CAJ | 학술논문

根据Ⅶ型新城疫病毒基质蛋白基因保守序列设计并合成特异性引物,建立快速检测Ⅶ型新城疫病毒的实时荧光定量RT-PCR方法。通过RT-PCR方法克隆Ⅶ型新城疫病毒M基因靶序列,并将其连入T-Easy载体,制备阳性标准品,优化反应条件,以10倍系列稀释的标准品绘制标准曲线,其相关系数为0.999。检测结果显示,该方法的灵敏度可达100 copies/μL,而且特异性良好,除Ⅶ型新城疫病毒外,对Ⅱ型/Ⅲ型/IV型/Ⅸ型新城疫病毒、H5亚型禽流感病毒、H9亚型禽流感病毒、鸡传染性支气管炎病毒检测结果均为阴性。本研究所建立的检测方法重复性好,批内和批间变异系数均小于5%。对40份实验感染样品的检测结果表明,其检出率为95%,而常规RT-PCR方法检出率仅为70%,表明该方法比常规RT-PCR检测方法灵敏度更高、特异性更强。
근거Ⅶ형신성역병독기질단백기인보수서렬설계병합성특이성인물,건립쾌속검측Ⅶ형신성역병독적실시형광정량RT-PCR방법。통과RT-PCR방법극륭Ⅶ형신성역병독M기인파서렬,병장기련입T-Easy재체,제비양성표준품,우화반응조건,이10배계렬희석적표준품회제표준곡선,기상관계수위0.999。검측결과현시,해방법적령민도가체100 copies/μL,이차특이성량호,제Ⅶ형신성역병독외,대Ⅱ형/Ⅲ형/IV형/Ⅸ형신성역병독、H5아형금류감병독、H9아형금류감병독、계전염성지기관염병독검측결과균위음성。본연구소건립적검측방법중복성호,비내화비간변이계수균소우5%。대40빈실험감염양품적검측결과표명,기검출솔위95%,이상규RT-PCR방법검출솔부위70%,표명해방법비상규RT-PCR검측방법령민도경고、특이성경강。
The real time SYBR Green Ⅰ fluorescence quantitative reverse transcription polymerase chain reaction（rRT-PCR） assay based on conserved region of matrix gene was developed for rapid detection of genotype Ⅶ Newcastle disease virus.The target sequence of M gene was cloned into the T-easy vector and a series of diluted recombinant plasmids were used to generate standard curve.The rRT-PCR assay had a detection limit of 100 copies of target DNA per-reaction and a correlation coefficient of 0.999.The assay showed good specificity and did not cross react with other viruses including genotype（Ⅱ,Ⅳ,Ⅸ） Newcastle disease virus,H5 Avian influenza virus,H9 Avian influenza virus and Infection bronchitis virus.The variation coefficients of intra assay were below 5%,which indicated good reproducibility.Forty experimentally infected samples were validated by the assay.The results showed that the detection rate was 95% compare with 70% by conventional RT-PCR.It showed better sensitivity and specificity of rRT-PCR than conventional RT-PCR.