摘要
为建立一种快速、灵敏、简便的猪繁殖与呼吸综合征病毒(PRRSV)早期检测方法,本研究利用逆转录环介导恒温扩增技术(RT-LAMP),针对PRRSV的ORF5基因片段设计了4条引物,利用Bst DNA聚合酶在65℃恒温条件下进行逆转录扩增,通过1.0%琼脂糖凝胶电泳和加入SYBR GreenⅠ染料肉眼判断结果,建立了PRRSV RT-LAMP检测方法。结果表明,该检测方法具有良好的特异性,与其他常见病毒如猪瘟病毒、猪圆环病毒2型、猪细小病毒、猪伪狂犬病病毒等无交叉反应,较普通RT-PCR灵敏性高100倍。采用RT-LAMP和RT-PCR分别对10份临床样本同时进行检测,符合率为100%。因此,本研究建立的RT-LAMP是一种可适用于临床PRRSV检测的快速、简单、灵敏、特异的检测方法。
In order to diagnose porcine reproductive and respiratory syndrome virus （PRRSV） early, a rapid, sensitive, simple reverse transcription-loop-mediated isothermal amplification （RT-LAMP） assay was established. Four primers targeting the ORF5 gene of PRRSV were de- signed. Reverse transcription and amplification of the viral cDNA using Bst DNA polymerase was optimal at a constant temperature of 65 ℃. The output of the RT-LAMP assay was visualized u- sing 1.0% agarose gel electrophoresis and color change after the addition of the SYBR Green I dye. The assay was also specific for PRRSV and did not cross react with classical swine fever vi- rus （CSFV）, porcine circovirus virus type 2 （PCV2）, porcine parvovirus （PPV）, porcine pseud- orabies virus （PRV）. The RT-LAMP method was approximately 100-fold more sensitive than RT-PCR for PRRSV detection. The clinical samples （n= 10） were identified both by RT-PCR and RT-LAMP, and the coincidece rate was 100%. Thus, the novel RT-LAMP assay was a rapid, simple, sensitive, specific test for PRRSV, and it could potentially be applied in clinical settings.
出处
《中国畜牧兽医》
CAS
北大核心
2017年第6期1630-1636,共7页
China Animal Husbandry & Veterinary Medicine
基金
四川省科技厅项目(2013JY0127)
四川省重点实验室项目(ESP1505)
四川省教育厅项目(16ZA0321)
