羊痘病毒环介导等温扩增检测方法的建立
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摘要
羊痘病毒(Capripoxvirus, CaPV)能引起山羊、绵羊和牛的皮肤、器官表面广泛性结节和水肿,病畜产乳量急剧减少,皮毛品质极大下降,造成巨大经济损失。不同毒株的毒力有较大差异,易感动物的致死率可达10%-58%或75%-100%不等,同时,因感染羊痘病毒造成山羊、绵羊和牛及制品的国际贸易受到限制,致使此病成为重要的经济疾病。羊痘病毒属(Capripoxvirus)由山羊痘病毒(Goatpox virus, GTPV)、绵羊痘病毒(Sheeppox virus, SPPV)和牛疙瘩皮肤病病毒(Lumpy skin disease virus, LSDV)组成,它们具有很强的宿主特异性,自然条件下,不会发生交叉感染。SPPV及GTPV呈世界性分布,现在广泛分布于非洲、中东、北欧、地中海各国等,我国近年来在如广西、贵州、黑龙江等地也有发生。1929年赞比亚首次发现牛疙瘩皮肤病(Lumpy skin disease, LSD),随后传入南非、坦桑尼亚、以色列及埃及,目前主要分布在非洲中南部地区,并有向欧洲及亚洲扩散的趋势。
传统的病毒学试验和血清学试验虽成本较低、操作简单,但存在耗费时间长、特异性和灵敏度不高等缺陷。随着分子检测技术的发展,PCR及实时荧光PCR也有较广泛的应用,但由于需要实时荧光定量PCR仪等昂贵的仪器设备、操作程序复杂等缺点,不利于现场快速诊断检测。目前,我国尚无LSDV流行的报导,同时也无检测该病的相关研究,因此,建立一种快速灵敏的检测方法,对防止牛疙瘩皮肤病传入和加强我国羊痘病毒的监测具有重要的现实意义。
环介导等温扩增方法(Loop-mediated isothermal amplification, LAMP)是Notomi等(2000)发明的一种新型的等温核酸扩增技术。该技术利用设计的两对特殊的内、外引物,特异性识别靶序列上的六个独立区域,利用Bst DNA聚合酶启动循环链置换反应,可以在1h之内,将靶DNA片段扩增109-1010倍。该方法具有简便快速、无需昂贵的扩增设备、高特异性及灵敏度、结果鉴定直观简便等特点,非常适合于基层及现场检疫工作。现已广泛用于如禽流感病毒,沙门氏茵及恶性疟原虫等病毒、细菌及寄生虫性病原体的检测。
本文通过分析GenBank中羊痘病毒的保守基因序列,选取末端重复序列(Inverted terminal repeat, ITR)为靶基因,运用在线设计软件Primer Explorer 4.0 (http://primerexplorer.jp/elamp4.0.0/index.html),设计LAMP引物。利用LAMP Real Time Turbidimeter LA-320仪对反应进程中的浊度进行实时监控,对不同引物组扩增的起始时间、最大扩增速率、达到最大扩增速率所需时间及进入平台期所需时间等参数进行分析,筛选出对羊痘病毒核酸高效、特异扩增的LAMP引物。针对影响LAMP反应效率的重要参数进行优化,确定CaPV-LAMP检测方法的最优反应体系,并经试验验证引物的特异性和最低检测限。通过采用同一批次和不同批次提取的DNA验证CaPV-LAMP的可重复性,并对不同感染组织进行检测,对开发LAMP法羊痘病毒检测试剂盒进行了初步性探索研究。主要研究成果如下：
1、根据上述优化试验,最佳的反应体系为：60 pmol FIP 1μL,60 pmol BIP 1μL,5 pmol F3 1μL,5 pmol B31μL,8 u Bst DNA Polymerase 1μL,2xReaction Mix 12,模板DNA2μL, Calcein 1μL, ddH2O4.5μL,总体积为25μL。优化反应条件为：62℃恒温反应60 mMin；80℃,5 min终止反应。
2、灵敏度试验表明,本文建立的LAMP法的灵敏度远远高于OIE推荐的普通PCR法,LAMP最低可检测到10-151TCID50,而PCR法最低检测限量为101.49TCID50。特异性试验未发现非特异性扩增,说明其具有较好的特异性。
3、在反应前添加经螯合处理的Calcein,肉眼可直接观察结果。避免了琼脂糖凝胶电泳检测LAMP产物的复杂操作,同时极大减少了浑浊度判断时的误差。
4、同一批次DNA和不同批次DNA分别进行LAMP检测,其变异系数(CV%)分别为1.65%、8.66%,符合率为98.35%和91.33%,表明该方法具有较好的稳定。
本文建立的CaPV-LAMP检测方法可在1.5 h内完成病毒核酸的提取和LAMP检测全过程,该方法只需水浴锅即可进行检测,具有快速高效、操作简便、成本低廉、设备要求低等特点。不需要昂贵的扩增仪,一台恒温水浴锅60 mMin内即可完成检测,结果判定简单直观,能满足临床样本大批量快速检测的需要。本研究建立的CaPV-LAMP检测方法,为CaPV的早期快速诊断提供了新途径,为CaPV的现场检疫和流行病学监测提供快速灵敏的技术手段。
Capripoxvirus can cause the extensive nodules and edema disease in the skin and organ surface of goats, sheep and cattle, which drastically reduce milkproduction and fur quality, and result in huge economic losses. According to the differences in the virulence between different strains, the fatality rate of susceptible animals is up to 10% to 58% or 75% to 100%.Besides, the goat, sheep, cattle and the product will be restricted in international trade, which infection from Capripoxvirus. It is composed of Goatpox virus (GTPV), sheeppox virus (SPPV), bovine lump skin disease virus (LSDV), which have strictly host specificity and will not occur cross-infection under natural conditions. SPPV and GTPV is worldwide distributed, and widely distributed in Africa, the Middle East, Northern Europe, Mediterranean countries now. In recent years, It happended at many areas in China, e.g. Guangxi, Guizhou, Heilongjiang. Lumpy skin disease was first found in Zambia and then spreay to South Africa, Tanzania, Israel and Egypt. Now LSD is Main distributed in central and southern Africa, and has the trend to spread to Europe and Asia.
Although the traditional virological tests and serological tests are lower-cost and simple, there are defects such as spending a long time, lower specificity and sensitivity. With the development of molecular techniques, PCR and real-time PCR are widely Application, however, because of the need of expensive PCR instruments and complex operating procedures, these methods are not conducive to the locale and fast diagnosis. Until now there are no report about the prevalence of the virus of LSDV and the detection research of this disease in China. Therefore, it is practically important to establish a rapid and sensitive method to prevent the introduction of LSDV and strengthen the monitoring of CaPV in China.
Loop-mediated isothermal amplification method (LAMP) is a new kind of isothermal nucleic acid amplification technology invented by Notomi et al (2000).This technology uses two pairs of special internal and external primers specifically to identification the six separate areas of target sequences, and Bst DNA polymerase to start the cycle strand displacement reaction. This method can amplify the target DNA fragmentsl09～1010 times within one hour. The method is simple, rapid amplification with high specificity, sensitivity and intuitive identification results, without expensive equipment, and very suitable for the grass-roots level and on-site quarantine. It is now widely used in the detection of viruses, bacteria and parasites such as avian influenza virus, Salmonella and Plasmodium falciparum.
In this study, we choose Inverted terminal repeat as Target genes, use Primer Explorer 4.0 (http://primerexplorer.jp/elamp4.0.0/index.html) to design primers by analyzing the conserved genes of sheep pox virus in the Gen Bank sequence. The study use LAMP Real Time Turbidimeter LA-320c instrument for real-time monitoring of turbidity in the reaction process, to analysis the parameters such as sets amplified the starting time, the maximum amplification rate, the time to arrive maximum amplification rate and the time required to enter the platform from different primers, and than screen out LAMP primers which have high effective and specific amplification. The study optimizes significant parameter to the efficiency of the reaction for LAMP, deign optimal reaction system, verify the specificity and lower limit of detection of primers. The study verify the repeatability of CaPV-LAMP by select DNA from same batch and different batch, and detection different infection group, make Preliminary Exploration to develop Detection kit of LAMP poxvirus.
The main study results are as follows:
1. According above optimize test, the optimal reaction system:60 pmol FIP 1μL,60 pmol BIP lμL,5 pmol F31μL,5 pmol B3 1μL,8 u Bst DNA Polymerase 1μL,2xReaction Mix 12.5μL, Simple DNA 2μL, ddH2O 5.5μL, total volume25μL. Optimize reaction condition:Constant temperature reaction in 62℃60min,80℃,5 min Reaction was terminated.
2. Sensitivity test show that Sensitivity of LAMP in this study is far more high than the PCR which OIE recommended, the lower detection limited was 10-1.51TCID50 in LAMP. However lower detection limited in PCR was 101.49TCID50.They isn't Non-specific amplification in specific reaction, it is show that the method has Good specificity.
3. The result can be observe by naked eye when added Calcein before reaction. It avoided the complex operate that use agarose gel electrophoresis detection LAMP LAMP product, and greatly reducing the error for turbidity judgment.
4. We detection the DNA from the same batch and different batch, coefficient of variatio (CV%) was 1.65% and 8.66%, the agreement was 98.35% and 91.33%. It is show that the method has Good stability.
The CaPV-LAMP detection method established in this study could complete the collection of sample,, the viral nucleic acid extraction and LAMP testing the entire process within 1.5 h。The method could detection in water bath, with advantages as fast, efficient, easy to operate, low cost, low equipment requirements. The detection do not require expensive thermal cycler, can complete the detection in a constant temperature water bath within 60 min, and it was simple and intuitive to determine results, can be able to meet the needs of large quantities of clinical samples for rapid detection. The CaPV-LAMP detection method established in this study provided a new way for the rapid diagnosis of CaPV in early stage and sensitive techniques for on-site quarantine and epidemiological surveillance of CaPV.
传统的病毒学试验和血清学试验虽成本较低、操作简单,但存在耗费时间长、特异性和灵敏度不高等缺陷。随着分子检测技术的发展,PCR及实时荧光PCR也有较广泛的应用,但由于需要实时荧光定量PCR仪等昂贵的仪器设备、操作程序复杂等缺点,不利于现场快速诊断检测。目前,我国尚无LSDV流行的报导,同时也无检测该病的相关研究,因此,建立一种快速灵敏的检测方法,对防止牛疙瘩皮肤病传入和加强我国羊痘病毒的监测具有重要的现实意义。
环介导等温扩增方法(Loop-mediated isothermal amplification, LAMP)是Notomi等(2000)发明的一种新型的等温核酸扩增技术。该技术利用设计的两对特殊的内、外引物,特异性识别靶序列上的六个独立区域,利用Bst DNA聚合酶启动循环链置换反应,可以在1h之内,将靶DNA片段扩增109-1010倍。该方法具有简便快速、无需昂贵的扩增设备、高特异性及灵敏度、结果鉴定直观简便等特点,非常适合于基层及现场检疫工作。现已广泛用于如禽流感病毒,沙门氏茵及恶性疟原虫等病毒、细菌及寄生虫性病原体的检测。
本文通过分析GenBank中羊痘病毒的保守基因序列,选取末端重复序列(Inverted terminal repeat, ITR)为靶基因,运用在线设计软件Primer Explorer 4.0 (http://primerexplorer.jp/elamp4.0.0/index.html),设计LAMP引物。利用LAMP Real Time Turbidimeter LA-320仪对反应进程中的浊度进行实时监控,对不同引物组扩增的起始时间、最大扩增速率、达到最大扩增速率所需时间及进入平台期所需时间等参数进行分析,筛选出对羊痘病毒核酸高效、特异扩增的LAMP引物。针对影响LAMP反应效率的重要参数进行优化,确定CaPV-LAMP检测方法的最优反应体系,并经试验验证引物的特异性和最低检测限。通过采用同一批次和不同批次提取的DNA验证CaPV-LAMP的可重复性,并对不同感染组织进行检测,对开发LAMP法羊痘病毒检测试剂盒进行了初步性探索研究。主要研究成果如下：
1、根据上述优化试验,最佳的反应体系为：60 pmol FIP 1μL,60 pmol BIP 1μL,5 pmol F3 1μL,5 pmol B31μL,8 u Bst DNA Polymerase 1μL,2xReaction Mix 12,模板DNA2μL, Calcein 1μL, ddH2O4.5μL,总体积为25μL。优化反应条件为：62℃恒温反应60 mMin；80℃,5 min终止反应。
2、灵敏度试验表明,本文建立的LAMP法的灵敏度远远高于OIE推荐的普通PCR法,LAMP最低可检测到10-151TCID50,而PCR法最低检测限量为101.49TCID50。特异性试验未发现非特异性扩增,说明其具有较好的特异性。
3、在反应前添加经螯合处理的Calcein,肉眼可直接观察结果。避免了琼脂糖凝胶电泳检测LAMP产物的复杂操作,同时极大减少了浑浊度判断时的误差。
4、同一批次DNA和不同批次DNA分别进行LAMP检测,其变异系数(CV%)分别为1.65%、8.66%,符合率为98.35%和91.33%,表明该方法具有较好的稳定。
本文建立的CaPV-LAMP检测方法可在1.5 h内完成病毒核酸的提取和LAMP检测全过程,该方法只需水浴锅即可进行检测,具有快速高效、操作简便、成本低廉、设备要求低等特点。不需要昂贵的扩增仪,一台恒温水浴锅60 mMin内即可完成检测,结果判定简单直观,能满足临床样本大批量快速检测的需要。本研究建立的CaPV-LAMP检测方法,为CaPV的早期快速诊断提供了新途径,为CaPV的现场检疫和流行病学监测提供快速灵敏的技术手段。
Capripoxvirus can cause the extensive nodules and edema disease in the skin and organ surface of goats, sheep and cattle, which drastically reduce milkproduction and fur quality, and result in huge economic losses. According to the differences in the virulence between different strains, the fatality rate of susceptible animals is up to 10% to 58% or 75% to 100%.Besides, the goat, sheep, cattle and the product will be restricted in international trade, which infection from Capripoxvirus. It is composed of Goatpox virus (GTPV), sheeppox virus (SPPV), bovine lump skin disease virus (LSDV), which have strictly host specificity and will not occur cross-infection under natural conditions. SPPV and GTPV is worldwide distributed, and widely distributed in Africa, the Middle East, Northern Europe, Mediterranean countries now. In recent years, It happended at many areas in China, e.g. Guangxi, Guizhou, Heilongjiang. Lumpy skin disease was first found in Zambia and then spreay to South Africa, Tanzania, Israel and Egypt. Now LSD is Main distributed in central and southern Africa, and has the trend to spread to Europe and Asia.
Although the traditional virological tests and serological tests are lower-cost and simple, there are defects such as spending a long time, lower specificity and sensitivity. With the development of molecular techniques, PCR and real-time PCR are widely Application, however, because of the need of expensive PCR instruments and complex operating procedures, these methods are not conducive to the locale and fast diagnosis. Until now there are no report about the prevalence of the virus of LSDV and the detection research of this disease in China. Therefore, it is practically important to establish a rapid and sensitive method to prevent the introduction of LSDV and strengthen the monitoring of CaPV in China.
Loop-mediated isothermal amplification method (LAMP) is a new kind of isothermal nucleic acid amplification technology invented by Notomi et al (2000).This technology uses two pairs of special internal and external primers specifically to identification the six separate areas of target sequences, and Bst DNA polymerase to start the cycle strand displacement reaction. This method can amplify the target DNA fragmentsl09～1010 times within one hour. The method is simple, rapid amplification with high specificity, sensitivity and intuitive identification results, without expensive equipment, and very suitable for the grass-roots level and on-site quarantine. It is now widely used in the detection of viruses, bacteria and parasites such as avian influenza virus, Salmonella and Plasmodium falciparum.
In this study, we choose Inverted terminal repeat as Target genes, use Primer Explorer 4.0 (http://primerexplorer.jp/elamp4.0.0/index.html) to design primers by analyzing the conserved genes of sheep pox virus in the Gen Bank sequence. The study use LAMP Real Time Turbidimeter LA-320c instrument for real-time monitoring of turbidity in the reaction process, to analysis the parameters such as sets amplified the starting time, the maximum amplification rate, the time to arrive maximum amplification rate and the time required to enter the platform from different primers, and than screen out LAMP primers which have high effective and specific amplification. The study optimizes significant parameter to the efficiency of the reaction for LAMP, deign optimal reaction system, verify the specificity and lower limit of detection of primers. The study verify the repeatability of CaPV-LAMP by select DNA from same batch and different batch, and detection different infection group, make Preliminary Exploration to develop Detection kit of LAMP poxvirus.
The main study results are as follows:
1. According above optimize test, the optimal reaction system:60 pmol FIP 1μL,60 pmol BIP lμL,5 pmol F31μL,5 pmol B3 1μL,8 u Bst DNA Polymerase 1μL,2xReaction Mix 12.5μL, Simple DNA 2μL, ddH2O 5.5μL, total volume25μL. Optimize reaction condition:Constant temperature reaction in 62℃60min,80℃,5 min Reaction was terminated.
2. Sensitivity test show that Sensitivity of LAMP in this study is far more high than the PCR which OIE recommended, the lower detection limited was 10-1.51TCID50 in LAMP. However lower detection limited in PCR was 101.49TCID50.They isn't Non-specific amplification in specific reaction, it is show that the method has Good specificity.
3. The result can be observe by naked eye when added Calcein before reaction. It avoided the complex operate that use agarose gel electrophoresis detection LAMP LAMP product, and greatly reducing the error for turbidity judgment.
4. We detection the DNA from the same batch and different batch, coefficient of variatio (CV%) was 1.65% and 8.66%, the agreement was 98.35% and 91.33%. It is show that the method has Good stability.
The CaPV-LAMP detection method established in this study could complete the collection of sample,, the viral nucleic acid extraction and LAMP testing the entire process within 1.5 h。The method could detection in water bath, with advantages as fast, efficient, easy to operate, low cost, low equipment requirements. The detection do not require expensive thermal cycler, can complete the detection in a constant temperature water bath within 60 min, and it was simple and intuitive to determine results, can be able to meet the needs of large quantities of clinical samples for rapid detection. The CaPV-LAMP detection method established in this study provided a new way for the rapid diagnosis of CaPV in early stage and sensitive techniques for on-site quarantine and epidemiological surveillance of CaPV.
引文
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[351邓朝阳.广西山羊痘防控研究[D].江苏,南京农业大学,2006：37-38.
[36]刘棋,黄夏,郭建刚,等.山羊痘病毒的分离鉴定及生物学特性的研究[J],中国预防兽医学报,2006,28(5)：494-498.
[371廖振续.山羊痘的暴发与防治[J],中国兽医杂志,1997,23(3)：31-32.
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[47]肖斌,朱永红,邹全明.简便敏感的环介导等温扩增基因诊断新技术[J].中华检验医学杂志,2005,28(7)：761-763.
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[63]Fumito M, T akehiko K, No buy asu Y, et al. Visualization and enumeration of bacteria carrying the specific gene sequence by in situ rolling circle amplification[J]. Applied And Environmental Microbiology,2005,71(12):7933-7940.
[64]Fumito Maruyama, Takehiko Kenzaka, Nobuyasu Yamaguchi, et al. Detection of Bacteria Carrying the stxA2 Gene by In Situ Loop-Mediated Isothermal Amplification[J]. APPLIED AND ENVIRONMENTAL MICROBIOLOGY,2003,69 (8):5023-5028.
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[48]Iwamoto T, Sonobe T, Hayashi K et al. Loop-mediated isothermal amplification for direct detection of Mycobacterium tuberculosis complex\Maviu and Mint racellulare in sput um samples[J]. Microbiol,2003,41(6):2616-2622.
[49]Han Feifei, Ge Beilei. Evaluation of a loop-mediated isothermal amplification assay for detecting Vibrio vulnificus in raw oysters [J]. Foodborne Pat hogens and Disease,2008,5(3): 311-320.
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[52]Horisaka T, Fujita K, Iwata T, et al. Sensitive and Specific Detection of Yersiniapseu by Loop-Mediated isothermal Amplification[J]. Journal of clinical Mobiology,2004,42(11): 5349-5352.
[53]Poon L L, Leung C S, Chan K H, et al. Detection of human influenza A viruses by loop mediated isothermal amplification [J]. J Clin Microbiol,2005,43(1):427-430.
[54]Pham H M, Nakajima C, Ohashi K, et al. Loop Mediated Isothermal Amplification for Rapid Detection of New castle Disease Virus[J]. Clin Microbiol,2005,43(4):1646-1650.
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[56]Enomoto Y, Yoshikawa T, Ihira M, et al. Rapid diagnosis of herpes simplex virus infection by a loop-mediated isothermal amplification method[J]. Journal of Clinical Microbiology, 2005,43(1):951-955.
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[63]Fumito M, T akehiko K, No buy asu Y, et al. Visualization and enumeration of bacteria carrying the specific gene sequence by in situ rolling circle amplification[J]. Applied And Environmental Microbiology,2005,71(12):7933-7940.
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[65]Hataoka Y, Zhang L, Mori Y, et al. Analysis of specific gene by integration of isothermal amplification and electrophoresis on poly microchips [J], Analytical Chemistry,2004,76 (13):3688-3693.
[66]周建胜,马慧玲,郭庆山.绵羊睾丸原代细胞培养及接种羊痘弱毒后的病变观察[J].中国兽医科技,2004,34(9)：71-74.
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