CAJ | 학술논문

根据GenBank公布的24株高致病性猪繁殖与呼吸综合征病毒毒株和5株PRRSV经典毒株的保守区基因序列,使用PrimerExpress 3.0软件设计并合成实时荧光定量PCR（Real-timeFluorescent Quantitative PCR,Real-time FQ-PCR）用引物和探针,建立了Real-time FQ-PCR检测方法以鉴别检测高致病性猪繁殖与呼吸综合征病毒。用建立的检测方法对已定量的10倍倍比稀释的质粒pGET-258为标准品进行检测,并与常规PCR进行比较。结果显示,该Real-time FQ-PCR方法敏感度可达1.5个拷贝,比常规PCR敏感度高100倍,且批内和批间重复性检测结果的变异系数均小于2%。用该方法与常规PCR方法及病毒分离方法对18份临床样品进行对比检测,显示该方法灵敏度高、成本低,并且能够对样品中病毒进行定量,为高致病性猪繁殖与呼吸综合征的快速鉴别诊断提供了有效的技术手段。
근거GenBank공포적24주고치병성저번식여호흡종합정병독독주화5주PRRSV경전독주적보수구기인서렬,사용PrimerExpress 3.0연건설계병합성실시형광정량PCR（Real-timeFluorescent Quantitative PCR,Real-time FQ-PCR）용인물화탐침,건립료Real-time FQ-PCR검측방법이감별검측고치병성저번식여호흡종합정병독。용건립적검측방법대이정량적10배배비희석적질립pGET-258위표준품진행검측,병여상규PCR진행비교。결과현시,해Real-time FQ-PCR방법민감도가체1.5개고패,비상규PCR민감도고100배,차비내화비간중복성검측결과적변이계수균소우2%。용해방법여상규PCR방법급병독분리방법대18빈림상양품진행대비검측,현시해방법령민도고、성본저,병차능구대양품중병독진행정량,위고치병성저번식여호흡종합정적쾌속감별진단제공료유효적기술수단。
Primers and probe were designed with PrimerExpress 3.0 and synthesized according to the conserved gene sequences of 24 highly pathogenic porcine reproductive and respiratory syndrome（HP-PRRSV） strains and 5 classical PRRSV strains available in GenBank.A real-time fluorescent quantitative PCR assay was established for detection,differentiation and quantitation of HP-PRRSV and classical PRRSV.A known concentration of 10-fold series of dilutions of pGET-258 plasmid DNA were detected by using the established real-time FQ-PCR assay,then compared to the detection result of routine PCR.The developed real-time FQ-PCR assay could detect 1.5 copies with plasmid DNA and its sensitivity was 100 times higher than that of the routine PCR.Three plasmid standards were examined using the real-time FQ-PCR repeatedly at three different time,the coefficient of variations were below 2%,and the results indicated that the real-time FQ-PCR assay was reproducible and could be used for the diagnosis and differentiation of HP-PRRSV infection.