根据对已报道的PCR检测方法灵敏性评价,以常用KHV病毒PCR检测的目的基因KHVSphI片段（AY568590）、KHV5/9（AF411803）和KHVTK基因（AJ535112）作为靶基因,设计并选择3对特异性引物建立的多重PCR检测体系用于KHV病毒多基因的检测。本研究建立的多重PCR体系具有较高的特异性,能够特异性扩增出KHVSphI片段290bp、KHV5/9片段484bp和KHVTK基因片段409bp,对锦鲤和鲤鱼的另外一种病毒性病原鲤春毒血症病毒检测结果为阴性。多重KHV病毒PCR体系检测KHVSphI、KHV5/9和KHVTK基因片段单一模板的检测下限分别为：10fg、100fg和100fg,在相同模板浓度的情况下,KHVSphI、KHV5/9和KHVTK基因片段同时被检出的检测下限为100fg。对KHV病毒感染组织的检测结果表明,多重KHV病毒PCR检测结果与常规PCR检测结果基本吻合,在多重PCR检测体系中KHVTK基因片段检测的灵敏度高于检验检疫行业标准方法。结果表明,多重KHV病毒PCR检测方法能够快速、准确和灵敏地检测KHV病毒基因。%A multiplex polymerase chain reaction （multi-PCR） was developed for simultaneously detection of 3 KHV viral DNA segments to improve the diagnosis of KHV infection.3 sets of primers were designed targeting specific sequences of KHVSphI（AY568590）,KHV5/9（AF411803）and KHV TK gene（AJ535112）,which were frequently used in PCR assay for detecting KHV viral DNA used in the assay and each of them could amplify and result in 290 bp,484 bp and 409 bp fragment with viral nucleic acids by PCR products with different size,the size of KHVSphI,KHV5/9and KHV TK gene.They were highly specific and no specific bands of the same size were amplified from SVCV rival cDNA.The sensitivity of the multiplex PCR was 10fg for KHVSphI,100 fg for KHV5/9and KHV TK gene.In the field application,the results were consistent with those results of the single PCR detection,indicating that this multi-PCR method was superior in terms of sensivity,specificity,rapidity and simplicity,and potentially a valuabable diagnostic tool for KHV infections.
展开▼
