本文選題：諾如病毒 + NoV�。� 參考：《江蘇大學(xué)》2017年碩士論文

【摘要】：目的:諾如病毒(Noroviruses,NoVs)是鎮(zhèn)江地區(qū)食源性疾病及突發(fā)公共衛(wèi)生事件的常見病原體,極易引起人群中的暴發(fā)流行。為明確NoV在鎮(zhèn)江地區(qū)的分子流行病學(xué)特征及其與腸道菌群的關(guān)系,本研究從分子生物學(xué)、生物信息學(xué)、基因組學(xué)等方面出發(fā),系統(tǒng)分析本地區(qū)NoV的分子流行病學(xué)特征、進(jìn)化、重組及對(duì)腸道菌群關(guān)系的影響。方法:1.為了分析NoV不同基因型在鎮(zhèn)江的流行,本研究采用實(shí)時(shí)熒光定量PCR(q RT-PCR)和逆轉(zhuǎn)錄PCR(RT-PCR)相結(jié)合的方法。收集2015.1-2016.12鎮(zhèn)江市食源性疾病常規(guī)腹瀉患者的糞便樣本及突發(fā)公共衛(wèi)生事件的病例樣本,使用NoV GI/GII雙重PCR試劑盒進(jìn)行q RT-PCR檢測(cè),再基于NoV基因組衣殼(Capsid)蛋白區(qū)設(shè)計(jì)引物實(shí)施RT-PCR和測(cè)序,進(jìn)行遺傳分型。2.擴(kuò)增RNA依賴的RNA聚合酶(Rd Rp)區(qū)并測(cè)序,結(jié)合Capsid區(qū)結(jié)果,檢索Gen Bank進(jìn)行相似性搜索。應(yīng)用Mega 6.06軟件鄰近歸并法(Neighbor-joining,N-J)基于核苷酸構(gòu)建進(jìn)化樹,進(jìn)行系統(tǒng)發(fā)育分析。同時(shí)運(yùn)用RDP 4.0軟件針對(duì)Rd Rp區(qū)和Capsid區(qū)序列分析鎮(zhèn)江地區(qū)NoV的系統(tǒng)進(jìn)化和重組。3.提取樣本總DNA,設(shè)計(jì)針對(duì)16S v4-v5區(qū)的特定引物進(jìn)行基因擴(kuò)增,得到擴(kuò)增片段后,加接頭,采用Hiseq 2500測(cè)序平臺(tái),通過拼接得到較長(zhǎng)序列,基于16S r RNA基因進(jìn)行種群、豐度及群落結(jié)構(gòu)等多樣性分析,闡明NoV感染與腸道菌群的關(guān)系。結(jié)果:1.感染狀況:2015年-2016年,共采集糞便及肛拭樣本1605份,NoV陽(yáng)性率5.30%(85/1605),其中GI組檢出率為0.87%(14/1605),GII組檢出率為4.55%(73/1605),有2份樣本同時(shí)感染GI/GII組,GI組和GII組檢出率差異有統(tǒng)計(jì)學(xué)意義(P0.05)。男女NoV的檢出率差異無統(tǒng)計(jì)學(xué)意義(P0.05)。NoV的流行高峰主要集中在11月至次年4月,以丹徒區(qū)的檢出率最高。本地區(qū)0歲~10歲的學(xué)齡兒童以及60歲以上的老年人主要感染NoV GII組,其他年齡組GI組和GII組均可感染。GII組在10歲~20歲的中青年中陽(yáng)性率最高,達(dá)17.80%,相對(duì)于其它年齡組,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。GI組的感染差異無統(tǒng)計(jì)學(xué)意義。2.NoV基因型:2015年NoV的基因型以GII.17所占比例最高為63.89%,與其它基因型的差異具有統(tǒng)計(jì)學(xué)意義,其它基因型為:GI.3、GII.3、GII.4、GII.13。2016年以GII.4 Sydney_2012所占比例最高為35%,其它基因型為:GI.3、GI.4、GI.5、GI.6、GI.7、GI.8、GII.2、GII.3、GII.14、GII.17。3.進(jìn)化與重組:系統(tǒng)進(jìn)化分析結(jié)果進(jìn)一步證實(shí)鎮(zhèn)江地區(qū)的31株GII.17型,有3株和1978年-2002年的GII.17型在同一進(jìn)化分支上,其他28株均為GII.17型變異株,與以往的GII.17型具有同源性,但存在一定的變異。還發(fā)現(xiàn)2個(gè)重組事件,GII.P12_GII.3型的ZJ086/2016/CHN和ZJ096/2016/CHN株等是由加拿大分離的U02030/Toronto/CA/GII.3株和英國(guó)分離的X86557/Lordsdale/UK/GII.4株重組而成;GII.P16_GII.2型的ZJ152/2016/CHN株由GII.P2_GII.2型ZJ124/2016/CHN株與GII.P16_GII.13型的ZJ105/2015/CHN株重組而成,重組位點(diǎn)為NoV重組的熱點(diǎn)區(qū)域:ORF1-ORF2交界區(qū)域。4.與腸道菌群關(guān)系:NoV感染人群可操作運(yùn)算分類單位(Operational Taxonomic Units,OTU)大于正常人群。在門分類水平上,NoV感染者的變形菌門數(shù)比正常人群顯著減少,厚壁菌門、擬桿菌門數(shù)顯著增多;在屬分類水平上,埃希菌屬、假單胞菌屬顯著減少,鏈球菌屬增多,擬桿菌屬顯著增多,另有未定位生物顯著增多。NoV感染者與正常人群有統(tǒng)計(jì)學(xué)意義的腸道菌屬達(dá)43種之多,其中單胞菌屬、小細(xì)菌屬、紅球菌屬等在NoV感染人群顯著降低,擬桿菌屬、韋榮球菌屬、纖毛菌屬、厭氧菌等顯著升高,并發(fā)現(xiàn)NoV感染者常導(dǎo)致多種菌屬的顯著增多。結(jié)論:1.NoV是鎮(zhèn)江地區(qū)食源性疾病及突發(fā)公共衛(wèi)生事件的常見病原體。本地區(qū)NoV的流行具有時(shí)間、地區(qū)、人群特征,NoV的流行高峰主要集中在11月至4月,以丹徒區(qū)的檢出率最高,感染者以NoV GII組為主。GII組在10歲~20歲的中青年中陽(yáng)性率最高,GI組的感染無明顯年齡差異。2.鎮(zhèn)江地區(qū)存在多種NoV基因型,并且處在動(dòng)態(tài)變化中,主要流行GII.17和GII.4基因型,其中GII.17基因型為新型變異株,GII.4基因型為最新的GII.4Sydney_2012株。3.本地區(qū)分到的NoV發(fā)現(xiàn)2個(gè)重組事件,分別是GII.P12_GII.3型由GII.3與GII.4型重組而成;GII.P16_GII.2型由GII.P2_GII.2與GII.P16_GII.13重組而成。4.基于16S r RNA基因的高通量序列測(cè)定發(fā)現(xiàn),NoV感染會(huì)伴隨多種腸道菌群的改變。
[Abstract]:Objective: Noroviruses (NoVs) is a common pathogen of food borne diseases and public health emergencies in Zhenjiang. It is very easy to cause the outbreak in the population. To clarify the molecular epidemiological characteristics of NoV in Zhenjiang and the relationship with the intestinal flora, the research from molecular biology, bioinformatics, genomics and so on. In order to analyze the molecular epidemiological characteristics, evolution, recombination and influence on the relationship of intestinal flora in the region of NoV, 1. in order to analyze the prevalence of different NoV genotypes in Zhenjiang, this study uses real-time fluorescence quantitative PCR (Q RT-PCR) and reverse transcriptase PCR (RT-PCR) to collect food borne diseases in 2015.1-2016.12 of Zhenjiang. Routine diarrhea patients' fecal samples and cases of public health emergencies were detected by NoV GI/GII double PCR kit for Q RT-PCR, and then RT-PCR and sequencing were designed based on NoV genome capsid (Capsid) protein region, and RNA polymerase (Rd) region was sequenced by genetic typing.2. amplification RNA dependence and sequenced. Fruit, retrieving Gen Bank for similarity search. Using the Mega 6.06 software adjacent merging method (Neighbor-joining, N-J) to construct the phylogenetic tree based on nucleotides to carry out phylogenetic analysis. At the same time, the RDP 4 software is used to analyze the system intake of NoV in Zhenjiang region and the total DNA of the.3. extraction samples for the Rd Rp region and Capsid region sequence. The specific primers of the region were amplified, and after the amplified fragment was amplified, the Hiseq 2500 sequencing platform was used to get a long sequence by splicing. The population, abundance and community structure based on the 16S R RNA gene were analyzed to clarify the relationship between the NoV infection and the intestinal flora. Results: 1. infection status: 2015 -2016, collecting fecal and feces. 1605 samples of anal swab, NoV positive rate 5.30% (85/1605), the detection rate of group GI was 0.87% (14/1605), the detection rate of GII group was 4.55% (73/1605), 2 samples were simultaneously infected with GI/GII group, GI and GII group detection rates were statistically significant (P0.05). The prevalence peak of male and female NoV was not statistically significant (P0.05) was mainly concentrated in November. By April the next year, the detection rate in the Dantu area was the highest. The school age children aged 0 and over 60 years old in the region were mainly infected with the NoV GII group. The positive rate of the GI group and the GII group in the other age groups was the highest in the group.GII group at the age of 10 years old, reaching 17.80%. Compared with its age group, the difference was statistically significant (P0.05).GI group. The difference in infection was not statistically significant.2.NoV genotypes: the highest proportion of the genotype of NoV in 2015 was 63.89%, and the difference between the genotype and other genotypes was statistically significant. The other genotypes were GI.3, GII.3, GII.4, and the highest proportion of GII.4 Sydney_2012 in GII.13.2016, and the other genotypes were GI.3, GI.4, GI.5, GI.5. II.2, GII.3, GII.14, GII.17.3. evolution and recombination: phylogenetic analysis further confirmed that 31 strains of GII.17 in Zhenjiang, 3 and GII.17 in the year -2002 were in the same evolutionary branch, and the other 28 were GII.17 variant, which were identical with the previous GII.17 type, but there were some variations. 2 recombination events, G, were found, G. The ZJ086/2016/CHN and ZJ096/2016/CHN strains of type II.P12_GII.3 were reorganized by the U02030/Toronto/CA/GII.3 strain isolated from Canada and the X86557/Lordsdale/UK/GII.4 strain isolated from the UK. The GII.P16_GII.2 type ZJ152/2016/CHN strain was reorganized from the ZJ124/2016/CHN strain of GII.P2_GII.2 type to the ZJ105/2015/CHN strain of the GII.P16_GII.13 type. Point is the hot area of NoV recombination: the relationship between.4. and intestinal flora in the ORF1-ORF2 junction area: the operation classification unit of NoV infected people (Operational Taxonomic Units, OTU) is larger than the normal population. At the portal classification level, the number of strains of the NoV infected persons is significantly less than that of the normal population, and the number of the thick wall bacteria and the bacteriobacteria increased significantly; in the genus At the level of classification, the genus and Pseudomonas genera were significantly reduced, Streptococcus increased, and Pseudomonas increased significantly, and there was a significant increase in the number of.NoV infected individuals with more than 43 species in the normal population. Among them, monomonas, small bacteria, and rhodotonas sp. were significantly reduced in NoV infected people. Genus, wehonia, cilium, anaerobes and other significant increases, and found that NoV infection often leads to a significant increase in the number of bacteria. Conclusion: 1.NoV is a common pathogen of food borne diseases and public health emergencies in Zhenjiang. The epidemic of NoV in the region has time, ground area, and population characteristics. The peak of NoV is mainly concentrated in November. In April, the detection rate in Dantu area was the highest. The positive rate of NoV GII group in group.GII was the highest in group.GII and ~20 years old, and there was no obvious age difference in GI group. There was a variety of NoV genotypes in.2. Zhenjiang region, and in dynamic changes, the main prevalent GII.17 and GII.4 genotypes, and GII.17 genotypes as new variant, GII. The 4 genotype was the latest GII.4Sydney_2012 strain.3. locally identified by NoV, which was found to be a recombination of the GII.P12_GII.3 type from GII.3 to GII.4, and the GII.P16_GII.2 type from GII.P2_GII.2 to GII.P16_GII.13 and.4. based on 16S R, found that the infection would accompany a variety of intestinal flora. Change.

【學(xué)位授予單位】：江蘇大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R512.5;R181.3

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