牡蛎诺如病毒多样性研究中病毒富集方法的评估
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摘要
牡蛎作为诺如病毒的载体,含有几乎所有的GI型和大部分GII型诺如病毒。为了有利于牡蛎中诺如病毒多样性的研究,对4种常用于宏基因组测序中病毒富集方法(PEG6000沉淀法、超速离心法、过滤法和直接提取法)进行比较评估,将含有GII. 4型诺如病毒基因的慢病毒(Lentivirus)加入阴性牡蛎中作为内参,利用反转录荧光定量PCR计算4种方法处理前后Lentivirus的数量,以回收率作为方法评估的指标,并以16S PCR来验证处理后微生物污染情况。重复本实验得到的两次结果均显示这4种方法回收率无明显差异,直接提取法回收率相对较高,但同时也具有较为严重的微生物污染。综合考虑其仍显示较好的优越性,因此直接提取方法更适用于后期牡蛎诺如病毒多样性研究中RNA的提取。
Oysters,the main vector of No Vs,contain almost all GI and most GII genogroup. In order to explore the diversity of the No Vs in oysters,this study aimed to evaluate four virus enrichment methods which were usually used in metagenomics study( PEG6000 precipitation method,ultracentrifugation method,filtration method and direct extraction method). Here,reverse transcription quantitative PCR( RT-qPCR) was adopted to calculate the Lentivirus( with GII. 4 No Vs gene) copies before and after treated with the four methods,and recovery rate of Lentivirus was the evaluation indicator. Besides that,16 S rRNA PCR was conducted to compare the microorganism contamination. Both results showed that these four methods had no obvious difference,and the direct extraction method had relatively higher recycle rate and more serious microorganism pollution. It demonstrated direct extraction method has relatively good superiority,which is more suitable to extract RNA for No Vs diversity in oysters.
Oysters,the main vector of No Vs,contain almost all GI and most GII genogroup. In order to explore the diversity of the No Vs in oysters,this study aimed to evaluate four virus enrichment methods which were usually used in metagenomics study( PEG6000 precipitation method,ultracentrifugation method,filtration method and direct extraction method). Here,reverse transcription quantitative PCR( RT-qPCR) was adopted to calculate the Lentivirus( with GII. 4 No Vs gene) copies before and after treated with the four methods,and recovery rate of Lentivirus was the evaluation indicator. Besides that,16 S rRNA PCR was conducted to compare the microorganism contamination. Both results showed that these four methods had no obvious difference,and the direct extraction method had relatively higher recycle rate and more serious microorganism pollution. It demonstrated direct extraction method has relatively good superiority,which is more suitable to extract RNA for No Vs diversity in oysters.
引文
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[2]VAN DUYNHOVEN Y T,DE JAGER C M,KORTBEEK L M,et al. A one-year intensified study of outbreaks of gastroenteritis in the Netherlands[J]. Epidemiology&Infection,2005,133(1):9-21.
[3]HALE A,MATTICK K,LEWIS D,et al. Distinct epidemiological patterns of Norwalk-like virus infection[J]. Journal of Medical Virology,2015,62(1):99-103.
[4]ITURRIZA-GOMARA M,ELLIOT A J,DOCKERY C,et al. Structured surveillance of infectious intestinal disease in pre-school children in the community:'The Nappy Study'[J]. Epidemiology and infection,2009,137(7):922-931.
[5]MIAO J,MING T,MING X,et al. Strain-specific interaction of a GII. 10norovirus with HBGAs[J]. Virology,2015,476:386-394.
[6]KRONEMAN A,VEGA E,VENNEMA H,et al. Proposal for a unified norovirus nomenclature and genotyping[J]. Archives of Virology,2013,158(10):2059-2068.
[7]吴琼,何玉林.诺如病毒的研究进展[J].中国人兽共患病学报,2014,30(12):1245-1251.
[8]DONALDSON E F,LINDESMITH L C,LOBUE A D,et al. Viral shapeshifting:norovirus evasion of the human immune system[J]. Nature Reviews Microbiology,2010,8(3):231-241.
[9]TAN M,JIANG X. Norovirus P particle:a subviral nanoparticle for vaccine development against norovirus,rotavirus and influenza virus[J].Nanomedicine,2012,7(6):889-897.
[10]高建.诺如病毒GⅡ. 4型Hunter504D株P粒子的表达、表征及免疫原性研究[D].长春:吉林大学,2013.
[11]宋维,李善爽,杨桂华,等.用大肠杆菌(Escherichia coli)表达诺如病毒P粒子[J].上海交通大学学报(农业科学版),2011,29(2):37-42.
[12]IMAMURA S,KANEZASHI H,GOSHIMA T,et al. Next-generation sequencing analysis of the diversity of human noroviruses in Japanese oysters[J]. Foodborne Pathogens&Disease,2017,14(8):465-471.
[13]SHI M,LIN X D,TIAN J H,et al. Redefining the invertebrate RNA virosphere[J]. Nature,2016,540(7634):539-543.
[14]IMAMURA S,HARUNA M,GOSHIMA T,et al. Application of nextgeneration sequencing to investigation of norovirus diversity in shellfish collected from two coastal sites in Japan from 2013 to 2014[J]. Japanese Journal of Veterinary Research,2016,64(2):113-122.
[15]IMAMURA S,HARUNA M,GOSHIMA T,et al. Application of nextgeneration sequencing to evaluate the profile of noroviruses in pre-and post-depurated oysters[J]. Foodborne Pathogens&Disease,2016,13(10):559-565.
[16]COTTEN M,KOOPMANS M. Next-generation sequencing and norovirus[J]. Future Virology,2016,11(11):719-722.
[17]钱明明,许海燕,宗晴,等.常规RT-PCR与荧光定量RT-PCR检测GⅠ、GⅡ型诺如病毒方法的建立及其应用[J].中国人兽共患病学报,2015,31(7):602-606.
[18]YU Y,CAI H,HU L,et al. Molecular epidemiology of oyster-related human noroviruses and their global genetic diversity and temporal-geographical distribution from 1983 to 2014[J]. Applied&Environmental Microbiology,2015,81(21):7615-7624.