猪传染性胃肠炎病毒RT-LAMP检测方法的建立及初步应用

发布时间：2018-04-16 17:40

  本文选题：猪传染性胃肠炎病毒 + 逆转录环介导等温扩增　； 参考：《东北农业大学》2015年硕士论文

【摘要】：猪传染性胃肠炎(TGE)是由猪传染性胃肠炎病毒(TGEV)引起的一种高度接触性传染性疾病,临床表现为呕吐、严重腹泻、脱水。TGEV对首次感染的猪群造成的危害尤其严重,2周龄以内仔猪死亡率可以达到90%-100%,严重威胁养猪业的健康发展。TGE临床症状与猪轮状病毒(PRV)感染和猪流行性腹泻(PED)等腹泻性疾病难以区分,而且经常出现混合感染,这为临床诊断带来了很大的困难。常规的实验室检测方法不仅耗时耗力还需要特殊的仪器,在基层中难以展开,所以建立一种TGEV的方便快速的检测方法十分必要。本试验建立了一种利用逆转录环介导等温扩增(RT-LAMP)技术来检测TGEV的快速检测方法,对该方法进行了温度与时间的优化,并对该检测方法的特异性与灵敏性进行了评价。针对TGEV N基因序列上的六个保守区域设计了四条RT-LAMP引物,建立针对TGEV N基因的RT-LAMP检测方法。该检测方法在63℃恒温下作用50 min,使TGEV N基因获得了最高效率的特异性扩增,未见与PEDV等其他猪源易感病毒出现交叉反应,具有很好的特异性;同时该检测方法具有极高的灵敏性,最低可检测到1.314fg/μL的目标病毒DNA,比普通的PCR灵敏度高三个数量级;反应结束后可以通过加入SYBR Green I荧光染料在紫外灯下观察反应管的颜色变化来判断实验结果,结果显示阳性扩增产物呈现绿色荧光,阴性扩增产物为橙色。从哈尔滨周边猪场收集47份临床样品RT-LAMP检测结果与已得到验证的RT-PCR和ELISA结果进行比对,结果显示符合率为100%。本研究建立的TGEV的RT-LAMP检测方法具有快速、特异、灵敏、简单易操作且设备要求低等特点,为基层实地检测TGEV提供了技术手段。
[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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