猪圆环病毒2型SYBR-Green 1实时荧光定量PCR检测方法的建立  

Development of a SYBR-Green 1 real-time fluorescent quantitative PCR method for detection of type 2 porcine circovirus

作　　者：贾艳艳[1,2] 李明凤[1] 王东方[1,2] 崔保安[1,2] 陈红英[1,2] 魏战勇[1] 吕晓丽[1,2] 郭显坡[1,2] 

机构地区：[1]河南农业大学牧医工程学院,河南郑州450002 [2]河南省动物性食品安全重点实验室,河南郑州450002

出　　处：《西北农林科技大学学报（自然科学版）》2009年第3期82-86,94,共6页Journal of Northwest A&F University(Natural Science Edition)

基　　金：河南省杰出人才创新基金项目(0621002100)

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PCV2ORF2基因组序列,设计并合成1对引物。通过常规PCR扩增PCV2ORF2基因,将鉴定正确的ORF2基因片段克隆入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,作为标准品模板建立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0.9988,最低检测的拷贝数为1拷贝/25μL,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。[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 Greenl fluorescent quanti- tative 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.

关 键 词：猪圆环病毒2型(PCV2) SYBR Greenl 实时定量PCR PCR检测方法 

分 类 号：S858.28[农业科学—临床兽医学]