| 摘要: |
| [目的]建立一种能够快速、灵敏地检测猪圆环病毒2型(PCV2)的SYBR-Green 1实时荧光定量PCR方法.[方法]根据已报道的PCV2 ORF2基因组序列,设计并合成1对引物.通过常规PCR扩增PCV2 ORF2基因,将鉴定正确的ORF2基因片段克隆人pTG19-T载体中,转化大肠杆菌JM109,经PCR及测序鉴定后得到阳性重组质粒,作为标准晶模板建立SYBR-Green 1荧光定量PCR标准曲线和溶解曲线,并做灵敏性、特异性和重复性试验.[结果] PCV2荧光定量PCR标准曲线循环阈值与模板浓度呈良好的线性关系.溶解曲线特异,相关系数为0.998 8.最低榆测的拷贝数为1拷贝/25 μL,特异性和重复性较好.[结论]建立了检测PCV2的SYBR-Green 1荧光定量PCR方法,为该病的早期诊断及定量分析PCV2感染程度奠定了基础. |
| 关键词: 猪圆环病毒2型(PCV2) 实时定量PCR PCR检测方法 |
| DOI： |
| 分类号: |
| 基金项目:河南省杰出人才创新基金 |
|
| Development of a SYBR-Green 1 real-time fluorescent quantitative PCR method for detection of type 2 porcine circovirus |
|
|
| Abstract: |
| 【Objective】 The study developed a SYBR Green 1 real-time quantitative PCR which can detect porcine circovirus type 2 quickly and flexibly.【Method】 According to genome sequences within ORF2 of porcine circovirus type 2 (PCV 2) published in GenBank,a pair of primers was designed.The ORF2 gene was amplified with traditional PCR.The PCR product was cloned into pTG19-T vector and sequenced.The positive recombinant plasmid was used as quantitative template to generate standard curve and melt curve.Sensitivity assay,reproducibility of the assay and specificity assay were determined.【Result】 The results demonstrated that standard curve established by recombinant plasmid was shown a fine linear relationship between threshold cycle and template concentration.Melt curve was specific and the correlation coefficient was 0.998 8;the detection limit of real-time PCR for PCV2 was 1 copy per 25 μL,and the quantitative PCR was more reproductire and specific than traditional PCR.【Conclusion】 A SYBR Green1 fluorescent quantitative PCR for detecting ORF2 gene of PCV2 was developed for the basis of the early and rapid detection and analysising the infect degree of PCV2 quantitatively. |
| Key words: Porcine circovirus 2(PCV2) SYBR-Green1 real-time quantitative PCR PCR method for detection |
