本文選題：副豬嗜血桿菌　切入點：16S　出處：《河南科技大學(xué)》2017年碩士論文

【摘要】：副豬嗜血桿菌(Haemophlius parasuis,Hps)危害嚴重,是目前養(yǎng)豬業(yè)的多發(fā)病之一,建立快速準(zhǔn)確的診斷方法,及時采取相應(yīng)的防治措施是防治成功的關(guān)鍵。但是副豬嗜血桿菌分離難度較大,常規(guī)的檢測方法比較繁瑣,且需要昂貴的儀器設(shè)備,很難在基層推廣。而環(huán)介導(dǎo)等溫擴增技術(shù)(loop-mediated isothermal amplification,LAMP)簡單方便,不需要特殊設(shè)備,所以建立基于LAMP的副豬嗜血桿菌的快速檢測方法十分必要,在臨床檢測中的應(yīng)用潛力非常大。本研究通過比對GengBank中已發(fā)表的Hps 16S r RNA序列,找出其高度保守區(qū)域,以此為靶基因并針對其6個特異性區(qū)域設(shè)計出4條特異性引物:兩條外引物F3、B3和兩條內(nèi)引物FIP、BIP,利用Bst DNA聚合酶的鏈置換作用對Hps進行LAMP擴增反應(yīng),通過優(yōu)化反應(yīng)體系和反應(yīng)條件建立了Hps的LAMP快速檢測方法,并檢測該方法的靈敏性與特異性。1.最佳反應(yīng)體系的篩選通過設(shè)置單因素梯度變化分別對反應(yīng)體系中的內(nèi)外引物比(F3∶FIP,B3∶BIP)、Mg2+終濃度、dNTPs終濃度進行優(yōu)化,內(nèi)引物比梯度為1∶1、1∶2、1∶4、1∶6、1∶8、1∶10,Mg2+終濃度梯度為0 mM、2 mM、4 mM、6mM、8 mM、10 mM,dNTPs終濃度梯度為0 mM、1 mM、2 mM、3 mM、4mM、5 mM。最終確立了LAMP反應(yīng)的最佳反應(yīng)體系:外引物各0.2μM,內(nèi)引物各0.8μM,Mg2+終濃度為6 mM,dNTPs終濃度為3 m M,10×Thermo Pol緩沖液2.5μL,Bst DNA聚合酶1μL,模板1μL,補水至25μL。2.最佳反應(yīng)條件的優(yōu)化通過設(shè)置單因素梯度變化分別對反應(yīng)溫度和反應(yīng)時間進行優(yōu)化,反應(yīng)溫度梯度為60℃、61℃、62℃、63℃、64℃、65℃、66℃,反時間梯度為30min、40 min、50 min、60 min、70 min。結(jié)果顯示反應(yīng)在63℃條件下1 h即可完成。3.LAMP靈敏性檢測對Hps基因組DNA進行10-n倍梯度稀釋,以100、10-1、10-2、10-3、10-4、10-5、10-6、10-7、10-8倍稀釋的基因組為模板分別進行常規(guī)PCR反應(yīng)和LAMP反應(yīng),通過比較兩種反應(yīng)的檢測結(jié)果來反映LAMP反應(yīng)的敏感性。結(jié)果顯示LAMP方法敏感性是PCR的100倍,最低檢出量為0.241 pg/μL。4.LAMP特異性檢測分別以副豬嗜血桿菌、豬鏈球菌(Streptococcus suis)、沙門氏菌(Salmonella enteriditis)、大腸桿菌(Escherichia coli)、枯草芽孢桿菌(Bacillus subtilis)、胸膜肺炎放線桿菌(Actinobacillus pleuropneumoniae)、金黃色葡萄球菌(Staphylocococcus aureus)為模板,用本研究所建立的方法進行LAMP反應(yīng),驗證該方法的特異性。結(jié)果表明該方法僅副豬嗜血桿菌能擴增出條帶,其他細菌均不能,說明該方法具有較好的特異性。本研究成功的建立了副豬嗜血桿菌LAMP快速檢測方法,該方法操作簡便、靈敏度高、特異型強,能夠用肉眼直觀的觀察實驗結(jié)果,適用于基層和現(xiàn)場快速診斷,為副豬嗜血桿菌的臨床診斷提供了重要的理論依據(jù)。
[Abstract]:Haemophlius parasuis (Hpss) is one of the most common diseases in swine industry at present. The key to success of prevention and treatment is to establish a rapid and accurate diagnosis method and to take corresponding prevention and cure measures in time.But the isolation of Haemophilus parasuis is difficult, the routine detection method is complicated, and expensive equipment is needed, so it is difficult to be popularized at the basic level.The loop-mediated isothermal amplification technique is simple and convenient, and does not need special equipment. Therefore, it is necessary to establish a rapid detection method for Haemophilus parais based on LAMP, which has great potential in clinical detection.By comparing the published Hps 16s r RNA sequences in GengBank, the highly conserved regions were found.Four specific primers were designed according to the target gene and six specific regions: two outer primers F3B3 and two internal primers FIP-BIP.The LAMP amplification reaction of Hps was carried out by using the chain replacement of Bst DNA polymerase.A rapid LAMP detection method for Hps was established by optimizing the reaction system and reaction conditions, and the sensitivity and specificity of the method were determined.The optimal reaction system was screened by setting a single factor gradient to optimize the final concentration of dNTPs in F3: FIPB3: BIP3: BIP2 final concentration.Finally, the optimal reaction system of LAMP reaction was established: external primer 0.2 渭 M, internal primer 0.8 渭 m mtMg2 final concentration 6 mMmmdNTPs final concentration 3 m MN 10 脳 Thermo Pol buffer 2.5 渭 L BST DNA polymerase 1 渭 L, template 1 渭 L, water up to 25 渭 L. 2.The optimum reaction conditions were optimized by setting single factor gradient. The reaction temperature gradient was 60 鈩，

本文編號：1693176