期刊论文

Establishment and optimization of a duplex PCR assay for simultaneous detection of Porcine circovirus types 2 and 3

中文

黑龙江畜牧兽医

1004-7034

2020

05

71-76

10.13881/j.cnki.hljxmsy.2019.04.0118

湖南省教育厅重点项目(15A086) 湖南省自然科学基金面上项目(2018JJ2177) 湖南农业大学2017年青年科学基金项目(17QN10) 湖南农业大学“大学生创新性试验计划”项目。

本校为第一机构

为了能够同时检测猪圆环病毒2型（Porcine circovirus type 2, PCV-2）和猪圆环病毒3型（Porcine circovirus type 3, PCV-3）,试验根据国内报道的PCV-2和PCV-3毒株全基因序列设计6对特异性引物（P3～P8）,并构建标准质粒进行引物特异性试验,然后通过优化扩增条件建立检测PCV-2和PCV-3混合感染的双重PCR方法。最后用PCV-2和PCV-3的感染性克隆同时转染PK-15细胞,盲传5代后用建立的双重PCR方法检测细胞培养液中的病毒核酸。结果表明:构建的PCV-2和PCV-3标准质粒浓度分别为290 ng/μL和380 ng/μL,双重PCR方法的最佳引物组合为P3/P6和P4/P8,最佳DNA聚合酶浓度为0.02 U/μL。使用引物组合P3/P6时,扩...

In order to simultaneously detect porcine circovirus type 2(PCV-2)and porcine circovirus type 3(PCV-3),six pairs of specific primers(P3-P8)were designed based on the published sequence of the complete genome of PCV-2 and PCV-3 in China,and standard plasmids were constructed for specificity test of primers,then duplex PCR methods were established by optimizing the amplification conditions to detect the co-infection of PCV-2 and PCV-3.Finally,the infectious clones of PCV-2 and PCV-3 were transfected into PK-15 cells simultaneously,after 5 passage...