摘要
目的建立基于TaqMan探针的猪圆环病毒2型(Porcine circovirus type 2,PCV2)荧光定量PCR检测方法。方法根据PCV2 ORF2的相对保守序列,设计两对特异性引物及TaqMan探针,制备重组质粒标准品;优化反应体系及反应条件,绘制标准曲线,建立PCV2 TaqMan荧光定量PCR检测方法,并验证其敏感性、特异性及重复性;用建立的方法对50份临床样本进行检测,并与普通PCR检测结果进行比较。结果重组质粒标准品经PCR及测序鉴定构建正确;建立的定量标准曲线Ct值与模板拷贝数对数之间呈良好的线性关系,相关系数R2为0.993 85;该方法的敏感性为6×101拷贝/μl,比普通PCR高2个数量级;检测猪繁殖与呼吸综合征病毒、猪伪狂犬病毒和猪瘟兔化弱毒均无扩增曲线;检测3份PCV2阳性模板的变异系数在0.07%～0.50%之间;临床样本的阳性检出率(64%)明显高于普通PCR(44%),差异有统计学意义(P<0.05)。结论建立的PCV2 TaqMan荧光定量PCR检测方法具有较高的敏感性、特异性和重复性,适用于临床样本中微量PCV2的快速检测及其组织亲嗜性、细胞培养特性的研究。
Objective To develop a TaqMan probe-based fluorescent quantitative PCR method for porcine cireovirus type 2 （PCV2）. Methods Two pairs of specific primers and TaqMan probe were designed according to the relatively conserved sequence of PCV20RF2 for preparation of standard for recombinant plasmid. The reaction system and condition were optimized, based on which the standard curve was plotted, and a TaqMan probe-based fluorescent quantitative PCR method was developed and verified for sensitivity, specificity and reproducibility. Fifty clinical samples were determined by the developed method, and the results were compared with those by routine PCR method. Results PCR and sequencing proved that the standard for recombinant plasmid was constructed correctly. The Ct value of plotted standard curve showed good linearity to the log of copy number of template, with a R2 value of 0. 993 85. The sensitivity of developed method was 6 × 10^1 copies/Ixl, which was two orders of magnitude higher than that of routine PCR method. No amplification curves were obtained from porcine reproductive and respiratory syndrome virus （PRRSV）, porcine pseudorabies virus or attenuated lapinized classical swine fever virus. The variation coefficients of determination results of three PCV2-positive templates by the developed method were O. 07% - 0. 50%. The positive rate of clinical samples by the developed method （64%） was significantly higher than that by routine PCR method （44%） （P 〈 0. 05 ）. Conclusion The developed TaqMan probe-based fluorescent quantitative PCR method showed high sensitivity, specificity and reproducibility, which was suitable for rapid detection of trace PCV2 in clinical samples as well as study on tissue tropism and cell culture characteristics of PCV2.
出处
《中国生物制品学杂志》
CAS
CSCD
2012年第4期487-491,共5页
Chinese Journal of Biologicals
基金
安徽省"十一五"科技攻关项目(08010302155)
安徽省生猪产业体系基金资助项目
安徽省长三角联合攻关项目(1101c0603065)
安徽省猪病监测工程技术研究中心(201106G01040)资助项目
