本文關(guān)鍵詞：湘瓊兩地蝙蝠攜帶星狀病毒與諾如病毒的調(diào)查研究　出處：《南方醫(yī)科大學(xué)》2011年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 蝙蝠 諾如病毒 星狀病毒 進(jìn)化樹分析 檢出率

【摘要】：研究背景和目的 研究表明,蝙蝠是多種人獸共患疾病病毒的自然宿主,并可以跨越物種間屏障,將病毒傳播給人類和其他動(dòng)物。目前研究人員已經(jīng)從蝙蝠身上發(fā)現(xiàn)或分離很多種病毒,約有18個(gè)病毒科包含70多種病毒。已知能從蝙蝠傳播給人類的病毒主要有狂犬病毒、尼巴病毒、亨德拉病毒,還有類SARS冠狀病毒,這些病毒都可以引起人類致命性的疾病。其他病毒,如甲病毒屬、黃病毒屬和布尼亞病毒屬病毒可通過節(jié)肢動(dòng)物傳播給蝙蝠,但還不確定蝙蝠是不是這類病毒所致疾病的播散宿主。可見,還需要大量研究,以明確蝙蝠在病毒性自然疫源性疾病中所扮演的角色。 我國(guó)約有120種蝙蝠,但國(guó)內(nèi)對(duì)蝙蝠的研究工作起步較晚,對(duì)其生態(tài)學(xué)、行為學(xué)研究尚不夠深入,尤其是對(duì)我國(guó)蝙蝠攜帶哪些種類病毒,以及病毒與蝙蝠生態(tài)學(xué)的關(guān)系,對(duì)人類健康的影響的研究還較少。迄今為止,我國(guó)科研工作者對(duì)蝙蝠所攜帶病毒的研究有以下發(fā)現(xiàn)：從蝙蝠體內(nèi)分離到羅斯河病毒、乙型腦炎病毒、基孔肯雅病毒、森林腦炎病毒、SARS類冠狀病毒、狂犬病病毒、星狀病毒、尼巴病毒等。這些發(fā)現(xiàn)表明我國(guó)蝙蝠可攜帶某些我國(guó)常見傳染病的病毒。 病毒性胃腸炎(viral gastroenteritis)又稱病毒性腹瀉,是一組由多種病毒引起的急性腸道傳染病。臨床特點(diǎn)為起病急、惡心、嘔吐、腹痛、腹瀉,排水樣便或稀便,也可有發(fā)熱及全身不適等癥狀,病程短,病死率低。病毒性腹瀉在兒童中更是一種常見病和多發(fā)病,是導(dǎo)致兒童死亡的重要原因。引起兒童腹瀉的病毒主要有輪狀病毒(rotavirus)、腸道腺病毒(adenovirus)、星狀病毒(astrovirus, Astv)、諾如病毒(norovirus, NoV)等。 患者與無癥狀帶毒者是病毒性胃腸炎的主要傳染源。糞-口途徑傳播是病毒性胃腸炎最重要的傳播途徑,少部分可以通過空氣傳播,其可通過被污染的食物或水引起人群胃腸炎暴發(fā)或散發(fā)。回顧上世紀(jì)90年代的亨德拉病毒和尼帕病毒的暴發(fā)事件,也是源于食源性感染,是由于蝙蝠病毒污染了水域、水果,再依次傳播給馬、豬、人,最后導(dǎo)致疾病暴發(fā)。 星狀病毒(Astrovirus, Astv)于1975年在胃腸炎患兒糞便標(biāo)本中首次發(fā)現(xiàn),由于電鏡下病毒顆粒呈星形,因此被命名為星狀病毒。此后世界各地均已有星狀病毒感染的報(bào)道,它既可散發(fā)也可以引起暴發(fā)流行,亦可引起醫(yī)源性感染。住院腹瀉患兒中星狀病毒檢出率為2.5%-9.0%,目前認(rèn)為星狀病毒是嬰幼兒病毒性胃腸炎的第二位病因,僅次于輪狀病毒。 星狀病毒既可感染人,也可感染牛、羊、豬、狗、鹿、火雞、鴨、鼠等多種動(dòng)物,導(dǎo)致不同程度的胃腸炎。人群感染的星狀病毒,傳統(tǒng)上可分為8個(gè)血清型。最近,研究者又在腹瀉病人中發(fā)現(xiàn)兩種的新星狀病毒,astrovirus MLB和astrovirus VA。研究還發(fā)現(xiàn),Astv MLB 1與新近發(fā)現(xiàn)的鼠星狀病毒很相近;Astv VA1在進(jìn)化樹上與感染羊和貂的星狀病毒最為接近。這提示新發(fā)現(xiàn)的人星狀病毒可能和動(dòng)物的星狀病毒存在一定的關(guān)系。 近年來,香港大學(xué)研究小組分別從香港和中國(guó)大陸的蝙蝠體內(nèi)檢出星狀病毒,檢出率高達(dá)46%和44.8%；基因分析顯示蝙蝠星狀病毒具有高度基因多態(tài)性,部分病毒可能與感染人的星狀病毒有系統(tǒng)進(jìn)化的關(guān)系。然而關(guān)于蝙蝠星狀病毒的生態(tài)學(xué)資料還很稀少,有必要進(jìn)一步擴(kuò)充不同地區(qū)和不同種類的蝙蝠樣本進(jìn)行研究。 諾如病毒(Norovirus, NoV)是一組杯狀病毒屬病毒,其原型株諾瓦克病毒于1972年在美國(guó)諾瓦克市被分離發(fā)現(xiàn)。由于該組病毒極易變異,此后在其他地區(qū)又相繼發(fā)現(xiàn)并命名了多種類似病毒,統(tǒng)稱為諾如病毒。NoV是一種高致病的、傳染性極強(qiáng)的腸胃病毒,能導(dǎo)致急性胃腸炎,一旦有人感染,通常會(huì)發(fā)展為群體性的大規(guī)模傳染。遺憾的是,由于病毒的高度變異性及其缺少合適的動(dòng)物研究模型,現(xiàn)今還未研發(fā)出理想的NoV疫苗,患者容易發(fā)生反復(fù)感染。 自然界中攜帶NoV的物種很多,包括有人、豬、牛、鼠、海獅等,可導(dǎo)致它們發(fā)生不同程度的癥狀。根據(jù)基因特征,NoV被分為五個(gè)遺傳群(Gene Group, GG):GGⅠ、GGⅡ、GGⅢ、GGⅣ和GGV。五個(gè)遺傳群中,GGⅠ、GGⅡ和GGIV主要感染人類,GGⅢ感染牛,GGV則在鼠類動(dòng)物內(nèi)檢出。 雖然研究已表明NoV在多種動(dòng)物體內(nèi)廣泛存在,但是關(guān)于動(dòng)物NoV感染的流行病學(xué)還不太清楚。動(dòng)物NoV在動(dòng)物體內(nèi)感染率差別較大,研究發(fā)現(xiàn)北美的實(shí)驗(yàn)鼠鼠類NoV的感染率高達(dá)22.1%,英國(guó)檢測(cè)發(fā)現(xiàn)牛的腹瀉病例中有11%比例為NoV感染所致,而豬NoV檢出率比較低,在日本豬GGⅡNoV的檢測(cè)率僅為0.35%,在荷蘭檢測(cè)率為2.0%。 盡管現(xiàn)今沒有證據(jù)表明動(dòng)物NoV可以感染人類,但是分子生物學(xué)分析表明動(dòng)物NoV和人類NoV的毒株相互之間有密切的關(guān)系,尤其是豬NoV的毒株可被歸入到與人類NoV毒株相同的基因群(GGⅡ)。新近研究發(fā)現(xiàn)豬體內(nèi)普遍存在人NoV的抗體,這進(jìn)一步加強(qiáng)了動(dòng)物可能充當(dāng)著人類NoV儲(chǔ)存庫(kù)的假設(shè)。此外,研究還發(fā)現(xiàn)貝殼類動(dòng)物可同時(shí)攜帶人類NoV和動(dòng)物NoV,這提示一種新的基因重組株可能出現(xiàn)。最近,在加拿大人們檢測(cè)到豬NoV和牛NoV的序列與人NoVGGⅡ.4毒株的序列非常接近。因此,將來或許要重新評(píng)估NoV種間傳播的可能性及其人畜共患的風(fēng)險(xiǎn)。 2009年,武漢病毒研究所在蝙蝠體內(nèi)檢測(cè)到另一種導(dǎo)致人腹瀉的病毒—腸道腺病毒,檢出率為8%(28/350),基因分析顯示蝙蝠腺病毒亦具有高度基因多樣性,這提示蝙蝠可能有攜帶一系列高變異病毒的能力。研究已經(jīng)在蝙蝠體內(nèi)發(fā)現(xiàn)了星狀病毒和腺病毒兩種腹瀉相關(guān)病毒,關(guān)于蝙蝠是否攜帶有NoV,暫未見有文獻(xiàn)報(bào)道,因而我們不禁疑問蝙蝠是否攜帶有可導(dǎo)致爆發(fā)流行的NoV。鑒于NoV的高度基因變異性和多宿主性,作為種類第二多的哺乳動(dòng)物(1000多種)一蝙蝠也很有可能攜帶此類病毒,甚至可能是它們的宿主。 因此,為進(jìn)一步了解不同地區(qū)不同種類蝙蝠攜帶星狀病毒的本底情況,探討其與感染人星狀病毒的關(guān)系,同時(shí)了解這些地區(qū)的蝙蝠是否攜帶諾如病毒,本研究擬在先前未見報(bào)道的湖南、海南部分地區(qū)為現(xiàn)場(chǎng),對(duì)當(dāng)?shù)仳饠y帶星狀病毒和諾如病毒情況進(jìn)行流行病學(xué)調(diào)查。 研究方法 1.樣本的采集和處理 2007年7月至2008年7月,于岳陽(yáng)、邵陽(yáng)、�？谌貐^(qū)蝙蝠長(zhǎng)期監(jiān)測(cè)點(diǎn)中選取4個(gè)蝙蝠自然棲息地,現(xiàn)場(chǎng)捕捉蝙蝠,進(jìn)行編號(hào)后,請(qǐng)蝙蝠研究專家進(jìn)行鑒定。標(biāo)本采集方式分兩種,分別為采集肛拭子和剪取直腸組織。采集的樣本存放于含200μl RNAlater儲(chǔ)存液的凍存管中,置放于密封的冰袋泡沫盒中當(dāng)天運(yùn)回實(shí)驗(yàn)室,放于-80℃冰箱貯存,待檢。 2.蝙蝠病毒的檢測(cè)與鑒定2.1星狀病毒 ①半巢式PCR篩檢樣本 參照文獻(xiàn),針對(duì)星狀病毒高度保守的RdRp基因,設(shè)計(jì)合成5條用于巢式PCR的引物,包括4條前引物和1條共同的后引物,應(yīng)用巢式PCR方法檢測(cè)標(biāo)本。 ②序列鑒定與分析 對(duì)陽(yáng)性PCR產(chǎn)物進(jìn)行切膠、純化和回收,然后送樣至公司測(cè)序。如遇陽(yáng)性PCR產(chǎn)物條帶很弱者,則先做克隆,再取接種培養(yǎng)后菌液送樣至公司測(cè)序鑒定。 ③將測(cè)序結(jié)果導(dǎo)入GenBank的BLAST軟件進(jìn)行在線同源性分析,然后在GenBank下載相關(guān)核酸序列,利用Clastal_X軟件對(duì)所要分析的序列進(jìn)行多序列比對(duì)分析,并用DNAstar軟件對(duì)核酸序列相似性進(jìn)行計(jì)算。使用MEGA4.1軟件,用Neighbor-Joining法構(gòu)建進(jìn)化樹進(jìn)行分析。 2.2諾如病毒 ①TaqMan real-time PCR檢測(cè)基因組Ⅱ型諾如病毒(GGⅡNoV) 參照文獻(xiàn)并合成用于檢測(cè)GGⅡNoV公認(rèn)的特異引物和探針,利用TaqMan real-time PCR方法對(duì)本進(jìn)行檢測(cè)。 ②SYBR Green real-time PCR檢測(cè)諾如病毒 參照文獻(xiàn)合成可檢測(cè)NoV的兩對(duì)通用引物P289/P290和12Y/13I,應(yīng)用SYBR Green real-time PCR方法分別檢測(cè)標(biāo)本。 ③GGV型NoV特異引物檢測(cè)標(biāo)本 參照文獻(xiàn)合成公認(rèn)的可檢測(cè)GGV型NoV引物,應(yīng)用普通RT-PCR方法檢測(cè)標(biāo)本。 ④對(duì)普通RT-PCR擴(kuò)增獲得的可疑陽(yáng)性PCR產(chǎn)物進(jìn)行切膠、純化和回收,然后送樣至公司測(cè)序。對(duì)TaqMan real-time PCR檢測(cè)的可疑陽(yáng)性PCR產(chǎn)物,送樣至公司克隆鑒定。 3.質(zhì)量控制 ①移液槍頭、凍存管、EP管等實(shí)驗(yàn)耗材均為一次性用品。 ②RNA提取和逆轉(zhuǎn)錄實(shí)驗(yàn)所采用的所有容器和配制試劑用DEPC水處理,高溫高壓處理,烘干待用,以去除RNA酶污染。 ③全程戴口罩、手套、帽子,在無菌室生物安全柜進(jìn)行操作,防止試劑和實(shí)驗(yàn)過程受到污染。 ④實(shí)驗(yàn)過程嚴(yán)格設(shè)立陽(yáng)性對(duì)照、陰性對(duì)照和空白對(duì)照。 結(jié)果 1蝙蝠基本情況 本次研究在湖南、海南兩省四個(gè)監(jiān)測(cè)點(diǎn)共采集蝙蝠321只,涉及2個(gè)蝙蝠亞目4個(gè)蝙蝠科9個(gè)種類的蝙蝠。共制備蝙蝠糞便標(biāo)本321份,包括肛拭子59份和直腸樣本262份。 2星狀病毒的檢測(cè) ①巢式RT-PCR檢測(cè)到陽(yáng)性標(biāo)本29例,總陽(yáng)性率為9.03%(29/321),在9種表面健康的蝙蝠中發(fā)現(xiàn)有3種蝙蝠攜帶有星狀病毒,陽(yáng)性蝙蝠種類包括長(zhǎng)翼蝠(22/187)、小黃蝠(6/38)和棕果蝠(1/59),以小黃蝠的檢出率最高。 ②以星狀病毒RdRp基因部分氨基酸序列構(gòu)建的進(jìn)化樹分析顯示,本研究檢測(cè)的蝙蝠星狀病毒不僅與來源于其他種類的星狀病毒差異較大,而且本身也有很大的差異。然而,有5個(gè)蝙蝠星狀病毒卻與羊星狀病毒(ovine Astv)、貂星狀病毒(mink Astv)和人新型星狀病毒Astv VA在進(jìn)化樹上最為相近,序列相似性為51.6%-63.1%。另外,有8株從邵陽(yáng)地區(qū)同一個(gè)洞穴長(zhǎng)翼蝠檢測(cè)到的星狀病毒相聚在一小分支,它們的氨基酸序列相似性為97%-99%。 3諾如病毒的檢測(cè) 本研究采用TaqMan real-time PCR、SYBR Green real-time PCR和普通RT-PCR三種實(shí)驗(yàn)方法,應(yīng)用多對(duì)引物分別篩查上述蝙蝠糞便樣本GGⅠ-Ⅳ型NoV攜帶情況,檢測(cè)結(jié)果均為陰性。 結(jié)論 ①巢式PCR檢測(cè)到星狀病毒陽(yáng)性率為9.03%(29/321),并首次在果蝠身上檢出星狀病毒(1/59),提示此類星狀病毒可能適應(yīng)于翼手目動(dòng)物,盡管不同種類的蝙蝠對(duì)它的易感程度不一。 ②本研究檢出的蝙蝠星狀病毒基因多態(tài)性豐富,這結(jié)果與先前研究一致。5株蝙蝠星狀病毒卻與羊星狀病毒、貂星狀病毒、人星狀病毒Astv VA在進(jìn)化樹上最為相近,并具有較高的氨基酸序列相似性。這提示蝙蝠星狀病毒可能跨種族傳播給人類或其他動(dòng)物,亦或是在它們之間相互傳播。 ③來自邵陽(yáng)同一個(gè)洞穴檢測(cè)得到的8株星狀病毒具有高度的基因相似性,進(jìn)一步提示蝙蝠星狀病毒在同一個(gè)洞穴內(nèi)傳播的可能。 ④本研究從所收集的321份蝙蝠糞便樣本中未檢測(cè)到諾如病毒,提示蝙蝠攜帶諾如病毒的可能性不大。由于諾如病毒高度遺傳變異,而且現(xiàn)今沒有一對(duì)單獨(dú)引物可同時(shí)檢測(cè)出全部五個(gè)基因組的諾如病毒。蝙蝠是否攜帶諾如病毒還不清楚,有待進(jìn)一步研究。
[Abstract]:Background and purpose of research
Research shows that bats are many zoonotic virus natural host, and can cross the species barrier and spread the virus to humans and other animal. At present, researchers have found that separation from the bats or many types of viruses, about 18 virus, including more than 70 kinds of virus. Known to spread to humans from bats the virus mainly has the rabies virus, Nipah virus, Hendra virus, and SARS coronavirus, these viruses can cause human fatal disease. Other viruses, such as a virus, and the virus flavivirus bunyavirus by arthropod animal spread to bat, but it is not clear whether bats are of this kind of disease caused by virus the spread of the host. Obviously, we still need a lot of research, in order to clear the bat in viral disease of natural focus in the role.
There are about 120 species of bats in China, but the domestic research work on bat started late, the ecology, behavioral research is still not enough, especially on what kind of virus carrying our bat, the relationship between virus and bat ecology, study the impact on human health is less. So far, the research in our country scientists are carrying the virus of bats are as follows: from separation to bat in the Ross River virus, Japanese encephalitis virus, chikungunya virus, Semliki Forest virus, SARS coronavirus, rabies virus, astrovirus, Nipah virus. These findings suggest that China's bat can carry some of common infectious diseases in China virus.
Viral gastroenteritis (viral gastroenteritis) also known as viral diarrhea, is a group of acute intestinal infectious disease caused by a variety of viruses. The clinical features of acute onset, nausea, vomiting, abdominal pain, diarrhea, drain or rare, also can have fever and malaise and other symptoms, short duration, low fatality rate. Viral diarrhea in children is a common disease, is an important cause of death in children. Children's diarrhea virus mainly caused by rotavirus (rotavirus), enteric adenovirus (adenovirus), astrovirus (astrovirus, Astv), norovirus (norovirus, NoV)..
Patients with asymptomatic carriers is the main source of infectious viral gastroenteritis. The fecal oral route transmission is viral gastroenteritis is the most important part, can be spread through the air, it can be through contaminated food or water caused by the crowd or sporadic events. An outbreak of gastroenteritis outbreaks were Hendra and Nipah viruses in the world in 90s, also due to foodborne infection, is due to bat virus contaminated water, fruit, then spread to the horse, pig, human, and finally lead to disease outbreaks.
Astrovirus (Astrovirus, Astv) in 1975 for the first time found in the stool samples of children with gastroenteritis, because the virus particles under the electron microscope is the star, it is named astrovirus. Then all over the world have astrovirus infection reported, it can also cause sporadic epidemic of violence, can also cause iatrogenic infection hospitalized children with diarrhea. Astrovirus detection rate was 2.5%-9.0%, the astrovirus is infant viral gastroenteritis second cause, second only to rotavirus.
Astrovirus can infect people, but also can infect cattle, sheep, pigs, dogs, deer, turkeys, ducks, rats and other animal, lead to different degrees of gastroenteritis. Astrovirus infection, traditionally can be divided into 8 serotypes. Recently, researchers have found that two kinds of star shaped virus in diarrhea patients, astrovirus MLB and astrovirus VA., the study also found that Astv and MLB 1 were astrovirus newly found very similar; Astv VA1 in evolutionary tree with infected sheep and mink astrovirus is most close. There is a certain relationship between the stellate virus suggesting human astrovirus and may the newly discovered animal.
In recent years, were detected astrovirus from Hongkong and Chinese, the bat in the University of Hong Kong research team, the detection rate of 46% and 44.8%; gene analysis showed that bat astrovirus has a high degree of polymorphism, relationship between system of astrovirus infection with the virus may some people. However ecological information about bats star the shape of the virus is still scarce, it is necessary to further expand and different kinds of bats samples in different regions.
Norovirus (Norovirus, NoV) is a group of calicivirus is a virus, the prototype strains of Novak virus in the United States in 1972, Novak was found. Because of this group of viruses isolated easily after variation, in other areas have been discovered and named several similar viruses, collectively referred to as norovirus is a highly pathogenic.NoV the highly contagious stomach virus, can cause acute gastroenteritis, once a person is infected, usually for the development of large-scale infectious group. Unfortunately, due to the high variability of the virus and the lack of suitable animal model, now has not yet developed the ideal NoV vaccine, patients prone to recurrent infections.
The nature of NoV carrying many species, including human, pig, cattle, rat, sea lions, they can lead to different degrees of symptoms. According to the characteristics of NoV gene, were divided into five genetic groups (Gene Group, GG): GG 1, GG II, GG III, GG I GG IV and GGV. five genetic groups, GG, 2 and GGIV can infect humans, GG infection in cattle, GGV is detected in rodent animal.
Although studies have shown that NoV is widely present in a variety of animal body, but epidemiological about animal NoV infection is not clear. NoV in different animal rate in infected animal, experimental study found that mouse class NoV North America infection rate as high as 22.1%, the British found cattle in 11% cases of diarrhea caused by NoV infection ratio but, pig NoV detection rate is relatively low, in the detection of Japanese pig GG II NoV rate was only 0.35% in Holland, the detection rate was 2.0%.
Although there is no evidence that the animal NoV can infect humans, but molecular analysis revealed that NoV animal and human NoV strains were closely related to each other, especially the swine NoV strain can be classified into the same group of genes with human NoV strains (GG II). Recent studies have found that the antibody against porcine is common in human NoV this, to further strengthen the animal may play the role of a human NoV storage hypothesis. In addition, the study also found that shellfish animal can simultaneously carry human and animal NoV NoV, suggesting a possible new gene recombinant strains. Recently, people in Canada are very close to the sequence detected in porcine NoV and bovine NoV sequences and NoVGG II.4 strain. Therefore, the future may have to re evaluate the risk possibility of the spread of NoV species and zoonoses.
In 2009, Wuhan virus Institute bats detected another cause diarrhea virus, enteric adenovirus, the detection rate was 8% (28/350), gene analysis showed that bat adenovirus also has high genetic diversity, suggesting that bats may have the ability to carry a series of highly variable virus research has found the star. Rotavirus and adenovirus two diarrhea related virus in bats, a bat is carrying NoV, yet see the reports in the literature, so we can not help but doubt whether bats carrying a outbreak of NoV. in height based NoV due to variability and multiple hosts, as the more than 2 species of mammals (1000 kinds) a bat is likely to carry the virus, or even their host.
Therefore, in order to further understand the different regions of different types of bats astrovirus the situation at the end of its relationship with human astrovirus infection, while understanding in these areas are carrying bats norovirus, this study has not been reported previously in Hunan, parts of Hainan for the scene, the local bats in the investigation of astrovirus and norovirus.
research method
Collection and processing of 1. samples
From July 2007 to July 2008, in Yueyang, Shaoyang, Haikou three areas of long-term monitoring of bats from 4 bats natural habitat in the scene to capture the bat, number, please bat research experts were identified. Samples were collected two hours, including rectal swab samples and cut from rectum tissue samples stored in water. 200 L RNAlater storage solution in freezing tube, placed in the ice box sealing foam when in the day back to the lab, put in the -80 C refrigerator storage, to be inspected.
Detection and identification of 2. bats virus 2.1 stellate viruses
1. Semi nested PCR screening sample
Referring to the literature, for the highly conserved RdRp gene of the stellate virus, we designed and synthesized 5 primers for nested PCR, including 4 primers and 1 common primers. Nested PCR was used to detect the samples.
Sequence identification and analysis
The positive PCR products were excised, purified and recovered, and then sent to the company for sequencing. In case of positive PCR products with very weak, then do cloning, then after inoculation of bacteria feeding to DNA sequencing.
The sequencing results into GenBank BLAST software for analysis of online source, and then download the related nucleic acid sequences in GenBank, use Clastal_X software to analyze the multiple sequence alignment of sequences to be analyzed, and the nucleic acid sequence similarity was calculated by DNAstar software. Using MEGA4.1 software, were analyzed by Neighbor-Joining method to construct phylogenetic trees.
2.2 norovirus
(1) TaqMan real-time PCR detection of geno type Nuo virus (GG II NoV)
The specific primers and probes used to detect GG II NoV were synthesized with reference to the literature, and the TaqMan real-time PCR method was used to detect the primers.
(2) SYBR Green real-time PCR detection of norovirus
Two pairs of universal primers P289/P290 and 12Y/13I were synthesized with reference to the literature, and the specimens were detected by SYBR Green real-time PCR method respectively.
GGV type NoV specific primer detection specimens
GGV type NoV primers were detected by reference to the literature, and the specimens were detected by the common RT-PCR method.
Fourth, the suspicious and positive PCR products obtained from common RT-PCR were cut, purified, recovered, then sent to the company for sequencing. The suspicious positive PCR products detected by TaqMan real-time PCR were sent to the company for cloning and identification.
3. quality control
(1) the experimental materials such as the liquid gun head, the cryopreservation tube and the EP tube are all disposable supplies.
(2) all the containers and reagents used in the RNA extraction and reverse transcriptase experiment were treated with DEPC water, high temperature and high pressure treatment, and dried to remove RNA enzyme pollution.
3. Wear masks, gloves and hats in the whole process, and operate in the asepsis room biological safety cabinet to prevent the reagent and the experimental process from being polluted.
4. Positive control was strictly set up in the experimental process, negative control and blank control.
Result
1 basic situation of bats
In this study, 321 bat species were collected from four monitoring sites in two provinces of Hunan and Hainan, involving 2 bat species, 4 bat families, 9 species of bats. Altogether 321 specimens of bat feces were prepared, including 59 59 anal swabs and 262 262 rectal samples.
Detection of 2 stellate viruses
The nested RT-PCR detected 29 positive samples, the total positive rate was 9.03% (29/321), in 9 apparently healthy bats were found in 3 species of bats carrying astrovirus, positive bat species including m.schreibersii (22/187), yellow bat (6/38) and brown (1/59), with yellow fruit bats the highest detection rate.
The star virus RdRp gene to construct phylogenetic tree of amino acid sequence part analysis shows that bat astrovirus in this study not only to detect astrovirus differences and from other types of large, but also have great differences. However, there are 5 bat astrovirus and sheep astrovirus (ovine Astv) mink astrovirus (mink, Astv) and a novel human astrovirus Astv VA in evolutionary tree is most similar to the sequence similarity of 51.6%-63.1%. and 8 strains from Shaoyang region in the same cave m.schreibersii detected astrovirus gathered on a small branch, their amino acid sequence similarity to 97%-99%.
Detection of 3 norovirus
In this study, TaqMan real-time PCR, SYBR Green real-time PCR and ordinary RT-PCR were used in three experimental methods. Multiple pairs of primers were used to screen the carrying condition of GG type I - IV NoV in the stool samples.

【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R373

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相關(guān)期刊論文 前1條

1 李文東,梁國(guó)棟,梁冰,胡志紅,石正麗,張樹義;蝙蝠攜帶病毒的研究進(jìn)展[J];中國(guó)病毒學(xué);2004年04期

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本文編號(hào)：1361285