牛结节性皮肤病病毒TaqMan-MGB荧光PCR检测方法的建立

Establishment of TaqMan-MGB real-time fluorescent PCR for rapid detection of lumpy skin disease virus

为建立快速、特异鉴别检测牛结节性皮肤病病毒(LSDV)的方法,本研究参照GenBank中发表的LSDV全基因组序列,设计并合成一对特异性引物和探针,经优化条件,建立了可鉴别LSDV的TaqMan-MGB荧光定量PCR检测方法。结果显示,该方法可特异性检测LSDV,而与山羊痘病毒、绵羊痘病毒、牛痘病毒和猪痘病毒均无交叉反应;标准曲线在各浓度范围内呈现良好的线性关系,该方法建立的标准曲线方程为y=-3.141x+32.10,线性相关系数为0.999,扩增效率达到108%,最低检出下限为1.48 copies/L;组内、组间重复性试验的变异系数均小于2%。采用此方法对52份模拟临床样品和112份临床样本的检测结果显示:32份模拟阳性样品检出LSDV核酸阳性,20份模拟阴性样品未检出LSDV;而用OIE推荐的普通PCR方法仅检出28份模拟阳性样品的LSDV核酸阳性,24份模拟样品的LSDV核酸阴性(其中20份为模拟阴性样品,4份为模拟阳性样品);两种方法均未从112份临床样品中检出LSDV核酸阳性。上述结果表明,荧光PCR的敏感性明显优于普通PCR,能够快速准确地检测LSDV,适用于临床样品的检测,为外来疫病防控提供了技术支撑。

To develop a rapid and sensitive method for lumpy skin disease virus（LSDV） detection,a TaqMan real-time PCR assay was established with a pair of primers and TaqMan probe for the conservative sequence of LSDV genome.Then,the reaction conditions were optimized with the recombinant plasmid containing the LSDV＇s gene for the establishment of a standard curve.In result,the method was able tospecifically amplify the target gene from LSDV,and had no cross-amplification from GTPV,SPPV,cow pox virus and swine pox virus.The standard curve of method was y=-3.141x＋32.10 and its linear correlation coefficient was 0.999.So,the standard curve presented the favorable linear correlation in various concentration.The amplification efficiency of the method was up to 108%.The minimum detectable limit was 1.48 copies/L of LSDV DNA.The variation coefficient of the intro-and inter-test assay were both less than 2%,indicating that the assay was reproducibile.The results were demonstrated by 52 simulated clinical samples and 112 clinical samples：32 simulated positive samples were found positive with LSDV,while no LSDV was detected in the 20 simulated negative samples.While using the PCR method recommended by OIE,only 28 of the total simulated positive samples were detected as positive,and 24 samples （including 20 negative samples and 4 positive samples） were found negative with LSDV.All of the 112 clinical sample were identified as negative with both of the OIE recommended PCR and our method.The above-mentioned results showed that this real-time PCR had the virtues of convenient,fast,specificity,high sensitivity and reproducibility,which could be used for clinical detection of LSDV infection.