猪圆环病毒2型LAMP检测方法的建立与评价

根据猪圆环病毒2型(Porcine circovirus type 2,PCV2)Rep和Cap基因保守区设计2对引物,1对外引物和1对内引物;利用设计的4条引物,在Bst大片段聚合酶的作用下,对PCV2 DNA进行恒温扩增;扩增条件为63℃恒温反应1h;建立了PCV2环媒恒温扩增技术(LAMP)检测方法。对检测方法特异性评价表明,检测方法只能检测PCV2 DNA,对猪圆环病毒1型(Porcine circovirus type 1,PCV1)、猪细小病毒(Porcine parvovirus,PPV)及猪伪狂犬病病毒(Porcine pseudorabies virus,PRV)检测无交叉反应。灵敏度评价表明,该检测方法可以检测到样品中10个拷贝的PCV2 DNA含量。

Establishment and evaluation of loop-mediated isothermal amplification for detecting of porcine circovirus type 2

A loop-mediated isothermal amplification(LAMP) method was developed for the detection of porcine circo-virus type 2(PCV2). A set of four primers,two outer and two inner primers,was designed based on the Rep and Cap genes for LAMP assay. The amplification could be finished in 60 min under isothermal condition at 63℃. With Bst DNA polymerase large fragment,ladder like DNA fragments can be seen with agarose gel electrophoresis. Good spe-cificity of the LAMP method was demonstrated as amplified product detected only from PCV2 DNA but not from other porcine DNA virus, such as porcine circovirus type 1 (PCV1), porcine parvovirus(PPV) and porcine pseudora-bies virus(PRV). The test limit was found to be 10 copies/μL PCV2 DNA in the detection of a seriesly diluted virus DNA samples. Because LAMP is rapid and easy to use,the developed LAMP is suitable for PCV2 monitoring and de-tection for screening large number of samples in farms.