副猪嗜血杆菌LAMP快速检测方法的建立

发布时间：2018-04-01 00:29

  本文选题：副猪嗜血杆菌　切入点：16S　出处：《河南科技大学》2017年硕士论文

【摘要】：副猪嗜血杆菌(Haemophlius parasuis,Hps)危害严重,是目前养猪业的多发病之一,建立快速准确的诊断方法,及时采取相应的防治措施是防治成功的关键。但是副猪嗜血杆菌分离难度较大,常规的检测方法比较繁琐,且需要昂贵的仪器设备,很难在基层推广。而环介导等温扩增技术(loop-mediated isothermal amplification,LAMP)简单方便,不需要特殊设备,所以建立基于LAMP的副猪嗜血杆菌的快速检测方法十分必要,在临床检测中的应用潜力非常大。本研究通过比对GengBank中已发表的Hps 16S r RNA序列,找出其高度保守区域,以此为靶基因并针对其6个特异性区域设计出4条特异性引物:两条外引物F3、B3和两条内引物FIP、BIP,利用Bst DNA聚合酶的链置换作用对Hps进行LAMP扩增反应,通过优化反应体系和反应条件建立了Hps的LAMP快速检测方法,并检测该方法的灵敏性与特异性。1.最佳反应体系的筛选通过设置单因素梯度变化分别对反应体系中的内外引物比(F3∶FIP,B3∶BIP)、Mg2+终浓度、dNTPs终浓度进行优化,内引物比梯度为1∶1、1∶2、1∶4、1∶6、1∶8、1∶10,Mg2+终浓度梯度为0 mM、2 mM、4 mM、6mM、8 mM、10 mM,dNTPs终浓度梯度为0 mM、1 mM、2 mM、3 mM、4mM、5 mM。最终确立了LAMP反应的最佳反应体系:外引物各0.2μM,内引物各0.8μM,Mg2+终浓度为6 mM,dNTPs终浓度为3 m M,10×Thermo Pol缓冲液2.5μL,Bst DNA聚合酶1μL,模板1μL,补水至25μL。2.最佳反应条件的优化通过设置单因素梯度变化分别对反应温度和反应时间进行优化,反应温度梯度为60℃、61℃、62℃、63℃、64℃、65℃、66℃,反时间梯度为30min、40 min、50 min、60 min、70 min。结果显示反应在63℃条件下1 h即可完成。3.LAMP灵敏性检测对Hps基因组DNA进行10-n倍梯度稀释,以100、10-1、10-2、10-3、10-4、10-5、10-6、10-7、10-8倍稀释的基因组为模板分别进行常规PCR反应和LAMP反应,通过比较两种反应的检测结果来反映LAMP反应的敏感性。结果显示LAMP方法敏感性是PCR的100倍,最低检出量为0.241 pg/μL。4.LAMP特异性检测分别以副猪嗜血杆菌、猪链球菌(Streptococcus suis)、沙门氏菌(Salmonella enteriditis)、大肠杆菌(Escherichia coli)、枯草芽孢杆菌(Bacillus subtilis)、胸膜肺炎放线杆菌(Actinobacillus pleuropneumoniae)、金黄色葡萄球菌(Staphylocococcus aureus)为模板,用本研究所建立的方法进行LAMP反应,验证该方法的特异性。结果表明该方法仅副猪嗜血杆菌能扩增出条带,其他细菌均不能,说明该方法具有较好的特异性。本研究成功的建立了副猪嗜血杆菌LAMP快速检测方法,该方法操作简便、灵敏度高、特异型强,能够用肉眼直观的观察实验结果,适用于基层和现场快速诊断,为副猪嗜血杆菌的临床诊断提供了重要的理论依据。
[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，

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