摘要
基于猪圆环病毒2型(porcine circocirus type 2,PCV-2)的ORF2基因建立了一种便捷、灵敏而又特异的可视化环介导等温核酸扩增(loop-mediated isothermal amplification,LAMP)方法。该方法通过针对PCV-2 6个位点的4条特异性引物,在Bst DNA聚合酶的作用下对PCV-2靶序列进行等温核酸扩增。经过对LAMP反应体系和反应条件的优化,所建立方法在61℃反应1 h能够成功的检测出PCV-2基因组DNA,且操作简单,不需要PCR仪等复杂仪器,结果可经电泳检测及肉眼观察;敏感性和特异性试验表明,所建立的方法能特异地检测PCV-2并且其敏感度可以达到0.5 pg的DNA分子;所建立LAMP方法的阳性检出率与国标PCR的阳性检出率符合度为100%。该研究为PCV-2的现场快速检测提供更加简便、快捷的方法,可以满足基层检疫的需要。
In this study, a convenient, sensitive, specific and appreciable LAMP （ loop-mediated isothermal amplifi- cation） assay for detection of PCV-2 （porcine circocirus type 2） based on the ORF2 gene was established. This method completed amplification of PCV-2 in the role of the Bst DNA polymerase, by four specific primers designed through six sites of target sequence. After optimization of the reaction mixture and conditions, the assay could suc- cessfully detect PCV-2 genomic DNA at 61% for 1 h, which was easy to operate, never need PCR instrument and other complex instruments, and the result can be obtained by agarose gel electrophoresis or naked eye. The sensitivi- ty and specificity tests showed that the method was able to detect PCV-2 specifically and its sensitivity could be up to 0. 5 pg PCV-2 DNA. The positive rate of LAMP method for PCV-2 was 100% in accordance with international stand- ard of PCR. This study provided an easier, faster way for rapid detection of PCV-2, which met the needs of grass- roots quarantine.
出处
《浙江农业学报》
CSCD
北大核心
2013年第1期21-26,共6页
Acta Agriculturae Zhejiangensis
基金
浙江省重大科技专项农业项目(2008C12049)
