鸡新城疫免疫胶体金诊断技术的研究
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摘要
鸡新城疫是鸡的一种高度感染和致死性疾病，是由一种侵害呼吸道、胃肠道以及中枢神经系统的新城疫病毒(NDV)所引起的。病毒存在于病鸡的血液、脑、内脏、粪便和口鼻中，国内外对本病的控制仍没有完全解决，鸡新城疫对养鸡业有着巨大的威胁。所以有必要建立一种快速、简便、准确的胶体金免疫层析法(GICA)用于鸡新城疫的诊断。胶体会技术最早是Faulk等于1971年报道的应用于电镜检查技术的一种示踪技术。与传统方法比较，由于胶体金具有肉眼可观察的红色，所以以这种技术为基础开发的检测试剂盒有快捷、准确、高特异性、高敏感性的特点，灵敏度可达0.5～1ng/mL，适用于各级医院、卫生所、家庭个人诊断、保健、体检。目前以此方法为基础发展起来的检测试剂盒涉及范围相当广泛，包括HCG等激素、肿瘤标志物、心血管标志物、传染病的抗原与抗体的检测等。采用柠檬酸钠还原法制备胶体金颗粒，标记纯化的鸡新城疫抗体，并包被在玻璃纤维素膜上，另外将纯化的鸡新城疫抗体和纯化的兔抗鸡抗体包被在硝酸纤维素膜上，组装成鸡新城疫快速诊断试纸条。当检样中含有鸡新城疫病毒抗原时，试纸条上出现两条红线，为阳性；当检样中不含有鸡新城疫病毒抗原时，试纸条出现一条红线，为阴性。用本试验研制的鸡新城疫快速诊断试纸条检测收集的20份样品，其结果与HA结果相对应，符合率100％。当病毒HA效价在1：32以上时，试纸条均能检测为阳性。鸡新城疫快速诊断试纸条与已知的禽流感病料和鸡传染性支气管炎病料的上清夜无交叉反应出现。进行热稳定性试验稳定性好。试验证明，本研究建立的胶体金免疫层析法(GICA)是一种快速、灵敏、特异的抗原检测方法，可用于鸡新城疫诊断，也可用于鸡新城疫的流行病学调查，具有推广价值。
Developing a rapid kit for the diagnostic of Chicken Newcastle disease Chicken Newcastle Disease is a highly infectious and deadly disease, it is caused by NDV (Newcastle disease virus) that violate the respiratory tract the digestive tract and the centrencephalic system. The viruses are in blood, brain, internal organs excrement mouth and nose, there is no complete solution for this disease at home and abroad, so it is a big menace for chicken cultivating industry. So it is necessary to establish a rapid, simple,self-performing GICA(gold immunochromatographic assay) for the diagnosis of Chicken Newcastle Disease. Colloidal gold technique firstly was a kind of the tracking technique , which was reported to apply to electron microscopy(IEM) detecting technique by persons like Faulk in 1971. By comparison with the traditional technique, since the colloidal gold embodies the property of red to be seen by nake eyes, so the detective test box developed basically on this technique has rapid, sensitive, special, se
nsitive features, colloidal golds were coupled with Chicken Newcastle Disease to prepare an GICA apparatus, marking the purified antibody of Chicken Newcastle Disease, wrapping them onto nitrocellose strips, and then wrapping them
and the purified rabbit antibody for chicken onto nitrocellose, assembled a rapid kit for the diagnostic of Chicken Newcastle Disease. If the sample contains virus antigen of Chicken Newcastle Disease and the kit appears two red lines, it is positive; if the sample contains no virus antigen of Chicken Newcastle Disease and the kit appears one red line, it is negative. When using the rapid diagnosis of kits for Chicken Newcastle Disease, which are developed in this experiment, to detect the accumulated 20 samples, their results correspond the result of HA, and the corresponding rate is 100%, When virus HA titer is above 1:32, the kits are all positive. The rapid diagnosis of kit for Chicken Newcastle Disease appears no intercross reaction with the clear liquid in Avian Ingluenza(AI) and Chicken Infectious Bronchitis(CIB). It was confirmed that the GICA(gold immunochromatographic assay) established in this experiment is a kind of rapid, senstive, and special antigen test method, it can be used to test Chicken
Newcastle Disease, and to investigate epidemiology of Chicken Newcastle Disease, and has the value to be generalized.
Developing a rapid kit for the diagnostic of Chicken Newcastle disease Chicken Newcastle Disease is a highly infectious and deadly disease, it is caused by NDV (Newcastle disease virus) that violate the respiratory tract the digestive tract and the centrencephalic system. The viruses are in blood, brain, internal organs excrement mouth and nose, there is no complete solution for this disease at home and abroad, so it is a big menace for chicken cultivating industry. So it is necessary to establish a rapid, simple,self-performing GICA(gold immunochromatographic assay) for the diagnosis of Chicken Newcastle Disease. Colloidal gold technique firstly was a kind of the tracking technique , which was reported to apply to electron microscopy(IEM) detecting technique by persons like Faulk in 1971. By comparison with the traditional technique, since the colloidal gold embodies the property of red to be seen by nake eyes, so the detective test box developed basically on this technique has rapid, sensitive, special, se
nsitive features, colloidal golds were coupled with Chicken Newcastle Disease to prepare an GICA apparatus, marking the purified antibody of Chicken Newcastle Disease, wrapping them onto nitrocellose strips, and then wrapping them
and the purified rabbit antibody for chicken onto nitrocellose, assembled a rapid kit for the diagnostic of Chicken Newcastle Disease. If the sample contains virus antigen of Chicken Newcastle Disease and the kit appears two red lines, it is positive; if the sample contains no virus antigen of Chicken Newcastle Disease and the kit appears one red line, it is negative. When using the rapid diagnosis of kits for Chicken Newcastle Disease, which are developed in this experiment, to detect the accumulated 20 samples, their results correspond the result of HA, and the corresponding rate is 100%, When virus HA titer is above 1:32, the kits are all positive. The rapid diagnosis of kit for Chicken Newcastle Disease appears no intercross reaction with the clear liquid in Avian Ingluenza(AI) and Chicken Infectious Bronchitis(CIB). It was confirmed that the GICA(gold immunochromatographic assay) established in this experiment is a kind of rapid, senstive, and special antigen test method, it can be used to test Chicken
Newcastle Disease, and to investigate epidemiology of Chicken Newcastle Disease, and has the value to be generalized.
引文
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2 林绍仪．鸡新城疫研究进展．国外兽医学—畜禽传染病，1982，(2)：41-43，
3 梁英．畜牧兽医月刊，1945，(5)：10-12
4 马闻天．畜牧兽医月刊，1947，(10)：24-28
5 叶章明．鸡新城疫的流行状况．中国兽医杂志，1986，(1)：45-47
6 潘玉明．鸡新城疫免疫程序浅谈．吉林畜牧与兽医，1984，(1)：22-25
7 彭匡时．畜禽主要病毒病世界地理分别概况．国外畜牧科技，1985，(1)58-61
8 柳纪省．免疫鸡群非典型鸡新城疫概概况．中国兽医科技，1990，(5)：18-20
9 温立斌，陈陆均．雏鸡接种新城疫Ⅱ系苗后细胞免疫状态及淋巴细胞转化试验较佳测定条件的研究．中国畜禽传染病，1996，(5)：53-56
10 崔卫东，山善智，丁玉勇等．鸡新城疫—传染性法氏囊炎母源抗体的监测．黑龙江畜牧兽医，1995，(9)：24-25
11 王克恭．鸡新城疫母源抗体对雏鸡免疫的影响．家畜传染病，1985，(1)：19
12 Lancaster J. E. World' s Poultry Science Journal, 1981,37(2):84-96
13 Wallcer J. W. Avian Diseases, 1973, (17): 486
14 徐应生，黄兴，陈振健等．监测雏鸡新城疫抗体水平探讨免疫适当时间和方法试验．福建畜牧兽医，1984，6(1)：1-3
15 于圣青，刘秀梵．单克隆抗体夹心ELISA试验检测鸡新城疫病毒抗原的 研究．江苏农学院学报，1990，4(2)：41-45
16 丁铲，于圣青，刘学贤．单一血清稀释度于圣青，定量检测鸡新城疫抗体水平的研究．中国畜禽传染病，1993，8(73)：40-43
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