›› 2013, Vol. 25 ›› Issue (1): 0-26.

• 论文 •    

猪圆环病毒2型LAMP检测方法的建立

申世川1,2,王一成1,*,袁秀芳1,徐丽华1,李军星1   

  1. 1浙江省农业科学院 畜牧兽医研究所,浙江 杭州310021;2南京农业大学 动物医学院,江苏 南京210095
  • 收稿日期:1900-01-01 修回日期:1900-01-01 出版日期:2013-01-25 发布日期:2013-01-25

Establishment of LAMP for detection of porcine circovirus type 2

SHEN Shichuan;WANG Yicheng;*;YUAN Xiufang;XYU Lihua;LI Junxing   

  1. 1Institute of Animal Husbandry and Veterinary, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China; 2College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China
  • Received:1900-01-01 Revised:1900-01-01 Online:2013-01-25 Published:2013-01-25

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。

关键词: 猪圆环病毒2型, 环介导等温扩增, 可视化, 快速检测

Abstract: In this study, a convenient, sensitive, specific and appreciable LAMP (loop\|mediated isothermal amplification) 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 successfully 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 sensitivity 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 standard of PCR. This study provided an easier, faster way for rapid detection of PCV-2, which met the needs of grass-roots quarantine.

Key words: PCV-2, LAMP, visual observation, quick detection