本文選題：鴨病毒性肝炎 + DHAV-1�。� 參考：《四川農(nóng)業(yè)大學(xué)》2015年碩士論文

【摘要】：鴨病毒性肝炎(DVH)是雛鴨的一種急性傳染性病,主要侵害4周齡以內(nèi)的雛鴨。該病由至少三種不同的病原引起,包括鴨甲肝病毒(DHAV).1型鴨星狀病毒(DastV-1)口2型鴨星狀病毒(DastV-2)。分布范圍最廣的D IAV屬于小RNA病毒科禽肝病毒屬�；谥泻驮囼�(yàn)和系統(tǒng)發(fā)育分析,DHAV已被分為三種基因型(DHAV-1、 DHAV-2、DHAV-3)。其中,DHAV-1分布范圍最廣泛, DHAV-2僅在臺灣發(fā)現(xiàn)報(bào)道,而近幾年DHAV-3已經(jīng)給我國大陸地區(qū)和韓國的養(yǎng)鴨業(yè)造成嚴(yán)重危害。DHAV-1與DHAV-2之間無交叉中和反應(yīng),DHAV-1和 DHAV-3之間交叉保護(hù)作用不明顯。通過對雛鴨的臨床癥狀和剖檢病變可以初步對DVH進(jìn)行診斷,但是該方法不能夠區(qū)分DHAV-1 和 DHAV-3.目前血清學(xué)診斷技術(shù)和分子生物學(xué)診斷技術(shù)已經(jīng)用DHAV的檢測和診斷,血清學(xué)診斷技術(shù)主要包括中和試驗(yàn)、間接血凝試驗(yàn)、瓊脂擴(kuò)散試驗(yàn)和免疫熒光抗體技術(shù)等,但是由于其繁瑣耗時和成本高,不能用于快速診斷。分子生物學(xué)診斷技術(shù)包括RT-PCR口RT-LAMP方法等,能夠快速特異地對DHAV分離株和臨床樣品進(jìn)行診斷,因此建立一種能夠快速鑒別診斷DHAV-1和DHAV-3的一步法雙重RT-PCR方法對我國DVH的防治有積極的作用。1.建立一步法雙重RT-PCR方法用于檢測DHAV-1和DHAV-3為了能夠快速鑒別診斷DHAV-1和DHAV-3,本研究根據(jù)GenBank中DHAV-1和DHAV-3的全基因組序列,在DHAV-1和DHAV-3的基因保守區(qū)域分別設(shè)計(jì)了2對特異性引物,并成功建立一步法雙重RT-PCR方法鑒別診斷DHAV-1和DHAV-3。該一步法雙重RT-PCR方法能夠分別針對DHAV-1和DHAV-3特異性地?cái)U(kuò)增出992bp和1533bp的目的片段,而對健康雛鴨肝臟組織、番鴨細(xì)小病毒、鴨瘟病毒、鴨圓環(huán)病毒、禽流感病毒、鴨疫里默氏菌、巴氏桿菌、金黃色葡萄球菌、大腸桿菌和沙門氏菌核酸的檢測結(jié)果為陰性。該方法最低能夠檢測出lOpg DHAV-(?)DHAV-3的RNA,具有較高的敏感性。2.應(yīng)用一步法雙重RT-PCR方法檢測DHAV-1和D HAV-3本研究中使用建立好的一步法雙重RT-PCR方法DHAV人工感染雛鴨和DHAV分離株進(jìn)行檢測,結(jié)果顯示該一步法雙重RT-PCR方法的檢測結(jié)果和病毒分離實(shí)驗(yàn)完全一致。應(yīng)用建立好的一步法雙重RT-PCR方法對從2012～2014年在四川、山東、湖北和廣西省采集的52份疑似DVH病料進(jìn)行檢測,結(jié)果顯示52份病料中有41份為DHAV-1陽性,11份為DHAV-3陽性,這些材料未發(fā)現(xiàn)混合感染情況。以上實(shí)驗(yàn)證明本研究中建立的一步法雙重RT-PCR檢測方法能夠用于DHAV-1(?)DHAV-3的臨床診斷和流行病學(xué)調(diào)查,并且檢測結(jié)果表明我國四川、山東、湖北和廣西省養(yǎng)鴨地區(qū)均有DHAV-1和DHAV-3的流行。綜上所述,本研究中建立的一步法雙重RT-PCR檢測方法可以快速地鑒別診斷DHAV-1和DHAV-3的感染情況。
[Abstract]:Duck viral hepatitis (DVH) is an acute sexually transmitted disease of ducklings, mainly infecting ducklings within 4 weeks of age. The disease is caused by at least three different pathogens, including DHAV1. 1 duck stellate virus (DastV-1) and DastV-2 stellate virus (DastV-2). The most widely distributed D IAV belongs to the genus Avian liver virus of small RNA virus family. Based on neutralization test and phylogenetic analysis, DHAV has been divided into three genotypes: DHAV-1, DHAV-2, DHAV-3. The distribution of DHAV-1 was the most widespread, and DHAV-2 was only reported in Taiwan. In recent years, DHAV-3 has caused serious harm to duck industry in mainland China and South Korea. There is no cross neutralization reaction between DHAV-1 and DHAV-2. The cross protection between DHAV-1 and DHAV-3 is not obvious. DVH can be diagnosed by clinical symptoms and pathological changes of ducklings, but this method can not distinguish DHAV-1 from DHAV-3. At present, serological diagnostic techniques and molecular biological diagnostic techniques have been used for detection and diagnosis of DHAV. Serological diagnostic techniques mainly include neutralization test, indirect hemagglutination test, Agar diffusion test and immunofluorescence antibody technique. However, because of its tedious, time-consuming and high cost, it can not be used for rapid diagnosis. Molecular biological diagnostic techniques, including RT-PCR oral RT-LAMP, can be used to diagnose DHAV isolates and clinical samples quickly and specifically. Therefore, the establishment of a one step double RT-PCR method for rapid differential diagnosis of DHAV-1 and DHAV-3 has a positive effect on the prevention and treatment of DVH in China. A one step double RT-PCR method was established to detect DHAV-1 and DHAV-3. In order to differentiate DHAV-1 and DHAV-3 quickly, two pairs of specific primers were designed in the conserved region of DHAV-1 and DHAV-3 according to the whole genome sequence of DHAV-1 and DHAV-3 in GenBank. One step double RT-PCR method was successfully established for differential diagnosis of DHAV-1 and DHAV-3. The two-step RT-PCR method could specifically amplify the target fragments of 992bp and 1533bp against DHAV-1 and DHAV-3, respectively, and could be applied to the liver tissue of healthy ducklings, Muscovy duck parvovirus, duck plague virus, duck circovirus, avian influenza virus and Riemer's disease virus. The nucleic acids of Pasteurella, Staphylococcus aureus, Escherichia coli and Salmonella were negative. This method can detect lOpg DHAV-(?)DHAV-3 at least, and has high sensitivity. 2. 2. One step double RT-PCR method was used to detect DHAV-1 and D HAV-3. In this study, a one step double RT-PCR method was used to detect artificially infected ducklings and DHAV isolates. The results show that the double RT-PCR method is in good agreement with the virus isolation test. A one step double RT-PCR method was used to detect 52 suspected DVH samples collected from 2012 to 2014 in Sichuan, Shandong, Hubei and Guangxi provinces. The results showed that 41 of 52 samples were DHAV-1 positive and 11 were DHAV-3 positive. No mixed infection was found in these materials. The results show that the two-step RT-PCR detection method can be used in the clinical diagnosis and epidemiological investigation of DHAV-1(?)DHAV-3, and the results show that DHAV-1 and DHAV-3 are prevalent in duck breeding areas of Sichuan, Shandong, Hubei and Guangxi provinces in China. In conclusion, the one-step double-step RT-PCR detection method established in this study can quickly differentiate the infection between DHAV-1 and DHAV-3.
【學(xué)位授予單位】：四川農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S852.65

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 楊發(fā)龍;張煥容;程方明;岳華;湯承;;基于基因C型鴨甲肝病毒VP1重組蛋白的鴨甲肝病毒抗體ELISA檢測方法的建立[J];畜牧獸醫(yī)學(xué)報(bào);2014年07期

2 張冰;張煥容;楊發(fā)龍;湯承;岳華;;基因C型鴨甲型肝炎病毒在鴨胚原代肝細(xì)胞中的增殖規(guī)律[J];中國獸醫(yī)科學(xué);2013年08期

3 孫慶歌;薛霜;肖愛芳;馬慧慧;朱薇;漆世華;溫文生;舒銀輝;謝紅玲;蘇敬良;;血清3型鴨甲型肝炎病毒實(shí)時熒光定量RT-PCR檢測方法的建立與初步應(yīng)用[J];中國獸藥雜志;2013年04期

4 李鑫;朱永梅;母曉宇;馬波;王君偉;;鴨肝炎病毒在鴨胚成纖維細(xì)胞上的適應(yīng)性研究[J];中國家禽;2013年04期

5 付玉志;張洪輝;李傳峰;陳宗艷;王超;陳鐵橋;劉光清;;I型鴨肝炎病毒在鴨胚成纖維細(xì)胞系中的增殖特性研究[J];中國預(yù)防獸醫(yī)學(xué)報(bào);2013年02期

6 ;Genetic Variation of the VP1 Gene of the Virulent Duck Hepatitis A Virus Type 1(DHAV-1) Isolates in Shandong Province of China[J];Virologica Sinica;2012年04期

7 黃秋雪;湯承;聶培婷;岳華;;鴨甲肝病毒基因A型和C型雙重RT-PCR檢測方法的建立[J];中國預(yù)防獸醫(yī)學(xué)報(bào);2012年02期

8 謝麗基;謝芝勛;劉加波;龐耀珊;鄧顯文;謝志勤;范晴;;鴨Ⅰ型肝炎病毒RT-LAMP可視化檢測方法的建立[J];中國預(yù)防獸醫(yī)學(xué)報(bào);2012年02期

9 李嬌;王金良;祖立闖;趙金花;沈志強(qiáng);;Ⅰ型鴨肝炎病毒巢式RT-PCR檢測方法的建立及應(yīng)用[J];中國獸醫(yī)科學(xué);2011年07期

10 孫濤;肖西志;徐彪;梁成珠;凌宗帥;張?zhí)?岳志芹;;Ⅰ型鴨肝炎病毒PCR-DHPLC檢測方法的建立[J];動物醫(yī)學(xué)進(jìn)展;2011年07期

相關(guān)會議論文 前2條

1 陳琳琳;徐倩;張瑞華;楊蕾;竇鵬斐;李井新;謝之景;姜世金;司興奎;;快速鑒別診斷鴨甲肝病毒1型和3型的雙重RT-PCR方法的建立和應(yīng)用[A];中國畜牧獸醫(yī)學(xué)會禽病學(xué)分會第十六次學(xué)術(shù)研討會論文集[C];2012年

2 付玉志;李傳峰;陳宗艷;劉光清;;鴨肝炎病毒1型“自殺性”DNA疫苗的構(gòu)建及其免疫評價(jià)[A];中國畜牧獸醫(yī)學(xué)會獸醫(yī)公共衛(wèi)生學(xué)分會第三次學(xué)術(shù)研討會論文集[C];2012年

相關(guān)碩士學(xué)位論文 前3條

1 邵澤香;Ⅰ型鴨肝炎病毒浙江分離株序列分析與RT-PCR檢測方法建立[D];山東農(nóng)業(yè)大學(xué);2009年

2 陳海軍;鴨肝炎病毒人工感染雛鴨病理發(fā)展規(guī)律及免疫組化與免疫熒光檢測病原侵染規(guī)律的研究[D];四川農(nóng)業(yè)大學(xué);2007年

3 楊金保;鴨病毒性肝炎綜合防治措施的探討及效果評價(jià)[D];山東農(nóng)業(yè)大學(xué);2006年

，

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