摘要
为了研究猪圆环病毒2型（PCV-2）在PK15细胞中的动态增殖，试验根据PCV-2ORF2基因保守区设计特异性引物，经PCR扩增和克隆目的基因制备阳性质粒，建立了基于SYBRGreenI的PCV-2荧光定量PCR方法，并利用该方法检测PCV-2在PK15细胞上的动态增殖情况。结果表明：建立的荧光定量PCR方法在2．0×10^2～2．0×10^9拷贝／IxL内有良好的线性关系，其相关系数为0．9989，扩增效率为99．50％；该方法的最低检测限为20拷贝／止，检测不出猪瘟病毒、猪繁殖与呼吸综合征病毒和猪细小病毒等；PCV-2感染PK15细胞后84小时病毒量达最高，为1．2×10^7拷贝／μL，该时间点为收获病毒的最佳时间。说明所建立的荧光定量PCR方法特异性好，灵敏度高，稳定可靠。
To investigate the dynamic Proliferation of Porcine circovirus type 2 （ PCV - 2 ） in porcine kidney 15 cell line （ PK15 ）, a pair of specific primers was designed based on PCV -2 open reading frame 2 （ORF2）. The target gene was amplified using PCR , and then cloned into pMD19 - T vector and indentified to prepare the positive plasmid. The fluorescent quantitative PCR assay for detection of PCV - 2 was estab- lished based on SYBR Green I , and then was used to detect the dynamic proliferation of PCV-2 in PK15 cell line. The results showed that the standard curve of the assay had a good liner relationship between threshold cycle values and template concentrations of 10 fold serially dilu- ted positive plasmids from 2.0 ~ 102 to 2.0 ~ 109 DNA copies per microliter. Its correlation coefficient was 0. 998 9, and the amplification effi- ciency was 99.50%. The detection limit of the assay was 20 DNA copies per microliter, and the classical swine fever virus, porcine reproduc- tive and respiratory syndrome virus and porcine parvovirus could not be detected. The highest amount of the PCV -2 DNA with 1.2 ×10^7 DNA copies per microliter was detected when the PCV -2 virus had been cultivated in PK15 cell line for 84 hours, which became the best time to harvest the virus. The results indicate that the developed SYBR Green I fluorescent quantitative PCR assay is stable and reliable with good spe- cificity and high sensitivity.
出处
《黑龙江畜牧兽医》
CAS
北大核心
2013年第11期13-17,共5页
Heilongjiang Animal Science And veterinary Medicine
基金
国家高技术研究发展计划项目(2012AA101304)
关键词
猪圆环病毒2型(PCV-2)
荧光定量PCR
PK15细胞
病毒增殖
porcine circovirus type 2 （PCV -2 ）
fluorescent.quantitative PCR assay
porcine kidneyl5 cell line
virus multiplication
