摘要
为了建立一种快速、特异、灵敏且能同时检测猪细小病毒(PPV)和猪伪狂犬病病毒(PRV)二重Luminex x-TAG方法,参照GenBank中PPV的NS-1基因序列和PRV的ICP8基因序列,设计了2对特异性引物,其中上游引物5′端加一段TAG序列,下游引物5′端用生物素标记,进行二重PCR扩增。然后将PCR产物与链霉亲和素-藻红蛋白、磁珠混匀,42℃孵育25min,混合物在Luminex200仪器上进行读数分析。并用该方法进行特异性、灵敏度、重复性及临床样品的检测。结果显示,该方法与其他猪的病原无交叉反应,两者的检测下限都可达1×10^2 copies/μL,批内批间的变异系数都在4%以下;在30份临床样品中,PPV和PRV的检出率分别为13.3%和60.0%,比普通PCR的检出率高。结果表明,本研究建立的二重Luminex x-TAG方法具有特异性强、灵敏度高、重复性好,可同时检测PPV和PRV两种病毒,可用于猪场中PPV和PRV的流行病学调查。
To develop a rapid,specific and sensitive duplex Luminex x-TAG beads-based array for simultaneous detection of porcine parvovirus(PPV)and porcine pseudorabies virus(PRV).According to the NS-1 gene sequences of PPV and the ICP8 gene sequences of PRV published in GenBank.Two pairs of specific primers were designed for amplified PPV and PRV,respectively.The forward primer was designed with a unique TAG sequence at 5′end.The reverse primer was biotin-labeled at 5′end.After amplifying the PCR fragment,the product,streptavidin R-phycoerythrin and magnetic beads were incubated at 42℃ for 25 min.The result was analyzed at Luminex 200 instrument.The specificity,sensitivity,reproducibility and clinical samples of this assay were also evaluated.The assay was specific for PPV and PRV,and there was no cross reactions to other porcine viruses.The limit of detection reached 1×102 copies/μL of plasmid DNA.The coefficient of variation of the intra-and inter-assayis was lower than 4%.In 30 clinical samples,the detection rates of PPV and PRV were 13.4% and 60.0%,the dectection rate of the assay was higher than that of conventional PCR.These results indicated that the developed assay for detecting PPV and PRV was specific,sensitive,reproduceable and simultaneous detection.The assay could be utilized to monitoring the presence of PPV and PRV in pigs industry.
作者
朱余军
徐宁
肖丽
伍妙梨
饶丹
徐凤娇
练月晓
黄韧
郭鹏举
陈梅丽
ZHU Yu-jun;XU Ning;XIAO Lia;WU Miao-li;RAO Dan;XU Feng-jiao;LIAN Yue-xiao;HUANG Ren;GUO Peng-ju;CHEN Mei-li(Guangdong Key Laboratory of Laboratory Animals,Guangzhou 510663,China;Liaoning Center for Animal Disease Emergency,Shenyang 110161,China;Guangdong Laboratory Animals Monitoring Institute ,Gruangzhou 510663,China)
出处
《中国兽医学报》
CAS
CSCD
北大核心
2018年第12期2258-2261,共4页
Chinese Journal of Veterinary Science
基金
广东省科技计划资助项目(2015B070701003,2016A040403060,2017A070702001,2017B030314171)
广州市科技计划资助项目(201707010440,2016201604030059)
