非洲猪瘟病毒非结构基因实时荧光LAMP检测方法的建立

Establishment of Real-time Fluorescence LAMP Assay for Detection of Non-structural DNA Polymerase Gene of African Swine Fever Virus

为建立非洲猪瘟病毒(ASFV)通用型高通量检测技术,利用高度保守的非结构DNA聚合酶G1211R基因,制备质粒标准品,建立实时荧光LAMP方法,出现典型的S形核酸扩增曲线,扩增产物具有特异的熔解曲线。G1211R基因质粒标准品在1.81×10~5拷贝数/μL^1.81×10~9拷贝数/μL对数值与Ct值之间的线性关系良好。以ASFV E70株病毒核酸为模板,LAMP检测灵敏度达到21pg,优于荧光定量PCR方法。重复性试验LAMP检测批内和批间变异系数均小于5%。LAMP方法检测ASFV特异性良好,与猪圆环病毒2型、伪狂犬病病毒、猪瘟病毒、猪繁殖与呼吸综合征病毒及昆虫核酸无交叉反应。以ASFV Arm 07株制备各种临床模拟样品,检出阳性率达到17.31%。检测方法的建立,可为非洲猪瘟防控提供新的技术手段,有利于不同基因型毒株检测和出入境快速筛查。

A universal real-time fluorescence LAMP detection method was established by cloning the DNA polymerase gene G1211R of African swine fever virus strain E70.A specific amplification was obtained and verified by a melt curve analysis.There were good linear relationships between Ct values and logarithm of input copy numbers over a range from 1.81×10^5 to 1.81×10^9 copies μL for G1211R gene.The sensitivity for detection of nucleic acid was greater than that of fluorescence PCR method.The coefficients of variation within a batch and between batches were both less than 5 %.There was no cross-reactivity with a variety of clinical related viruses. The total positive rate of spiked samples with ASFV strain Arm 07 was up to 17.31%. This study can provide an important reference for the prevention and control of ASF, and needs for further verification and application with different genotypes.