水貂阿留申病毒微滴式数字PCR检测方法的建立与应用

Establishment and application of droplet digital PCR method for detection of Aleutian mink disease virus

为建立一种准确且灵敏的水貂阿留申病毒(AMDV)微滴式数字PCR检测方法(dd PCR),本研究根据AMDV-G基因保守区,设计特异性引物及探针,并对反应条件进行优化,初步建立检测AMDV的dd PCR方法。同时对该方法的特异性、敏感性和重复性进行分析。结果显示,dd PCR的退火温度为55℃,最佳引物和探针终浓度均为10μmol/μL。绘制的标准曲线结果显示,核酸标准物质稀释倍数的对数与对应检出拷贝数的对数之间呈良好的线性关系(R^(2)=0.9945>0.99)。建立的dd PCR方法的特异性强,除能检测到AMDV以外,对相关病原如水貂犬瘟热病毒、犬细小病毒、貂肠炎病毒、犬冠状病毒、犬腺病毒I型、狂犬病毒的检测结果均为阴性。对AMDV核酸标准物质的检测限为6.2拷贝/μL,敏感性高,比荧光定量PCR(q PCR)敏感性高10倍。对不同稀释度AMDV核酸标准物质的批内和批间重复性试验的变异系数均<5%,重复性好。采用建立的dd PCR方法和荧光定量PCR方法对貂场采集的40份水貂脏器组织样品进行检测,两种检测结果基本一致,但dd PCR敏感性更高。上述结果表明,本研究建立了一种特异性强、灵敏度高、重复性好的AMDV定量检测方法,为水貂阿留申病毒的有效防控提供技术支持。

To establish an accurate and sensitive droplet digital PCR method(dd PCR)for the detection of Aleutian mink disease virus(AMDV),specific primers and probes were designed according to the conserved region of AMDV G gene,and the reaction conditions were optimized.Meanwhile,the specificity,sensitivity and repeatability were analyzed.The results showed that the annealing temperature of dd PCR was 55℃,and the optimal final concentration of primers and probes was 10μmol/μL.The standard curve plotted demonstrated a good linear relationship between the logarithm of the dilution factor of the nucleic acid standard substance and the logarithm of the detected copy number(R~2=0.9945>0.99).The established dd PCR method had strong specificity and did not amplify other common diseases.The lower limit of detection for the AMDV nucleic acid standard substance was 6.2 copies/μL.The coefficient of variation for both intra-batch and inter-batch repeatability tests were less than 5%,indicating good repeatability.When the established dd PCR method and fluorescence quantitative PCR method was used to detect 40 mink organs collected from mink farm,the results of the two methods were generally consistent,but dd PCR showed higher sensitivity.These findings indicated that this study established a quantitative detection method of AMDV with strong specificity,high sensitivity and good repeatability,provided technical support for effective prevention and control of AMDV.