摘要
针对猪圆环病毒-Ⅱ型(porcine circovirus typeⅡ,PCV-Ⅱ)、猪瘟病毒(swine fever virus,CSFV)和猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndrome virus,PRRSV)的保守区域设计用于构建荧光定量PCR体系的引物和探针。以构建的重组质粒标准品为模板建立三重荧光定量PCR体系,并对三重荧光定量PCR体系进行优化和特异性检测、灵敏性检测、重复性试验、标准曲线制作。最后利用构建的三重荧光定量PCR体系和普通PCR体系同时进行临床检测,并对两者的检测结果进行对比。结果显示,3种病毒在单重和三重荧光定量PCR体系中均无非特异性扩增;3种病毒在单重和三重荧光定量PCR体系中的灵敏度均可达到10拷贝/μL;单重和三重荧光定量PCR体系组内和组间重复性变异系数均在5%以内;单重和三重荧光定量PCR在稀释度范围内呈现良好的线性关系。应用本研究构建的三重荧光定量PCR体系对106份临床组织样品进行检测,其中PCV-Ⅱ阳性检出率为74.5%,CSFV阳性检出率为34.9%,PRRSV阳性检出率为18.9%,普通PCR检测中PCV-Ⅱ阳性检出率为37.7%,CSFV阳性检出率为16.9%,PRRSV阳性检出率为7.5%。
Primers and probes for establishing a fluorescent quantitative PCR method were designed based on the conserved regions of porcine circle virus-Ⅱ(PCV-Ⅱ),swine fever virus(CSFV)and porcine reproductive and respiratory syndrome virus(PRRSV).The recombinant plasmid was constructed as standard product and used as a template to establish a triple fluorescent quantitative PCR method,and then the triple fluorescent quantitative PCR method was optimized,the detection of specificity,sensitivity,repeatability,and standard curve of production were performed.Finally,the triple fluorescence quantitative PCR and ordinary PCR method were used for clinical testing at the same time,and the test results of the two methods were compared.The results as following:the three viruses had no non-specific amplification in single and triple fluorescence quantitative PCR methods;the sensitivity of the three viruses in single and triple fluorescent quantitative PCR could reach 10copies/μL;the single and triple fluorescent quantitative PCR system and the repeatability coefficient of variation within and between groups were all within 5%;the single and triple fluorescence quantitative PCR showed a good linear relationship within the dilution range.The triple fluorescence quantitative PCR system established in this study was used for clinical detection of 106tissue samples.The positive detection rate of PCV-Ⅱwas 74.5%,the positive detection rate of CSFV was 34.9%,and the positive detection rate of PRRSV was 18.9%.In the PCR test,the positive detection rate of PCV-Ⅱwas 37.7%,the positive detection rate of CSFV was 16.9%,and the positive detection rate of PRRSV was 7.5%.
作者
刘斌
赵翠青
刘立明
沙万里
董文龙
刘嘉琳
李国江
LIU Bin;ZHAO Cuiqing;LIU Liming;SHA Wanli;DONG Wenlong;LIU Jialin;LI Guojiang(College of Animal Science and Technology,Jilin Agricultural University,Changchun 130118,China;College of Animal Science and Technology,Jilin Agricultural Science and Technology College,Jilin City,Jilin 132109,China;Jilin Province Key Laboratory of Preventive Veterinary Medicine,Jilin City,Jilin 132109,China)
出处
《中国兽医学报》
CAS
CSCD
北大核心
2022年第7期1309-1314,共6页
Chinese Journal of Veterinary Science
基金
吉林省科技发展计划资助项目(20200602048ZP)。
关键词
猪圆环病毒-Ⅱ型
猪瘟病毒
猪繁殖与呼吸综合征病毒
荧光定量PCR
porcine circovirus typeⅡ
swine fever virus
porcine reproductive and respiratory syndrome virus
fluorescence quantitative PCR
