓦克病毒携带情况调查[J].中国卫生检验杂志,2011,21(9):2310-2311.
[13]苏来金,马丽萍,徐仰丽,等.山东半岛地区贝类中诺如病毒污染状况与病毒鉴定初报[J].中国人兽共患病学报,2010,26(5):451-454.
[14]周德庆,苏来金,赵峰,等.诺如病毒在贝类中的富集特性与机制研究进展[J].病毒学报,2015,31(3):313-317.
[15]郭志刚,杨作升,范德江,等.长江口泥质区的季节性沉积效应[J].地理学报,2003,58(4):591-597.