黑龙江畜牧兽医Issue(1)：120-125,6.DOI:10.13881/j.cnki.hljxmsy.2024.09.0089
猪圆环病毒2型微滴数字PCR检测方法的建立及临床应用
Establishment and clinical application of droplet digital PCR detection method for Porcine circovirus type 2
摘要
Abstract
In order to establish a droplet digital PCR detection method for Porcine circovirus type 2(PCV-2)with high specificity,sensitivity,and repeatability,the experiment designed primers and probes based on the conserved region of PCV-2 ORF1.Droplet digital PCR method was established by optimizing the concentrations of primers and probes as well as the annealing temperature,tests for specificity,sensitivity,and repeatability,and this method was used to detect 90 clinical samples.The results showed that the optimal concentrations of primers and probes for the established PCV-2 droplet digital PCR method were 18 μmol/L and 9 μmol/L respectively,with an annealing temperature of 61℃;it could specifically detect PCV-2 and had no cross-reaction with a variety of common swine pathogens(PCV-3,PRV,PRRSV,ASFV,CSFV and PEDV);the minimum detection limit of the virus was 3.48 copies/μL.In the repeatability test,the intra-group and inter-group coefficients of variation of the amplified copy numbers ranged from 0.91%to 6.13%and 1.44%to 8.53%respectively,both of which were less than 10%.Among 90 clinical samples,the established droplet digital PCR method detected 43 PCV-2-positive samples,with a detection rate of 47.78%.Statistics showed that the positive samples detected by the droplet digital PCR method included all positive samples detected by the real-time fluorescent quantitative PCR method,and the coincidence rate with the real-time fluorescent quantitative PCR method was 88.89%.These results indicated that the PCV-2 droplet digital PCR detection method established in this study had strong specificity,high sensitivity,and good repeatability,and was suitable for the detection of clinical samples(especially samples with low viral load).关键词
猪圆环病毒2型/微滴数字PCR/病毒检测/检测方法/特异性试验/敏感性试验/重复性试验Key words
Porcine circovirus type 2/droplet digital PCR/virus detection/detection method/specificity test/sensitivity test/repeatability test分类
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刘武函,杨青,胡巧云,唐小明,彭志,张坤,谢怡灵,范仲鑫,王卫国,尚玲,张梦凡..猪圆环病毒2型微滴数字PCR检测方法的建立及临床应用[J].黑龙江畜牧兽医,2026,(1):120-125,6.基金项目
湖南省重点领域研发计划项目"畜禽疫病诊断及疫苗免疫评价技术研究与示范"(2023NK2017) （2023NK2017）
