摘要
为了建立一种定量检测猪圆环病毒1型(PCV1)的实时荧光定量PCR方法,根据PCV1基因序列设计了1对特异性引物,并构建含有PCV1基因片段的重组质粒,以系列稀释后的重组质粒作为模板建立了定量检测PCV1的SYBR GreenⅠreal-time PCR方法,并绘制标准曲线,进行特异性、敏感性和重复性试验。结果显示,该方法的标准曲线的决定系数为0.999,显示出优良的线性关系;最低可准确检测320copies/μL的核酸模板,重复性试验的变异系数小于2%;该方法对PCV2、猪细小病毒、伪狂犬病病毒、猪繁殖与呼吸综合征病毒及猪瘟病毒等的检测结果均为阴性;对临床样品的检出率高于常规PCR方法。结果表明,建立的real-time PCR特异性强、灵敏度高、重复性好,可用于PCV1的检测与定量分析。
The objective of this study was to develop a real-time quantitative fluorescence PCR assay for quantitative detection of porcine circovirus type 1(PCV1).A pair of primers specific to the PCV1 gene was designed to construct a recombinant plasmid,and the positive recombinant plasmid was diluted serially as templates to establish a real-time PCR assay based on SYBR GreenⅠ for quantitative detection of PCV1.Meanwhile,the corresponding standard curve was generated,and the specificity,sensitivity and reporducibility of this assay were determined.The coefficient of determination for the developed standard curve was 0.999,which indicated an excellent linear relationship.The assay had an accurate detectable limit of 320copies/μL,and its coefficient of variation was less than 2%in the reproducible assays.No amplification was detected by this method from unrelated swine virus samples,including PCV2,porcine parvovirus,pseudorabies virus,porcine reproductive and respiratory syndrome virus and classical swine fever virus.The detectable rate of several clinical samples with this assay was higher than that of conventional PCR assay.The results indicated that this real-time PCR assay was of high specificity,sensitivity and reproducibility,and could be used for the detection and quantitative analysis of PCV1.
出处
《中国兽医科学》
CAS
CSCD
北大核心
2014年第10期1036-1041,共6页
Chinese Veterinary Science
基金
北京市属高等学校高层次人才引进与培养计划项目(CIT&TCD201304092)
