摘要
应用通用反转录试剂盒和环介导等温核酸扩增技术(LAMP)建立了猪繁殖与呼吸综合征病毒(PRRSV)的快速检测方法,同时评价了该检测方法的特异性、稳定性和灵敏度。结果表明,根据猪繁殖与呼吸综合征病毒M蛋白基因保守区设计的LAMP引物能够在64.5℃60min内实现对目标核酸片段的大量扩增,检测结果可通过直接观察反应副产物(焦磷酸镁)进行判读,该检测系统具有很高的特异性,与猪瘟病毒、猪乙型脑炎病毒等均无交叉反应;通过PRRSV不同毒株疫苗、模拟样品和临床样品确定,该检测系统具有很好的稳定性;通过质粒确定该检测系统可以检测到10拷贝/μL的病毒核酸模板。结果表明成功建立了猪繁殖与呼吸综合征病毒LAMP快速检测方法,为临床提供了一种很好的检测手段。
A rapid detection method for porcine reproductive and respiratory syndrome virus was established by using the universal reverse transcription kit and loop-mediated isothermal amplification technology,and the specificity,stability,sensitivity of the detection were evaluated.The results showed that the target nucleic acid fragment can be amplified in 64.5℃ within 60 min using the LAMP primers designed according to porcine reproductive and respiratory syndrome virus M protein gene conservative region,the test results can be directly observed by the naked eye through the reaction-products（magnesium pyrophosphate）,the detection system has high specificity,and viruses such as classical swine fever virus,porcine Japanese encephalitis virus showed no cross reaction;the good stability of the detection system was confirmed by different strains of vaccines,simulated samples and clinical samples;the plasmid-template which is just 10copies/μL in concentration was detected by the detection system.The test results proved that the LAMP method of rapid detection for porcine reproductive and respiratory syndrome virus was successfully established.
出处
《动物医学进展》
北大核心
2016年第3期1-5,共5页
Progress In Veterinary Medicine
基金
"十二五"国家科技支持计划项目(2013BAD12B04)
