【目的】建立检测新型阿卡斑病毒(AKAV)的SYBR Green Ⅰ荧光定量RT-PCR,并结合序列测定开展广西边境地区蚊携带新型AKAV调查,了解其流行趋势及遗传进化特征,为建立重要蚊媒病毒病预警机制提供技术支持。【方法】分别针对新型AKAV的S基因...【目的】建立检测新型阿卡斑病毒(AKAV)的SYBR Green Ⅰ荧光定量RT-PCR,并结合序列测定开展广西边境地区蚊携带新型AKAV调查,了解其流行趋势及遗传进化特征,为建立重要蚊媒病毒病预警机制提供技术支持。【方法】分别针对新型AKAV的S基因和M基因主要变异位点设计引物,建立SYBR Green Ⅰ荧光定量RT-PCR并通过Ct值、标准曲线斜率、相关系数、扩增效率及扩增准确性等指标确定最佳反应体系及扩增程序;以优化的SYBR Green Ⅰ荧光定量RT-PCR检测2019年6-10月在广西边境地区采集的139份蚊样品,获得的新型AKAV经测序后采用ClustalW构建遗传进化树。【结果】SYBR Green Ⅰ荧光定量RT-PCR最佳反应体系:SYBR Premix Ex Taq Ⅱ 10.0μL,上、下游引物(4μmol/L)各1.0μL,标准品或反转录产物2.0μL,双蒸水补齐至20.0μL。扩增程序:95℃预变性30 s;95℃30 s,64℃(S基因)/66℃(M基因)30 s,72℃30 s,进行40个循环。SYBR Green Ⅰ荧光定量RT-PCR检测新型AKAV S基因和M基因的极限为1.0×101copies/μL和1.0×102copies/μL,对应的扩增效率分别为98.61%和105.81%,但不能扩增蓝舌病病毒、鹿流行性出血热病毒、竹鼠圆环病毒和布鲁氏杆菌;检测S基因和M基因的批内重复试验、批间重复试验变异系数(CV)均低于5.00%。采用建立的SYBR Green I荧光定量RT-PCR成功从广西边境地区防城港市、东兴市、宁明县、大新县及北海市的阿蚊和库蚊中检出新型AKAV,且均属于基因Ia型,尤其与广东和湖南蠓虫、中华按蚊、致倦库蚊分离毒株具有较高的相似性。【结论】针对新型AKAV S基因和M基因建立的SYBR Green Ⅰ荧光定量RT-PCR具有特异性强、灵敏度高、重复性好的特点,更适用于检测病毒拷贝数低的样品。此外,从广西边境地区蚊样品中检出的AKAV毒株均为新型AKAV,属于基因Ia型,与广东和湖南蠓虫、中华按蚊、致倦库蚊分离毒株具有较高的相似性,推测新型AKAV已在广西周边省份流行。展开更多
Based on the CCD images,IIM data and DEM data of China＇s lunar exploration project （Chang＇E-Ⅰ） and related processed and analytic results,an integrated study of regional geology of Sinus Iridum and its adjacent a...Based on the CCD images,IIM data and DEM data of China＇s lunar exploration project （Chang＇E-Ⅰ） and related processed and analytic results,an integrated study of regional geology of Sinus Iridum and its adjacent area was conducted,and a series of relevant researches and analyses were carried out,including analysis of impact craters and their extrusive and accumulative materials,division of stratigraphic and tectonic units and classification of rock types,integrated analysis of chronology and lunar evolution history.In consideration of crater＇s shape features,quantity and preserving status of filling materials,the lunar impact craters can be divided into 7 types and 11 subtypes,and the accumulative materials of craters are divided into 6 types and 9 accumulative groups.According to the content and distribution of TiO2 and image characteristics,the basalts are divided into high-TiO2,medium-TiO2 and low-TiO2 basalts.Discussion was made on division of tectonic units and evolution features in the study area.The geological map of the Sinus Iridum Quadrangle （LQ-4） at a scale of 1∶2.5 M was preliminarily compiled with the ArcGIS system,and the spatial database of the map was established.Related technical specification,procedure and method for lunar geological mapping have been worked out,so as to lay a foundation for the forthcoming geological mapping of the global Moon in China by using the data of Chang＇E-Ⅱ and also for comprehensive study and geological mapping of other celestial bodies in the future.展开更多
文摘【目的】建立检测新型阿卡斑病毒(AKAV)的SYBR Green Ⅰ荧光定量RT-PCR,并结合序列测定开展广西边境地区蚊携带新型AKAV调查,了解其流行趋势及遗传进化特征,为建立重要蚊媒病毒病预警机制提供技术支持。【方法】分别针对新型AKAV的S基因和M基因主要变异位点设计引物,建立SYBR Green Ⅰ荧光定量RT-PCR并通过Ct值、标准曲线斜率、相关系数、扩增效率及扩增准确性等指标确定最佳反应体系及扩增程序;以优化的SYBR Green Ⅰ荧光定量RT-PCR检测2019年6-10月在广西边境地区采集的139份蚊样品,获得的新型AKAV经测序后采用ClustalW构建遗传进化树。【结果】SYBR Green Ⅰ荧光定量RT-PCR最佳反应体系:SYBR Premix Ex Taq Ⅱ 10.0μL,上、下游引物(4μmol/L)各1.0μL,标准品或反转录产物2.0μL,双蒸水补齐至20.0μL。扩增程序:95℃预变性30 s;95℃30 s,64℃(S基因)/66℃(M基因)30 s,72℃30 s,进行40个循环。SYBR Green Ⅰ荧光定量RT-PCR检测新型AKAV S基因和M基因的极限为1.0×101copies/μL和1.0×102copies/μL,对应的扩增效率分别为98.61%和105.81%,但不能扩增蓝舌病病毒、鹿流行性出血热病毒、竹鼠圆环病毒和布鲁氏杆菌;检测S基因和M基因的批内重复试验、批间重复试验变异系数(CV)均低于5.00%。采用建立的SYBR Green I荧光定量RT-PCR成功从广西边境地区防城港市、东兴市、宁明县、大新县及北海市的阿蚊和库蚊中检出新型AKAV,且均属于基因Ia型,尤其与广东和湖南蠓虫、中华按蚊、致倦库蚊分离毒株具有较高的相似性。【结论】针对新型AKAV S基因和M基因建立的SYBR Green Ⅰ荧光定量RT-PCR具有特异性强、灵敏度高、重复性好的特点,更适用于检测病毒拷贝数低的样品。此外,从广西边境地区蚊样品中检出的AKAV毒株均为新型AKAV,属于基因Ia型,与广东和湖南蠓虫、中华按蚊、致倦库蚊分离毒株具有较高的相似性,推测新型AKAV已在广西周边省份流行。
基金financially supported by the National "863" Project (Grant No.:2010AA12220105)the Standard Research of Lunar mapping of Geology and Tectonics (Grant No.:Y2ZZ031000-02) from Institute of Geochemistry, Chinese Academy of Sciences
文摘Based on the CCD images,IIM data and DEM data of China＇s lunar exploration project （Chang＇E-Ⅰ） and related processed and analytic results,an integrated study of regional geology of Sinus Iridum and its adjacent area was conducted,and a series of relevant researches and analyses were carried out,including analysis of impact craters and their extrusive and accumulative materials,division of stratigraphic and tectonic units and classification of rock types,integrated analysis of chronology and lunar evolution history.In consideration of crater＇s shape features,quantity and preserving status of filling materials,the lunar impact craters can be divided into 7 types and 11 subtypes,and the accumulative materials of craters are divided into 6 types and 9 accumulative groups.According to the content and distribution of TiO2 and image characteristics,the basalts are divided into high-TiO2,medium-TiO2 and low-TiO2 basalts.Discussion was made on division of tectonic units and evolution features in the study area.The geological map of the Sinus Iridum Quadrangle （LQ-4） at a scale of 1∶2.5 M was preliminarily compiled with the ArcGIS system,and the spatial database of the map was established.Related technical specification,procedure and method for lunar geological mapping have been worked out,so as to lay a foundation for the forthcoming geological mapping of the global Moon in China by using the data of Chang＇E-Ⅱ and also for comprehensive study and geological mapping of other celestial bodies in the future.
