While functioning as a boundary line,urban waterfront space has a unique charm created by the collision of two different geographical features.WATER has always played a vital role in the birth and progress of human ci...While functioning as a boundary line,urban waterfront space has a unique charm created by the collision of two different geographical features.WATER has always played a vital role in the birth and progress of human civilization,whether it was in the Mesopotamia,the Nile Valley or the Yellow River basin.Water was not only helpful in solving the problems of irrigation,drinking.展开更多
为了能够同时检测猪圆环病毒2型(Porcine circovirus type 2,PCV-2)和猪圆环病毒3型(Porcine circovirus type 3,PCV-3),试验根据国内报道的PCV-2和PCV-3毒株全基因序列设计6对特异性引物(P3~P8),并构建标准质粒进行引物特异性试验,然...为了能够同时检测猪圆环病毒2型(Porcine circovirus type 2,PCV-2)和猪圆环病毒3型(Porcine circovirus type 3,PCV-3),试验根据国内报道的PCV-2和PCV-3毒株全基因序列设计6对特异性引物(P3~P8),并构建标准质粒进行引物特异性试验,然后通过优化扩增条件建立检测PCV-2和PCV-3混合感染的双重PCR方法。最后用PCV-2和PCV-3的感染性克隆同时转染PK-15细胞,盲传5代后用建立的双重PCR方法检测细胞培养液中的病毒核酸。结果表明:构建的PCV-2和PCV-3标准质粒浓度分别为290 ng/μL和380 ng/μL,双重PCR方法的最佳引物组合为P3/P6和P4/P8,最佳DNA聚合酶浓度为0.02 U/μL。使用引物组合P3/P6时,扩增得到的目的片段大小分别为308 bp和608 bp,最佳退火温度为58℃,最佳循环次数为28个;使用引物组合P4/P8时,扩增得到的目的片段大小分别为291 bp和645 bp,最佳退火温度为60℃,最佳循环次数为26个。运用建立好的双重PCR方法评估感染细胞模型,能有效鉴别PCV-2和PCV-3的单一感染和混合感染。说明优化得到的双重PCR检测方法可用于PCV-2和PCV-3的同时检测。展开更多
文摘While functioning as a boundary line,urban waterfront space has a unique charm created by the collision of two different geographical features.WATER has always played a vital role in the birth and progress of human civilization,whether it was in the Mesopotamia,the Nile Valley or the Yellow River basin.Water was not only helpful in solving the problems of irrigation,drinking.