CAJ | 학술논문

根据GenBank中猪伪狂犬病毒（PRV ）gE基因序列设计特异性的引物，PCR扩增获得PRV gE基因片段，构建重组质粒pMD18 T gE ，作为阳性标准品。对SYBR Green Ⅰ实时荧光定量PCR反应条件进行优化，建立猪PRV SYBR Green Ⅰ实时荧光定量PCR诊断方法，研制诊断试剂盒。扩增产物的熔解曲线分析结果显示只出现单特异峰，无引物二聚体，对猪圆环病毒（PC V 2）、猪细小病毒（PPV ）、PRV （gE基因缺失株）、猪源 E ．coli、猪瘟病毒（CSFV ）、猪繁殖与呼吸障碍综合征病毒（PRRSV ）均无阳性信号扩增，且重复性好，灵敏度可达2．3×101拷贝/μL。结果表明，研制的PRV野毒株SYBR Green Ⅰ实时荧光定量PCR试剂盒特异、灵敏、快速、重复性好，适于伪狂犬病毒临床样品的检测。
근거GenBank중저위광견병독（PRV ）gE기인서렬설계특이성적인물，PCR확증획득PRV gE기인편단，구건중조질립pMD18 T gE ，작위양성표준품。대SYBR Green Ⅰ실시형광정량PCR반응조건진행우화，건립저PRV SYBR Green Ⅰ실시형광정량PCR진단방법，연제진단시제합。확증산물적용해곡선분석결과현시지출현단특이봉，무인물이취체，대저원배병독（PC V 2）、저세소병독（PPV ）、PRV （gE기인결실주）、저원 E ．coli、저온병독（CSFV ）、저번식여호흡장애종합정병독（PRRSV ）균무양성신호확증，차중복성호，령민도가체2．3×101고패/μL。결과표명，연제적PRV야독주SYBR Green Ⅰ실시형광정량PCR시제합특이、령민、쾌속、중복성호，괄우위광견병독림상양품적검측。
According to the gene sequences of gE gene of PRV in GenBank ,one pairs of specific primer was designed for amplifying the specific fragments of gE gene .Then gE gene of PRV amplified by PCR was cloned into pMD18-T vector and it was used as positive standard .After optimization of annealing temperature and primers concentrations ,a SYBR GreenⅠ real-time quantitative PCR kit was developed for detection of PRV wild-type strains .The melting curve analysis using SYBR Green Ⅰdye showed one specific peak ,and primer-dimers peak was not observed .No fragment was amplified from PRV strains depleted of gE gene ,PPV ,PCV-2 ,Escherichia coli ,CSFV ,PRRSV by the SYBR Green Ⅰ real-time quantitative PCR .The PCR kit was highly sensitive in 2.3 × 101 copies/μL DNA .The results revealed that the PCR kit was sensitive ,specific and it could be used to detect PRV wild-type strains in clinical samples .