【摘要】：禽流感病毒(Avianinfluenzavirus, AIV).禽白血病病毒(Avianleukosisvirus,ALV)、禽傳染性支氣管炎病病毒(Infectiousbronchitisvirus, IBV)、雞傳染性法氏囊病病毒(Infectiousbursaldiseasevirus, IBDV)是四種嚴重危害家禽養(yǎng)殖業(yè)的禽類傳染病重要病原。建立AIV. ALV. IBV和IBDV特異靈敏的檢測技術(shù),對于早期發(fā)現(xiàn)傳染源,及盡早采取措施消除傳染源,切斷疾病傳播途徑,降低疾病危害具有重要意義。本研究根據(jù)ALV病毒M基因的保守序列,利用Primer Premier 5.0設(shè)計ALV簡并引物,參照文獻選擇AIV、IBV、IBDV的引物,以從AIV、ALV、IBV和IBDV的細胞培養(yǎng)液中抽提的病毒RNA作為模板,分別RT-PCR擴增了他們各自特異的基因片段,成功構(gòu)建陽性對照質(zhì)粒pMD-18T-AIV、pMD-18T-ALV、pMD-18T-IBV和pMD-18T-IBDV。通過退火溫度、引物比例、引物濃度、dNTP濃度、Mg2+濃度和PCR buffer濃度等條件的優(yōu)化,確定AIV、ALV、IBV和IBD V四重PCR最佳反應(yīng)條件為：AIV與IBV引物濃度為0.125μM, ALV與IBDV引物濃度為0.1875μM, PCR Buffe濃度為1.5×, Taq DNA聚合酶用量為0.075U/μL, Mg2+濃度為2.5mM, dNTP濃度為200μM。最佳退火溫度為52℃。以10倍系列稀釋的pMD-18T-AIV、pMD-18T-ALV、pMD-18T-IBV、作為模板擴增結(jié)果顯示,四重PCR檢測ApMD-18T-IBDV的靈敏度分別為102拷貝、103拷貝、102拷貝、103拷貝。以10倍系列稀釋的AIV、IV、ALV、IBV和IBDV和IBDV作為模板擴增結(jié)果顯示,四重RT-PCR檢測AIV、ALV、IBVALV、IBV和IBDV的靈敏度分別為四重PCR擴增0.0001TCID50、100fg、0.00001TCID50和0.001TCID50。或其中2、3和4種質(zhì)粒組合時,均可相應(yīng)產(chǎn)生1、2、3或4條特異性條帶；而檢pMD-18T-AIV.pMD-18T-ALV.pMD-18T-IBV和pMD-18T-IBDV測其它幾種禽類病毒基因克隆質(zhì)粒時,均無特異性條APV、EDSV、MDV和NDV帶產(chǎn)生。四重擴增RT-PCR核酸或其中2、3和4種核酸AIV.ALV、IBV和IBDV組合時,也均相應(yīng)產(chǎn)生1、2、3和4條特異性條帶；而檢測其它幾種禽類病毒APV、和NDV核酸時,均無特異性條帶產(chǎn)生。表明本研究建立的EDSV、MDV四重RT-PCR具有高度特異性,一次擴增可檢測可以檢測出AIV、ALV、 IBV和IBDV中任1種,或其中2、3和4種病毒的混合。AIV、ALV、 IBV和IBDV將細胞培養(yǎng)的單獨或其中2、3或4種病毒混合添加AIV、ALV、IBV、IBDV到經(jīng)單一PCR檢測陰性的禽類糞便制備人工陽性樣品,四重RT-PCR擴增結(jié)果顯示,該方法可準確檢測出四種病毒中之1種,或其中2、3和4種病毒混合的人工陽性樣品。表明本研究建立的AIV、ALV、IBV和IBDV四重RT-PCR不僅可以用于實驗室培養(yǎng)病毒的檢測,也適用于禽類組織和排泄物材料等臨床樣品中AIV、ALV、IBV和IBDV的檢測。在武漢市虎泉集貿(mào)市場采集雞、鴨和鴿子糞便樣品各10份,在湖北省武漢市、大冶市采集六科7屬9種野生鳥類67只,分別提取病毒RNA四重RT-PCR檢測,結(jié)果在雞、鴨和鴿子糞便樣品中未檢測到AIV、ALV、IBV和BDV,67只野生鳥類組織樣品中,自大冶市采集的6只斑鳩及武漢市采集的1只領(lǐng)雀嘴鵯IBDV檢測陽性,AIV、ALV和IBV檢測陰性。檢測結(jié)果為IBDV的分子流行病學及分子溯源提供了依據(jù)。
[Abstract]:Avian influenza virus (Avianinfluenzavirus, AIV). Avian Leukosis virus (Avianleukosisvirus,ALV), Avian Infectious bronchitis virus (Infectiousbronchitisvirus, IBV), Chicken Infectious bursal Disease virus (Infectiousbursaldiseasevirus, IBDV) is one of the four important pathogens of avian infectious diseases which seriously harm the poultry industry. Establish AIV. ALV. The specific and sensitive detection of IBV and IBDV is of great significance for early detection of infection source, and early measures to eliminate infection source, cut off the transmission route of disease and reduce the harm of disease. In this study, according to the conserved sequence of M gene of ALV virus, the degenerate ALV primers were designed by Primer Premier 5.0.According to the literature, the primers of AIV,IBV,IBDV were selected and the virus RNA extracted from the cell culture medium of AIV,ALV,IBV and IBDV was used as the template. Their specific gene fragments were amplified by RT-PCR respectively, and the positive control plasmids pMD-18T-AIV,pMD-18T-ALV,pMD-18T-IBV and pMD-18T-IBDV. were constructed successfully. Through the optimization of annealing temperature, primer ratio, primer concentration, dNTP concentration, Mg2 concentration and PCR buffer concentration, the optimum reaction conditions of AIV,ALV,IBV and IBD V quadruple PCR were determined as follows: the concentration of AIV and IBV primer was 0.125 渭 M, The primer concentration of ALV and IBDV was 0.1875 渭 M, the concentration of, PCR Buffe was 1.5 脳, Taq DNA polymerase dosage was 0.075U/ 渭 L, the concentration of Mg2 was 2.5 mm, the concentration of dNTP was 200 渭 M. The optimum annealing temperature is 52 鈩，

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