本文選題：豬傳染性胃腸炎病毒 + 逆轉(zhuǎn)錄環(huán)介導(dǎo)等溫?cái)U(kuò)增��； 參考：《東北農(nóng)業(yè)大學(xué)》2015年碩士論文

【摘要】：豬傳染性胃腸炎(TGE)是由豬傳染性胃腸炎病毒(TGEV)引起的一種高度接觸性傳染性疾病,臨床表現(xiàn)為嘔吐、嚴(yán)重腹瀉、脫水。TGEV對首次感染的豬群造成的危害尤其嚴(yán)重,2周齡以內(nèi)仔豬死亡率可以達(dá)到90%-100%,嚴(yán)重威脅養(yǎng)豬業(yè)的健康發(fā)展。TGE臨床癥狀與豬輪狀病毒(PRV)感染和豬流行性腹瀉(PED)等腹瀉性疾病難以區(qū)分,而且經(jīng)常出現(xiàn)混合感染,這為臨床診斷帶來了很大的困難。常規(guī)的實(shí)驗(yàn)室檢測方法不僅耗時(shí)耗力還需要特殊的儀器,在基層中難以展開,所以建立一種TGEV的方便快速的檢測方法十分必要。本試驗(yàn)建立了一種利用逆轉(zhuǎn)錄環(huán)介導(dǎo)等溫?cái)U(kuò)增(RT-LAMP)技術(shù)來檢測TGEV的快速檢測方法,對該方法進(jìn)行了溫度與時(shí)間的優(yōu)化,并對該檢測方法的特異性與靈敏性進(jìn)行了評價(jià)。針對TGEV N基因序列上的六個(gè)保守區(qū)域設(shè)計(jì)了四條RT-LAMP引物,建立針對TGEV N基因的RT-LAMP檢測方法。該檢測方法在63℃恒溫下作用50 min,使TGEV N基因獲得了最高效率的特異性擴(kuò)增,未見與PEDV等其他豬源易感病毒出現(xiàn)交叉反應(yīng),具有很好的特異性;同時(shí)該檢測方法具有極高的靈敏性,最低可檢測到1.314fg/μL的目標(biāo)病毒DNA,比普通的PCR靈敏度高三個(gè)數(shù)量級;反應(yīng)結(jié)束后可以通過加入SYBR Green I熒光染料在紫外燈下觀察反應(yīng)管的顏色變化來判斷實(shí)驗(yàn)結(jié)果,結(jié)果顯示陽性擴(kuò)增產(chǎn)物呈現(xiàn)綠色熒光,陰性擴(kuò)增產(chǎn)物為橙色。從哈爾濱周邊豬場收集47份臨床樣品RT-LAMP檢測結(jié)果與已得到驗(yàn)證的RT-PCR和ELISA結(jié)果進(jìn)行比對,結(jié)果顯示符合率為100%。本研究建立的TGEV的RT-LAMP檢測方法具有快速、特異、靈敏、簡單易操作且設(shè)備要求低等特點(diǎn),為基層實(shí)地檢測TGEV提供了技術(shù)手段。
[Abstract]:Transmissible gastroenteritis (TGEV) is a highly contagious disease caused by transmissible gastroenteritis virus (TGEV). It is characterized by vomiting and severe diarrhea.Dehydration. TGEV is especially harmful to the first infected pigs. The mortality rate of piglets within 2 weeks of age can reach 90 to 100, which is a serious threat to the healthy development of pig industry. The clinical symptoms of TGE and PRV infection of rotavirus in pigs and epidemic diarrhea in pigs (PED), etc.Diarrhoeal diseases are difficult to distinguish.And often mixed infection, which brings great difficulties in clinical diagnosis.Conventional laboratory testing methods not only time-consuming and labor-consuming, but also need special instruments, so it is very necessary to establish a convenient and rapid detection method for TGEV.A rapid detection method for TGEV by reverse transcription loop mediated isothermal amplification (RT-LAMPP) was established. The temperature and time of the method were optimized and the specificity and sensitivity of the method were evaluated.Four RT-LAMP primers were designed for six conserved regions of TGEV N gene to establish a RT-LAMP detection method for TGEV N gene.The TGEV N gene was amplified with the highest efficiency for 50 min at 63 鈩，

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