RT-LAMP技术快速检测禽流感病毒的研究

发布时间：2018-04-19 13:08

本文选题：逆转录环介导等温扩增 + 快速检测　；参考：《安徽大学》2014年硕士论文

【摘要】：禽流感(Avian influenza, AI)是由正粘病毒科流感病毒属A型禽流感病毒(Avian influenza virus, AIV)所引起的一种禽类呼吸道疾病综合征,1878年首次报道于意大利,然后在世界范围内广泛存在,对很多国家和地区的经济增长以及公共卫生安全造成了严重的影响。近年来,由H5N1亚型高致病性禽流感病毒引发的禽流感疫情影响严重,对全球经济增长和人类公共安全已构成了重大威胁,比如1997年香港首次报道的感染人的H5N1亚型禽流感病毒,自2003年以来又在全球15个国家内造成了648人感染、384人死亡的大流行。另外,2005年在中国青海湖爆发的H5N1亚型高致病性禽流感则造成了6000多只迁徙的候鸟死亡。而H7亚型禽流感病毒引起的禽流感大爆发也对许多国家和地区造成了重大危害,导致了七千五百多万的家禽死亡,而且也感染了很多人甚至导致其死亡。此外,2013年中国爆发的人感染H7N9亚型高致病性禽流感病毒的疫情,是全球首次报道,随之在中国多省份相继暴发,到目前为止已经造成超过100人死亡的悲剧。目前禽流感病毒的检测方法主要包括病毒学、血清学和分子生物学等方法,但是其中很多方法操作繁琐,且费时费力,又需要昂贵的仪器设备,因此不适合在基层快速的检测禽流感病毒,也很不利于对禽流感疫情的有效监测和控制。2000年日本Notimi T博士在普通PCR方法的基础上发明了一种新型核酸扩增技术,即环介导等温扩增技术(loop-mediated isothermal amplification, LAMP),它根据靶序列的六个特定区域设计特异的四个引物,在具有链置换活性的Bst DNA聚合酶的作用下,65℃恒温条件反应一小时就可完成目的基因的扩增,并且扩增结果仅用肉眼就能直接判断实验结果,因此LAMP技术具有简便、快速、高特异性和高灵敏度等优点,特别适合在设备不完善的基层实验室使用。本研究在前期LAMP研究的基础上,运用了一种新的扩增产物结果判断方法,即通过实时定量PCR仪观察反应荧光强度变化,并根据此结果判定及优化最佳反应体系。本实验所使用的荧光染料是钙黄绿素,肉眼观察时阳性反应管内反应液颜色会由橙黄色变为浅绿色。本研究以H5亚型禽流感病毒和H7亚型禽流感病毒的各自HA基因序列以及禽流感病毒的保守M基因序列作为靶基因序列,对它们进行序列比对并找出各自的高度保守区域,然后利用PrimerExplorerV4在线引物设计软件分别设计了针对每个靶基因的多组LAMP特异性引物,每组引物都包括一对外引物F3和B3、一对内引物FIP和BIP以及一对环引物LF和LB。对每个靶基因的两组引物进行重复试验,选取最佳的一组引物。然后对反应温度、Mg++浓度、dNTP浓度和甜菜碱浓度等扩增条件进行优化,根据荧光强度变化强弱和1.5%琼脂糖凝胶电泳条带的宽度和亮度进行结果筛选,成功建立了每组引物的最优LAMP反应体系,即M-RT-LAMP的的最优反应体系是：6mM/L Mg2+,1.6mM/L dNTP,0.6M/L Betaine,1.6μM/L FIP,BIP,0.2μM/L F3,B3,0.8μM/L LF,LB,最适温度63℃；H5-RT-LAMP的最优反应体系为：6mM/L Mg2+,1.4mM/L dNTP,0.8M/L Betaine,1.6μM/L FIP,BIP,0.4μM/L F3,B3,0.8μM/L LF, LB,最适温度65℃；H7-RT-LAMP的最优反应体系是：6mM/L Mg2+,1.4mM/L dNTP,0.8M/L Betaine,1.6μM/L FIP,BIP,0.2μM/L F3,B3,0.8μM/L LF, LB,最适温度65℃。在反应前将荧光染料混合液(Calcein/MnCl2)加入到反应体系中,然后将每组反应体系在其最适温度反应60min,然后80℃、5min以终止反应。反应结束后,通过荧光强度变化、肉眼直接观察颜色变化或经1.5%琼脂糖凝胶电泳进行扩增结果的判断。利用优化后的反应体系,对H1, H3, H4, H5, H6, H7, H9亚型禽流感病毒和新城疫病毒进行RT-LAMP特异性检测,试验结果只能检测到目的基因的存在,说明所建立的AIV-RT-LAMP方法具有良好的特异性。将各亚型禽流感病毒RNA进行十倍倍比稀释,然后用同一RNA进行RT-LAMP和RT-PCR实验以比较RT-LAMP方法的灵敏性,结果表明本研究建立的M-RT-LAMP体系的灵敏度为0.01pg,是RT-PCR方法的1000倍；H5-RT-LAMP体系其最低检测限为0.1pg,是RT-PCR方法的100倍；H7-RT-LAMP体系其最低检测限是O.1pg,比RT-PCR方法灵敏100倍。用80份临床样品对所建立的AIV-RT-LAMP检测方法进行技术评价,并与RT-PCR方法进行检测阳性率的比较,结果说明本研究所建立的AIV-RT-LAMP技术是有效的且可行的。综上所述,本研究建立的AIV-RT-LAMP快速检测禽流感病毒的方法是可行的,并且该方法具有快速、简便、灵敏度高和特异性好等优势,特别适合在基层及现场使用,为禽流感疫情的预警和早期诊断提供了便利。
[Abstract]:Avian influenza (AI) is a kind of avian respiratory disease syndrome caused by the A avian influenza virus (Avian influenza virus, AIV), which is first reported in Italy in 1878, and then widely existed worldwide. It is an economic growth and public health security for a large number of countries and regions. In recent years, the epidemic of avian influenza caused by the H5N1 subtype highly pathogenic avian influenza virus has been seriously affected, and has posed a major threat to global economic growth and human public safety. For example, the first reported avian influenza virus (H5N1), which was first reported in Hongkong in 1997, has been caused in 15 countries in the world since 2003. 648 people were infected with 384 deaths. In addition, the H5N1 subtype of highly pathogenic avian influenza, which broke out in Qinghai Lake in China in 2005, resulted in the death of more than 6000 migratory migratory birds, and the outbreak of avian influenza caused by the H7 subtype avian influenza virus was also a major hazard to many countries and regions, resulting in the death of about seventy-five million poultry. In addition, the outbreak of the H7N9 subtype highly pathogenic avian influenza virus in China in 2013 was the first report in the world, and the outbreak of the outbreak in many provinces in China has caused more than 100 deaths so far.
At present, the detection methods of avian influenza virus mainly include virology, serology and molecular biology, but many of them are complicated, time-consuming and costly, and need expensive equipment. Therefore, it is not suitable for rapid detection of avian influenza virus at the grass-roots level, and it is also not conducive to the effective monitoring and control of the epidemic of avian influenza in.2000 years. On the basis of the common PCR method, Dr. Notimi T invented a new type of nucleic acid amplification technology, namely, loop-mediated isothermal amplification (LAMP), which designed four specific primers based on six specific regions of the target sequence, and at 65 C under the action of Bst DNA polymerase with chain replacement activity. The amplification of the target gene can be completed in one hour by the temperature condition, and the result of the amplification can be directly judged by the naked eye. Therefore, the LAMP technology has the advantages of simple, rapid, high specificity and high sensitivity, and is especially suitable for the basic laboratory of imperfect equipment.
On the basis of the earlier LAMP study, a new method of determining the result of the amplification product was used to determine the change of the fluorescence intensity by real-time quantitative PCR instrument, and to determine and optimize the optimal reaction system according to the results. Color will change from orange yellow to light green. In this study, the sequence of H5 subtype avian influenza virus and H7 subtype avian influenza virus and the sequence of the conservative M gene of avian influenza virus were used as the target gene sequence, and the sequence alignment of the avian influenza virus was compared and the highly conserved regions were found. Then the PrimerExplorerV4 online primer was used to design the software. LAMP specific primers for each target gene were designed for each target gene. Each primer consisted of one external primer F3 and B3, a pair of primers FIP and BIP, and a pair of ring primers LF and LB. to repeat two primers for each target gene and select the best set of primers. Then the reaction temperature, Mg++ concentration, dNTP concentration and betaine were selected. Optimization of the concentration and other amplification conditions, according to the intensity of fluorescence intensity change and the width and brightness of the 1.5% agarose gel electrophoresis strip, the optimal LAMP reaction system for each primer was established successfully, that is, the optimal reaction system of M-RT-LAMP was 6mM/L Mg2+, 1.6mM/ L dNTP, 0.6M/L Betaine, 1.6 Mu M/L FIP, BIP, 0.2 micron. 3,0.8 M/L LF, LB, the optimum temperature at 63 degrees C. The optimal reaction system for H5-RT-LAMP is 6mM/L Mg2+, 1.4mM/L dNTP, 0.8M/L Betaine, 1.6 micron M/L. F, LB, the optimum temperature at 65 C. The fluorescent dye mixture (Calcein/MnCl2) was added to the reaction system before the reaction. Then each reaction system was reacted at 60min at its optimum temperature, and then at 80, 5min to terminate the reaction. After the reaction ended, the color changes were observed directly by the naked eye, or by 1.5% agarose gel electrophoresis. H1, H3, H4, H5, H6, H7, H9 subtype of avian influenza virus and Newcastle disease virus were detected by the optimized reaction system. The result of the test can only detect the existence of the target gene, which shows that the established AIV-RT-LAMP method has a good specificity. Ten avian influenza virus RNA is carried out. Double times dilution, then using the same RNA for RT-LAMP and RT-PCR experiments to compare the sensitivity of the RT-LAMP method. The results show that the sensitivity of the M-RT-LAMP system is 0.01pg, 1000 times the RT-PCR method, and the minimum detection limit for H5-RT-LAMP system is 0.1pg, 100 times the RT-PCR square method, and the lowest detection limit for H7-RT-LAMP system is that O.1pg is 100 times more sensitive than the RT-PCR method. A technical evaluation of the established AIV-RT-LAMP detection method with 80 clinical samples and the comparison with the positive rate of the RT-PCR method are compared. The results show that the AIV-RT-LAMP technology established in this study is effective and feasible.
To sum up, the method of rapid detection of avian influenza virus by AIV-RT-LAMP in this study is feasible, and the method has the advantages of fast, simple, high sensitivity and good specificity. It is especially suitable for use in the grass-roots and site, and provides convenience for early warning and early diagnosis of avian influenza epidemic.

【学位授予单位】：安徽大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R511.7

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