本文選題：蝙蝠 + 狂犬病毒　； 參考：《南方醫(yī)科大學(xué)》2009年博士論文

【摘要】：1.研究背景和目的: 狂犬病(Rabies)是人畜共患的自然疫源性疾病,可引起人和所有溫血?jiǎng)游锛毙灾滤佬阅X脊髓炎,一旦發(fā)病,患者或病畜幾乎100%死亡�？袢】稍谑澜绶秶鷥�(nèi)分布,是全球性的嚴(yán)重公共衛(wèi)生問(wèn)題�？袢《�(Rabies virus)是人和動(dòng)物狂犬病的病原,已知?jiǎng)游锼拗鞅姸?且隨著研究的深入,越來(lái)越多的物種都能分離出狂犬病毒。自1953年美國(guó)首次發(fā)現(xiàn)食蟲蝙蝠咬傷引起的人狂犬病以來(lái),蝙蝠攜帶狂犬病毒及其與人狂犬病的關(guān)系引起人類的廣泛關(guān)注,近年來(lái)許多國(guó)家和地區(qū)不斷地從蝙蝠體內(nèi)分離出狂犬病毒。 狂犬病的主要傳染源是野生動(dòng)物,主要包括:狼、狐、豹、野狗、猴、貓、臭鼬、浣熊和蝙蝠等�？袢〉牧餍幸话阆瘸霈F(xiàn)在野生動(dòng)物中間,通過(guò)家養(yǎng)動(dòng)物波及人類。家養(yǎng)動(dòng)物是野生動(dòng)物狂犬病和人類狂犬病的聯(lián)系環(huán)節(jié)。犬雖然非最敏感動(dòng)物,但是由于世界大多數(shù)地區(qū)的城市和農(nóng)村養(yǎng)犬密度很高,所以犬已成為病毒的長(zhǎng)期宿主和媒介。家貓亦有同樣的趨勢(shì)。 狂犬病的傳染源在不同地區(qū)有所不同,如非洲85%以上的國(guó)家、南美大多數(shù)國(guó)家和亞洲地區(qū)國(guó)家(除以色列和孟加拉以外)以犬狂犬病為主,而南非、歐洲、美國(guó)和加拿大等北美地區(qū)則主要是野生動(dòng)物狂犬病。圭亞那、巴拿馬和多哥則是以蝙蝠傳播狂犬病。荷蘭、德國(guó)和西班牙的蝙蝠狂犬病僅次于野生動(dòng)物狂犬病。我國(guó)絕大多數(shù)的狂犬病為犬所致,偶有貓、牛等其他動(dòng)物的報(bào)道。 近年來(lái),野生動(dòng)物狂犬病的比重不斷增加,時(shí)有野生動(dòng)物狂犬病傳給人的報(bào)道出現(xiàn),野生動(dòng)物攜帶狂犬病毒帶來(lái)的危害日趨嚴(yán)重。野生動(dòng)物不僅直接傳播疾病,而且可能通過(guò)破壞畜牧業(yè)間接地影響人類健康,而野生動(dòng)物的管理比較困難,一般地難于對(duì)其進(jìn)行免疫預(yù)防。尤其是某些蹄類野生動(dòng)物具有遷徙的本能,能周期性地、替換方向地改變棲息地以獲得最佳的環(huán)境條件。鑒于這個(gè)特點(diǎn),野生動(dòng)物作為狂犬病毒的貯存宿主和媒介,能給傳播狂犬病毒帶來(lái)更大地危害。 蝙蝠屬翼手目(Chiroptera)動(dòng)物,是哺乳類中分布最廣、數(shù)量最多的動(dòng)物之一,全世界大約有17科180屬,約960種,其中我國(guó)有7科29屬107種。翼手目分兩個(gè)亞目:大蝙蝠亞目(Megachiroptera),又稱狐蝠,我國(guó)有1科5屬7種;小蝙蝠亞目(Microchiroptera),即通常所說(shuō)的蝙蝠,我國(guó)有6科24屬100種。 除南北極外,蝙蝠在世界各地都有分布,包括在高緯度地區(qū)、荒涼的沙漠和孤立的島嶼上,甚至在撒哈拉大沙漠也有蝙蝠的活動(dòng)。然而,大多數(shù)蝙蝠種類生活在熱帶和亞熱帶地區(qū)。蝙蝠物種豐富,分布廣泛,壽命長(zhǎng),并有很強(qiáng)的飛行能力,其中一些蝙蝠種類還有長(zhǎng)途遷移的習(xí)性。 蝙蝠與人類接觸密切。有些蝙蝠就棲息在屋檐下,或是房屋的裂縫中;另外蝙蝠在覓食時(shí)經(jīng)常會(huì)誤闖入居民家中;一些旅游景點(diǎn)的巖洞中也居住有蝙蝠。在中醫(yī)里面,菊頭蝠的糞便被做成“夜明沙”用于治療眼疾;在廣東,果蝠被一些餐館作為野味擺上餐桌;湖南一些地區(qū)則相信蝙蝠可以用來(lái)治療癲癇。 狂犬病病毒(Rabies virus)為彈狀病毒科(Rhabdoviridae)狂犬病毒屬(Lyssavirus),形態(tài)呈彈狀(60～400 nm×60～85 nm),一端純園,一端平凹,有囊膜,內(nèi)含衣殼呈螺旋對(duì)稱。核酸是單股負(fù)鏈不分節(jié)段RNA,基因組長(zhǎng)約12 kb,從3′到5′端依次為N、P、M、G、L的5個(gè)基因,各個(gè)基因間還含非編碼的間隔序列。分別編碼核蛋白(N)、磷蛋白(P)、基質(zhì)蛋白(M)、糖蛋白(G)、轉(zhuǎn)錄酶大蛋白(L)。完整的病毒顆粒由核衣殼及包膜兩部分構(gòu)成,核衣殼由RNA與核蛋白、磷蛋白和轉(zhuǎn)錄酶大蛋白構(gòu)成,而包膜由糖蛋白和基質(zhì)蛋白構(gòu)成。在狂犬病毒的5個(gè)基因中,N基因有高度保守性。1993年Bourhy等根據(jù)核蛋白基因N端500個(gè)堿基的同源性將狂犬病毒屬分為6個(gè)基因型,基因1～4型分別對(duì)應(yīng)于血清Ⅰ～Ⅳ,而血清Ⅴ型的歐洲狂犬病毒EBL1株和EBL2株為基因型5、6。1998年澳大利亞報(bào)道分離出新的蝙蝠狂犬病病毒,被定為基因7型,澳大利亞蝙蝠狂犬病病毒(ABLV)。近年來(lái),新基因型別的狂犬病病毒(Lyssavirus)不斷發(fā)現(xiàn),在蝙蝠身上分離到Aravan virus,Khujand virus,Irkutsk virus,以及WestCaucassian bat virus(WCBV)幾種狂犬相關(guān)病毒�；�1型病毒為狂犬病毒,其他基因型病毒稱為狂犬病相關(guān)病毒。這些基因型又進(jìn)一步合并為2個(gè)遺傳譜系(phylogroup):基因型1、4、5、6和7為遺傳譜系Ⅰ;基因型2和3為遺傳譜系Ⅱ。蝙蝠是狂犬病較獨(dú)特的宿主和媒介,除基因3型狂犬相關(guān)病毒外狂犬病毒屬各基因型均可在蝙蝠體內(nèi)分離得到。 我國(guó)是受狂犬病危害最為嚴(yán)重的國(guó)家之一,我國(guó)華南地區(qū)及部分華中地區(qū)近幾年狂犬病的發(fā)病數(shù)呈快速上升趨勢(shì)。這些地區(qū)蝙蝠分布數(shù)量較大,種類多,且蝙蝠夏天活動(dòng)冬天冬眠的特點(diǎn)與狂犬病春夏秋發(fā)病多季節(jié)特點(diǎn)有關(guān)。這些現(xiàn)象提示我國(guó)蝙蝠可能作為狂犬病毒的宿主。然而,目前我國(guó)這一方面的研究還比較少。我國(guó)蝙蝠攜帶狂犬病毒的情況尚不清楚,因此有必要開(kāi)展研究。 2研究方法 2.1建立狂犬病毒免疫學(xué)和分子生物學(xué)檢測(cè)方法 2.1.1直接免疫熒光技術(shù)(The direct fluorescent antibody test,DFA)檢測(cè)狂犬病毒 ①用狂犬病毒減毒活疫苗顱內(nèi)接種小鼠,選擇抗體工作濃度。 ②用選擇的抗體濃度對(duì)蝙蝠腦組織進(jìn)行檢測(cè)。 2.1.2巢式RT-PCR檢測(cè)狂犬病毒 ①參照論文,以特異性擴(kuò)增核蛋白(N)基因最保守區(qū)域?yàn)槟康幕?比對(duì)了7個(gè)基因型的狂犬病毒,設(shè)計(jì)了套式RT-PCR檢測(cè)引物。 ②用狂犬病毒減毒活疫苗作為陽(yáng)性對(duì)照,進(jìn)行巢式RT-PCR最低檢測(cè)濃度試驗(yàn)。 ③巢式RT-PCR對(duì)蝙蝠腦組織狂犬病毒的檢測(cè)。 2.1.3初步建立RT-LAMP(RT-loop-mediated isothermal amplification)快速檢測(cè)方法檢測(cè)狂犬病毒 ①參照論文,以特異性擴(kuò)增核蛋白(N)基因最保守區(qū)域?yàn)槟康幕?比對(duì)了7個(gè)基因型的狂犬病毒,自行設(shè)計(jì)RT-LAMP檢測(cè)引物。 ②用狂犬病毒減毒活疫苗作為陽(yáng)性對(duì)照,進(jìn)行RT-LAMP最低檢測(cè)濃度試驗(yàn)。 2.2蝙蝠標(biāo)本的采集 2007年5月至2007年8月之間,2008年7月至2008年8月之間,在海南、廣東和湖南部分地區(qū)共收集蝙蝠1057只,進(jìn)行編號(hào)后,請(qǐng)廣州大學(xué)生命科學(xué)院蝙蝠研究專家吳毅教授進(jìn)行鑒定。采集蝙蝠的地方主要有廢棄的房屋,或者房屋的縫隙、屋檐,以及野外陰濕的山洞。通過(guò)無(wú)菌操作解剖蝙蝠,取完整腦組織標(biāo)本,一份標(biāo)本分為三種保存方式。分別保存在含有RNAlater、PBS及含10%甲醛的PBS緩沖鹽溶液的凍存管中,4℃運(yùn)輸,回實(shí)驗(yàn)室后,放入-70℃保存。 2.3蝙蝠腦組織樣本狂犬病毒的檢測(cè) 用上述建立的DFA和巢式RT-PCR方法對(duì)采集的蝙蝠腦組織進(jìn)行檢測(cè)。 2.4測(cè)序和基因分析 ①對(duì)于擴(kuò)增獲得的目的基因進(jìn)行測(cè)序; ②測(cè)序結(jié)果利用GeneBank的BLAST軟件進(jìn)行在線同源性分析; ③在GeneBank下載其他狂犬病毒序列,利用Clustal W進(jìn)行多序列比對(duì)分析; ④用MEGA 4.0軟件,采用Neighbor-Joining法,Jukes-Cantor模型做進(jìn)化樹分析。 2.5病毒分離培養(yǎng) 檢出陽(yáng)性樣本用Vero細(xì)胞進(jìn)行病毒分離培養(yǎng),結(jié)果用流式細(xì)胞術(shù)和細(xì)胞免疫熒光方法進(jìn)行鑒定。 2.6質(zhì)量控制 ①移液槍頭、EP管、凍存管、手套等實(shí)驗(yàn)耗材均為一次性用品; ②逆轉(zhuǎn)錄過(guò)程采用的移液槍頭、EP管和研磨器用DEPC處理,高溫高壓消毒,烤干后待用,以去除RNA酶污染; ③RNA的提取,RT-PCR的反應(yīng)體系的試劑加樣均在無(wú)菌間生物安全Ⅱ級(jí)超凈臺(tái)進(jìn)行操作,防止環(huán)境中RNA酶的污染; ④操作過(guò)程中,全程戴口罩、帽子,勤換手套,防止樣本和試劑受到來(lái)自操作者攜帶的RNA酶污染; ⑤在RNA提取,RT-PCR,直接免疫熒光過(guò)程中均設(shè)立陽(yáng)性對(duì)照和陰性對(duì)照。 3結(jié)果 3.1狂犬病毒直接免疫熒光檢測(cè)方法的建立:直接免疫熒光法檢測(cè)狂犬病毒,篩選免疫熒光抗體工作濃度為1:1×10~2,該濃度下所有陽(yáng)性對(duì)照均能檢出,而陰性對(duì)照未檢出�？稍隍鹉X組織中檢出狂犬病毒。 3.2巢式RT-PCR檢測(cè)方法的建立:應(yīng)用巢式RT-PCR方法對(duì)狂犬病毒進(jìn)行檢測(cè),能檢測(cè)到狂犬病毒減毒活疫苗(20.0 LD_(50)/0.03 ml)最低濃度為1:1×10~4�？瞻讓�(duì)照及陰性對(duì)照均未檢出,靈敏度、特異度均較高�？蓹z出蝙蝠腦組織中的狂犬病毒。 3.3 RT-LAMP檢測(cè)的初步建立:應(yīng)用RT-LAMP方法對(duì)狂犬病毒進(jìn)行檢測(cè),能檢測(cè)到狂犬病毒減毒活疫苗(10.0 LD_(50)/0.03 ml)最低濃度為1:1×10~4。空白對(duì)照及陰性對(duì)照均未檢出,靈敏度、特異度均較高。初步建立狂犬病毒的快速檢測(cè)方法,用于蝙蝠腦組織檢測(cè),與巢式RT-PCR相比有較高的靈敏度,且大大地縮短了檢測(cè)時(shí)間。 3.4病毒分離培養(yǎng)后鑒定:2007年采集的30例狂犬陽(yáng)性樣本懸液轉(zhuǎn)染Vero細(xì)胞7 d后,作流式細(xì)胞術(shù)檢測(cè),加入熒光標(biāo)記的狂犬病毒單克隆抗體后,平均熒光強(qiáng)度增強(qiáng)5.34～27.22,95%可信區(qū)間10.62～14.66;熒光細(xì)胞結(jié)合百分率增加3.48%～11.74%,95%可信區(qū)間5.52%～7.36%。2008年采集的9例陽(yáng)性腦組織樣本制成懸液后,轉(zhuǎn)染細(xì)胞用熒光標(biāo)記抗體結(jié)合后,在顯微鏡下可見(jiàn)蘋果綠熒光,而未加入熒光抗體細(xì)胞空白對(duì)照未見(jiàn)熒光。 3.5現(xiàn)場(chǎng)調(diào)查應(yīng)用:應(yīng)用直接免疫熒光和巢RT-PCR兩種檢測(cè)方法對(duì)采集的蝙蝠腦組織標(biāo)本進(jìn)行檢測(cè),在普通長(zhǎng)翼蝠、中菊頭蝠和小黃蝠中都檢測(cè)出了狂犬病毒。巢式RT-PCR法檢測(cè)陽(yáng)性率為3.69%(39/1057),其中第一年陽(yáng)性率為6.41%(30/468),第二年陽(yáng)性率為1.53%(9/589),檢出自湖南,均從普通長(zhǎng)翼蝠和中菊頭蝠中檢出。對(duì)2008年采集的589份蝙蝠腦組織樣本,直接免疫熒光方法檢出率為9.85%(58/589)。陽(yáng)性樣本檢出自湖南的普通長(zhǎng)翼蝠、中菊頭蝠和海南的小黃蝠。 3.6對(duì)序列進(jìn)一步的系統(tǒng)進(jìn)化分析表明:盡管樣本間存在基因多態(tài)性,但是構(gòu)建的系統(tǒng)進(jìn)化樹顯示本次所檢測(cè)的蝙蝠狂犬病毒聚集成一個(gè)分支,都屬于狂犬病毒基因Ⅰ型。 4結(jié)論 4.1蝙蝠腦組織攜帶狂犬病檢測(cè)方法的建立:建立了直接免疫熒光和巢式RT-PCR兩種方法實(shí)驗(yàn)室檢測(cè)狂犬病毒,建立的方法可以用于蝙蝠腦組織狂犬病毒的檢測(cè)。兩種方法在檢測(cè)狂犬病毒上均有較高的特異度;初步比較直接免疫熒光方法和巢式RT-PCR方法檢測(cè)蝙蝠腦組織攜帶狂犬病毒靈敏度有所不同,直接免疫熒光較高。初步建立的RT-LAMP方法能夠快速檢測(cè)蝙蝠腦組織狂犬病毒。 4.2用所建立的兩種方法對(duì)中國(guó)南方三省的蝙蝠進(jìn)行現(xiàn)場(chǎng)流行病學(xué)調(diào)查應(yīng)用,檢出湖南中菊頭蝠、普通長(zhǎng)翼蝠和海南小黃蝠三種體內(nèi)存在狂犬病毒。但不能排除其他種類蝙蝠攜帶狂犬病毒的可能。 4.3本次研究檢測(cè)出的狂犬病毒序列差異不大,全部包括在CHINA-1分支,屬于基因1型狂犬病毒。與亞洲流行的基因型別一致。
[Abstract]:1. background and purpose of the study:
Rabies (Rabies) is a natural epidemic disease of zoonotic disease, which can cause acute fatal encephalomyelitis in human and all warm blooded animals. Once the disease occurs, the patient or the sick animal is almost 100%. The rabies can be distributed worldwide and is a global serious public health problem. The Rabies virus is the pathogen of human and animal rabies. There are many known animal hosts, and more and more species are able to separate rabies virus with the study. Since the first discovery of the human rabies caused by the bite of insectivorous bats in the United States in 1953, the bats carrying rabies virus and its relationship with human rabies have aroused widespread concern in human beings. In recent years, many countries and regions have continuously been from many countries and regions. The bats are isolated from the rabies virus.
The main source of rabies infection is wild animals, including wolves, foxes, leopards, wild dogs, monkeys, cats, skunk, raccoons and bats. The epidemic of rabies is usually found in the middle of wild animals and through domesticated animals to humans. Domesticated animals are the link between wild animal rabies and human rabies. Although dogs are not the most sensitive animals, but not the most sensitive animals, but Due to the high density of dogs in urban and rural areas in most parts of the world, dogs have become the long-term host and vector of viruses.
The source of rabies is different in different regions, such as more than 85% of Africa, most countries in South America and Asian countries (except Israel and Bengal) are mainly canine rabies, while South Africa, Europe, the United States and Canada are mainly wild rabies. Guyana, Panama and Togo are the most important ones. Bats spread rabies. Holland, Germany and Spain are second only to wild animal rabies. Most of the rabies in our country are caused by dogs. There are occasional reports of other animals, such as cats and cattle.
In recent years, the proportion of wild animal rabies has been increasing, and the reports of wild animal rabies have been reported to people, and the harm of wild animals carrying rabies virus is becoming more serious. Wild animals not only direct the disease directly, but also may indirectly affect the human health by destroying animal husbandry, and the management of wild animals is difficult, In general, it is difficult to immunize them. Especially, some hoofed wild animals have a migratory instinct, which can periodically change their habitats in a replacement direction to obtain the best environmental conditions. In view of this, wildlife as the storage host and medium of the rabies virus can cause more harm to the spread rabies virus.
The Chiroptera animal is one of the most widely distributed and most abundant mammals in mammals. There are about 17 families and 180 genera in the world, about 960 species, of which there are 7 families, 29 genera and 107 species in our country. The bats are divided into two suborders: the large bat suborder (Megachiroptera) and the fox bat, our country has 1 families, 5 genera and 7 species; the small bat suborder (Microchiroptera) is usually the common species. There are 6 families, 24 genera and 100 species of bats.
In addition to the north and south poles, bats are distributed all over the world, including in high latitudes, desolate deserts and isolated islands, and even in the Sahara desert. However, most bats live in tropical and subtropical areas. Bats are rich in species, widely distributed, long life, and have strong flying abilities. Some bats have a long migration habit.
Bats live in close contact with humans. Some bats perch under eaves, or cracks in houses; and bats often break into homes in foraging; bats are also inhabited in caves in some tourist attractions. In traditional Chinese medicine, the dung of chrysanthemum bat is made into "night clear sand" for treatment of eye diseases; in Guangdong, fruit bats are in restaurants. In Hunan, some areas believe that bats can be used to treat epilepsy.
Rabies virus is a rabies virus (Rhabdoviridae) rabies virus (Rhabdoviridae) rabies virus (Lyssavirus), with a projectile shape (60~400 nm x 60~85 nm), one end pure garden, a flat end, a capsule, and a spiral symmetry in the capsid. The nucleic acid is a single strand of negative segment RNA, and the gene group is about 12 KB, and the 3 'to 5' ends are N, P, M, G, 5 bases. Because each gene contains an uncoded interval sequence, encoding nucleoprotein (N), protein (P), matrix protein (M), glycoprotein (G), and transcriptional large protein (L). The complete virus particles are composed of two parts of the nucleocapsid and envelope, and the nucleocapsid is composed of RNA and nuclear egg white, protein and transcriptional protein, and the envelope is composed of glycoprotein and matrix protein. Of the 5 genes of rabies virus, the N gene has a highly conserved.1993 year Bourhy and so on, according to the homology of the 500 bases of the nucleoprotein gene, the rabies virus is divided into 6 genotypes, and the gene 1~4 corresponds to the serum I to IV respectively, while the serotype V type European rabies virus EBL1 and EBL2 strain is the genotype 5,6.1998 Australian Australian Dali. Subreports isolated the new bat rabies virus (ABLV), the Australian bat rabies virus (ABLV). In recent years, the new gene type of rabies virus (Lyssavirus) has been found to separate Aravan virus, Khujand virus, Irkutsk virus, and WestCaucassian bat virus (WCBV) virus related viruses in the bats. The gene type 1 virus is a rabies virus, and the other genotypes are called rabies related viruses. These genotypes are further merged into 2 genetic lineages (phylogroup): genotype 1,4,5,6 and 7 are genetic lineage I; genotype 2 and 3 are genetic lineage II. Bats are the more specific hosts and vectors of rabies, except for the virus type 3 rabies related viruses. All rabies virus genotypes can be isolated from bat.
China is one of the countries most seriously affected by rabies. The number of rabies in Southern China and central China has been rising rapidly in recent years. These areas have a large number of bats and many species, and the characteristics of the winter hibernation in the summer activities of bats are related to the seasonal characteristics of rabies in spring, summer and autumn. Bats in our country may be used as the host of rabies virus. However, there are few studies on this aspect in our country at present. It is not clear that bats are carrying rabies virus in our country, so it is necessary to carry out the research.
2 research methods
2.1 to establish rabies virus immunology and molecular biology detection methods.
Detection of rabies virus by 2.1.1 The direct fluorescent antibody test (DFA)
First, the live attenuated rabies virus vaccine was injected into the mice and the antibody concentration was selected.
(2) using the selected antibody concentration to detect the brain tissue of bat.
Detection of rabies virus by 2.1.2 nested RT-PCR
(1) according to the paper, using the most conservative region of the specific amplified nucleoprotein (N) gene as the target gene, a set of primers for RT-PCR detection were designed to compare the 7 genotypes of rabies virus.
(2) using the live attenuated rabies vaccine as the positive control, the nested RT-PCR minimum concentration test was conducted.
Nested RT-PCR for detection of rabies virus in bat brain tissue.
2.1.3 preliminary establishment of RT-LAMP (RT-loop-mediated isothermal amplification) rapid detection method for rabies virus detection
(1) according to the paper, the most conservative region of the specific amplified nucleoprotein (N) gene was used as the target gene, and the 7 genotypes of rabies virus were compared, and RT-LAMP primers were designed by ourselves.
(2) using the live attenuated rabies vaccine as the positive control, the minimum RT-LAMP concentration test was carried out.
Collection of 2.2 bats
From May 2007 to August 2007, between July 2008 and August 2008, 1057 bats were collected in parts of Hainan, Guangdong and Hunan. After numbering, Professor Wu Yi, a bat research expert at the Guangzhou University Life Science Academy, was asked to identify the places where the bats were mainly abandoned, or the gaps, eaves, and wilderness of the houses. The whole brain tissue specimens were dissected by aseptic operation. A specimen was divided into three preservation methods. They were stored in the cryopreservation tube containing RNAlater, PBS and 10% formaldehyde containing PBS buffer solution, and were transported to the laboratory at 4, and stored at -70 C after returning to the laboratory.
Detection of rabies virus in 2.3 bats brain tissue samples
The DFA and nested RT-PCR methods were used to detect the brain tissues of the bat.
2.4 sequencing and gene analysis
(1) sequencing of the target genes obtained by amplification;
(2) sequencing results were analyzed by online homology analysis using GeneBank's BLAST software.
(3) downloading other rabies virus sequences in GeneBank, and using Clustal W for multiple sequence alignment analysis.
(4) using MEGA 4 software, Neighbor-Joining method and Jukes-Cantor model were used to analyze phylogenetic tree.
2.5 virus isolation and culture
The positive samples were detected by Vero cells. The results were identified by flow cytometry and cellular immunofluorescence.
2.6 quality control
(1) all the experimental consumables such as pipette head, EP tube, freezing tube and gloves are disposable goods.
(2) the pipette head used in the reverse transcription process, the EP tube and the grinder were treated with DEPC, then sterilized at high temperature and high pressure, dried and then used to remove RNA enzyme pollution.
(3) the extraction of RNA and the reagent addition of RT-PCR reaction system are operated at the aseptic biosafety stage II super clean platform to prevent the pollution of RNA enzymes in the environment.
During the operation, wearing masks, hats and gloves should be worn throughout the operation to prevent samples and reagents from being contaminated by RNA enzymes from operators.
5. Positive control and negative control were established in RNA extraction, RT-PCR and direct immunofluorescence.
3 Results
The establishment of 3.1 rabies virus direct immunofluorescence detection methods: direct immunofluorescence detection of rabies virus, screening immunofluorescence antibody working concentration of 1:1 x 10~2, the concentration of all positive controls can be detected, but the negative control is not detected.
3.2 the establishment of nested RT-PCR detection method: the nested RT-PCR method was used to detect rabies virus, and the lowest concentration of rabies virus attenuated vaccine (20 LD_ (50) /0.03 ml) was detected, and the lowest concentration was 1:1 x 10~4. blank control and negative control, and the sensitivity and specificity were all high.
The preliminary establishment of 3.3 RT-LAMP detection: using RT-LAMP method to detect rabies virus, the lowest concentration of rabies virus attenuated vaccine (10 LD_ (50) /0.03 ml) was detected, the minimum concentration was 1:1 x 10~4. blank control and negative control, and the sensitivity and specificity were all high. Weaving detection has higher sensitivity compared with nested RT-PCR, and greatly reduces detection time.
3.4 virus isolation and culture identification: 30 cases of rabies positive sample suspension collected in 2007 were transfected to Vero cells for 7 d and were detected by flow cytometry. The average fluorescence intensity increased from 5.34 to 27.22,95% confidence interval 10.62 to 14.66, and the fluorescein binding percentage increased 3.48% to 11.74%, 95% trusted. After a suspension of 9 positive brain tissue samples collected in the interval from 5.52% to 7.36%.2008, the transfected cells were combined with fluorescent labeled antibodies, and the apple green fluorescence was observed under the microscope, but no fluorescence was found in the blank control of the fluorescent antibody cells.
3.5 field investigation application: using direct immunofluorescence and nest RT-PCR two detection methods to detect the collected bats' brain tissue specimens, the rabies virus was detected in ordinary long wing bats, middle chrysanthemum bats and small yellow bats. The positive rate of nested RT-PCR method was 3.69% (39/1057), and the positive rate of the first year was 6.41% (30/468) and second year positive. The sex rate was 1.53% (9/589), detected from Hunan, all from the common long wing bats and the middle chrysanthemum bats. The direct immunofluorescence detection rate of 589 bats collected in 2008 was 9.85% (58/589). The positive samples were detected from the common long wing bats, the middle chrysanthemum bats and the Yellow bats in Hainan.
3.6 further phylogenetic analysis of sequences showed that although polymorphisms existed among samples

【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2009
【分類號(hào)】：R181.3

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