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检测鼠或鼠源细胞中坦布苏病毒荧光定量PCR方法的建立 被引量:2

Development of real-time PCR assay to detect Tembusu virus in mice or murine cells
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摘要 利用DNAStar对GenBank上发布的坦布苏病毒(TMUV)的E基因进行序列分析,根据分析结果选择其保守区域设计1对特异性引物。按照相同的方法,设计另1对鼠源内参基因β-actin的引物。通过PCR扩增获得E和β-actin的部分片段,分别与pMD18-T载体连接,构建重组质粒,经鉴定、纯化后作为阳性标准品,分别用于实时荧光定量PCR中E基因及内参基因β-actin标准曲线的构建。通过对反应条件进行优化,建立了以β-actin为内参基因的检测鼠或鼠源细胞中TMUV的SYBR GreenⅠ实时荧光定量PCR方法。该方法对TMUV的最低检出量为10个拷贝,比普通PCR高1 000倍;批内和批间重复性试验的变异系数均小于0.45%;特异性试验的结果表明,该方法只能检测到TMUV的扩增曲线。对50份样品进行检测,该方法的检出率为100.00%(普通PCR为87.50%),并对组织中病毒载量进行了相对定量,进一步说明了该方法的敏感性高,可用于试验样品的检测,为研究该病毒对哺乳动物的致病特性提供特异性的检测方法。 A pair of specific primes was designed by analyzing that Tembusu virus(TMUV)E gene sequences from GenBank were aligned with DNAStar software.According to the same method,a pair of specific primers for miceβ-actin genes was designed.The E gene andβ-actin gene amplified by PCR were separately cloned into pMD18-T vector and the recombinant plasmids were used as positive standards.The real-time PCR with miceβ-actin as a reference gene was developed by optimizing the reaction conditions.The detection limit of the real-time PCR assay was approximately10copies/μL.The intra-assay CVs and inter-assay CVs were separately equal or less than 0.45%.No cross-amplication was observed with other pathogens.The real-time PCR was used to detect 15 test samples and the tissue viral load was relatively quantified.The establishment of the assay provided a specific detection method for the study on TMUV pathogenicity to mammals.
出处 《中国兽医学报》 CAS CSCD 北大核心 2016年第6期917-922,共6页 Chinese Journal of Veterinary Science
基金 国家自然科学基金资助项目(31272583 31472199) 国家水禽产业技术体系资助项目(CARS-43-10)
关键词 坦布苏病毒 荧光定量PCR Tembusu virus mice real-time PCR
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