摘要:
参照GenBank收录的 PCV2 ORF2基因设计合成引物和Taqman探针,建立PCV2 TaqMan荧光定量PCR检测方法,对临床确诊PCV2的病料和30份临床疑似感染PCV2的病料进行检测,同时与常规PCR检测方法进行比较。结果显示,TaqMan荧光定量PCR检测方法灵敏度可达4.5103拷贝 L-1,比普通PCR检测方法高10倍;对30份疑似感染PCV2病料的检测表明,TaqMan荧光定量PCR和普通PCR检测阳性率分别为66.7%和56.7%,两者符合率90%。应用TaqMan荧光定量PCR检测方法检测PRV、 CSFV、 PRRSV结果均为阴性,无交叉反应;表明该方法具有灵敏度高、特异性强、重复性好等优点,可用于PCV2的流行病学调查和临床诊断。
Abstract:
The probes and primers were designed according to the nucleotide sequence of porcine circovirus type 2(PCV2) available in GenBank, and real-time TaqMan fluorescence quantitative PCR for detection of PCV2 was established successfully.Clinical diagnosis of PCV2 samples and the 30 suspected samples were detected by using the established quantitative PCR which was compared with that of routine PCR.The results indicated that the established quantitative PCR assay could detect 4.5103 copysL-1 of plasmid DNA.Sensitivity and positive rate for clinical samples of TaqMan fluorescent quantitative PCR were higher than routine PCR, and its sensitivity was 10 times higher than that of the routine PCR.The 30 suspected samples were detected by TaqMan fluorescence quantitative PCR and routine PCR, respectively, the positive detection rate were 66.7% and 56.7%, the coincidence rate was 90%.Classical swine fever virus(CSFV), porcine reproductive and respiratory syndrome virus(PRRSV) and pseudorabies virus(PRV) were detected by using the established quantitative PCR and the result indicated that CSFV, PRRS and PRV were negative, and had no cross reaction.The real-time TaqMan fluorescence quantitative PCR assay which is specific,sensitive and accurate can be used for the clinical diagnosis and epidemiological investigation of PCV2.
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