小鵝瘟病毒PCR檢測(cè)方法的建立和山東濟(jì)寧地區(qū)小鵝瘟流行病學(xué)調(diào)查
發(fā)布時(shí)間：2018-10-21 11:52
【摘要】：小鵝瘟是由鵝細(xì)小病毒引起的一種急性傳染病,主要侵害30日齡以內(nèi)的雛鵝和雛番鴨,以發(fā)病急,死亡率高,傳播快為特點(diǎn)。發(fā)病率和死亡率隨著日齡的增加而下降,主要侵害7日齡以內(nèi)的雛鵝和雛番鴨,根據(jù)發(fā)病情況分為3種類型,最急性型,急性型和亞急性型(殷震and劉景華,1997)。發(fā)病鵝主要以腸粘膜脫落,壞死,腸栓,脫水等為癥狀。1966年,匈牙利學(xué)者Derszy用鵝胚也分離到病毒,他是國(guó)外第一個(gè)用鵝胚分離到病毒的人,并于1967年對(duì)本病進(jìn)行了詳盡的描述,但當(dāng)時(shí)他卻把該病誤認(rèn)為流感,直到1971年Schettler確定這種病是由細(xì)小病毒引起。1978年建議把這種病稱為鵝細(xì)小病毒感染,1974年世界禽類學(xué)會(huì)為紀(jì)念Derzsy的先期工作而將此病命名為Derzsy氏病(Derzsy D,1975)。1995年Zadori等首次報(bào)道了GPV全基因序列,并將其與番鴨細(xì)小病毒基因序列進(jìn)行了同源性比較(Zadori Z,1995)。本病大流行具有一定周期性。在大流行以后當(dāng)年余下的鵝群都獲得了主動(dòng)免疫,使次年的雛鵝具有天然被動(dòng)免疫力,故本病不會(huì)在同一地區(qū)連續(xù)兩年發(fā)生大流行。由此可見,本病的發(fā)病率和死亡率一方面和日齡有關(guān),另一方面取決于親代母鵝的特異性抗體水平。免疫預(yù)防是控制小鵝瘟的重要措施。目前已建立了PCR、ELISA、間接免疫熒光技術(shù)、微量中和試驗(yàn)等檢測(cè)小鵝瘟的方法,但是這幾種檢測(cè)方法均需特殊的儀器設(shè)備,且耗時(shí)長(zhǎng),不適于在基層推廣應(yīng)用。因此,建立一種更加快速、靈敏的病毒檢測(cè)方法迫在眉睫。本研究建立的聚合酶鏈?zhǔn)椒磻?yīng),具有操作簡(jiǎn)單,快速,適合基層推廣的特點(diǎn)。近年來小鵝瘟在我國(guó)的一些省、市、自治區(qū)均有流行,給養(yǎng)漖業(yè)造成嚴(yán)重的危害,同時(shí)也給飼主帶來巨大的經(jīng)濟(jì)損失,因此受到國(guó)內(nèi)外學(xué)者和工作者的廣泛關(guān)注。本研究通過工作之便,對(duì)所在地區(qū)鵝養(yǎng)殖現(xiàn)狀和小鵝瘟發(fā)病情況做了相應(yīng)的調(diào)查,并對(duì)其流行情況做了匯總,以期對(duì)養(yǎng)鵝業(yè)小鵝瘟疾病的發(fā)生做出相關(guān)的預(yù)防措施。本研究根據(jù)GENBANK上發(fā)表的小鵝瘟的vp3的序列信息,設(shè)計(jì)了一對(duì)特異性引物,通過優(yōu)化各種條件,最終擴(kuò)增出與預(yù)期片段大小一致的314bp的核酸片段,在通過相應(yīng)的特異性實(shí)驗(yàn)發(fā)現(xiàn)此引物僅能擴(kuò)增出小鵝瘟的特異片段,能夠用來檢測(cè)小鵝瘟的發(fā)生。本研究中對(duì)于分離到的10株病毒進(jìn)行測(cè)序分析,結(jié)果顯示分離株之間,同源性最高達(dá)100%,最低也在95.2%以上,說明小鵝瘟病毒的擴(kuò)增片段核苷酸變異很小,VP3是小鵝瘟的主要抗原,所以本研究對(duì)于免疫預(yù)防具有指導(dǎo)性的意義。依據(jù)本研究中調(diào)查的小鵝瘟的發(fā)病日齡等基本信息,尤其每個(gè)鵝場(chǎng)的免疫預(yù)防程序進(jìn)行了調(diào)查,發(fā)現(xiàn)小鵝瘟的發(fā)病日齡集中在12-2月份之間,根據(jù)小鵝瘟的發(fā)病情況,對(duì)其免疫程序作出了相應(yīng)的調(diào)整,結(jié)果顯示小鵝瘟的發(fā)病概率降低了,說明合適的免疫程序是預(yù)防該病的比較有效的措施。
[Abstract]:Gosling plague is an acute infectious disease caused by goose parvovirus, which mainly infects goslings and young muscovy ducks within 30 days of age. It is characterized by rapid onset, high mortality and rapid transmission. The morbidity and mortality decreased with the increase of the age of the day, mainly affecting the goslings and young muscovy ducks within 7 days of age, which were divided into three types according to the incidence, the most acute type, the acute type and the subacute type (Yin Zhen and Liu Jinghua, 1997). In 1966, Hungarian scholar Derszy also isolated the virus from goose embryos. He was the first person in foreign countries to isolate the virus from goose embryos, and described the disease in detail in 1967. But at the time he mistook the disease for the flu, It was not until 1971 that Schettler determined that the disease was caused by parvovirus. In 1978, it was suggested that the disease should be called goose parvovirus infection. In 1974, the World Avian Society named the disease Derzsy's disease (Derzsy Dl1975) to commemorate the advance work of Derzsy. Zadori et al. The entire GPV gene sequence, The gene sequence of muscovy duck parvovirus was compared with that of muscovy duck parvovirus (Zadori ZJ 1995). The epidemic has a certain periodicity. After the pandemic, the remaining geese got active immunity, which made the next goslings have natural passive immunity, so the disease would not occur in the same area for two consecutive years. Therefore, the morbidity and mortality of the disease are related to the age of day, on the other hand, it depends on the specific antibody level of the parent goose. Immune prevention is an important measure to control goose plague. At present, PCR,ELISA, indirect immunofluorescence technique and micro neutralization test have been established to detect Gosling plague. However, these methods all need special instruments and equipment, and take a long time, so they are not suitable for popularization and application in basic level. Therefore, it is urgent to establish a more rapid and sensitive method for virus detection. The polymerase chain reaction established in this study has the characteristics of simple operation, fast operation and suitable for basic level popularization. In recent years, Gosling plague has been prevalent in some provinces, cities and autonomous regions of China, which has caused serious harm to the industry of feeding and feeding, and at the same time has brought huge economic losses to the breeders, so it has been widely concerned by scholars and workers at home and abroad. In this study, the current situation of goose breeding and the incidence of Gosling plague in the region were investigated, and the epidemic situation was summarized, in order to make relevant preventive measures to the occurrence of goose plague in the goose industry. In this study, a pair of specific primers were designed based on the sequence information of vp3 of Gosling plague published on GENBANK. By optimizing various conditions, the nucleic acid fragments of 314bp with the expected size were amplified. It was found that the primer could only amplify the specific fragment of Gosling plague and be used to detect the occurrence of Gosling plague. In this study, 10 isolates were sequenced. The results showed that the highest homology was 100% and the lowest was over 95.2% among the isolates, indicating that the nucleotide variation of the amplified fragments of Gosling plague virus was very small, and VP3 was the main antigen of Gosling plague. Therefore, this study has instructive significance for immune prevention. According to the basic information of Gosling plague in this study, especially the immune prevention program in every goose farm, it was found that the incidence age of Gosling plague was between December and February, and according to the incidence of Gosling plague, The results showed that the incidence probability of Gosling plague had been reduced, which indicated that the proper immunization procedure was an effective measure to prevent the disease.
【學(xué)位授予單位】：山東農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S858.33
本文編號(hào)：2285013
[Abstract]:Gosling plague is an acute infectious disease caused by goose parvovirus, which mainly infects goslings and young muscovy ducks within 30 days of age. It is characterized by rapid onset, high mortality and rapid transmission. The morbidity and mortality decreased with the increase of the age of the day, mainly affecting the goslings and young muscovy ducks within 7 days of age, which were divided into three types according to the incidence, the most acute type, the acute type and the subacute type (Yin Zhen and Liu Jinghua, 1997). In 1966, Hungarian scholar Derszy also isolated the virus from goose embryos. He was the first person in foreign countries to isolate the virus from goose embryos, and described the disease in detail in 1967. But at the time he mistook the disease for the flu, It was not until 1971 that Schettler determined that the disease was caused by parvovirus. In 1978, it was suggested that the disease should be called goose parvovirus infection. In 1974, the World Avian Society named the disease Derzsy's disease (Derzsy Dl1975) to commemorate the advance work of Derzsy. Zadori et al. The entire GPV gene sequence, The gene sequence of muscovy duck parvovirus was compared with that of muscovy duck parvovirus (Zadori ZJ 1995). The epidemic has a certain periodicity. After the pandemic, the remaining geese got active immunity, which made the next goslings have natural passive immunity, so the disease would not occur in the same area for two consecutive years. Therefore, the morbidity and mortality of the disease are related to the age of day, on the other hand, it depends on the specific antibody level of the parent goose. Immune prevention is an important measure to control goose plague. At present, PCR,ELISA, indirect immunofluorescence technique and micro neutralization test have been established to detect Gosling plague. However, these methods all need special instruments and equipment, and take a long time, so they are not suitable for popularization and application in basic level. Therefore, it is urgent to establish a more rapid and sensitive method for virus detection. The polymerase chain reaction established in this study has the characteristics of simple operation, fast operation and suitable for basic level popularization. In recent years, Gosling plague has been prevalent in some provinces, cities and autonomous regions of China, which has caused serious harm to the industry of feeding and feeding, and at the same time has brought huge economic losses to the breeders, so it has been widely concerned by scholars and workers at home and abroad. In this study, the current situation of goose breeding and the incidence of Gosling plague in the region were investigated, and the epidemic situation was summarized, in order to make relevant preventive measures to the occurrence of goose plague in the goose industry. In this study, a pair of specific primers were designed based on the sequence information of vp3 of Gosling plague published on GENBANK. By optimizing various conditions, the nucleic acid fragments of 314bp with the expected size were amplified. It was found that the primer could only amplify the specific fragment of Gosling plague and be used to detect the occurrence of Gosling plague. In this study, 10 isolates were sequenced. The results showed that the highest homology was 100% and the lowest was over 95.2% among the isolates, indicating that the nucleotide variation of the amplified fragments of Gosling plague virus was very small, and VP3 was the main antigen of Gosling plague. Therefore, this study has instructive significance for immune prevention. According to the basic information of Gosling plague in this study, especially the immune prevention program in every goose farm, it was found that the incidence age of Gosling plague was between December and February, and according to the incidence of Gosling plague, The results showed that the incidence probability of Gosling plague had been reduced, which indicated that the proper immunization procedure was an effective measure to prevent the disease.
【學(xué)位授予單位】：山東農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S858.33
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