Development of SYBR-Green Ⅰ real-time quantitative PCR assay for detection of pseudorabies virus | 猪伪狂犬病毒SYBR-GreenⅠ实时定量PCR检测方法的建立
2009
Li Mingfeng, Henan Agricultural University, Zhengzhou(China), College of Animal Husbandry and Veterinary Science | Wei Zhanyong, Henan Agricultural University, Zhengzhou(China), College of Animal Husbandry and Veterinary Science | Guo Xianpo, Henan Agricultural University, Zhengzhou(China), College of Animal Husbandry and Veterinary Science
Chino. 利用PCR技术扩增出猪伪狂犬病毒gH基因中187 bp的片段,并克隆到pGEMT载体上,纯化的质粒作为PCR检测的标准模板,以10倍梯度稀释质粒为标准模板,进行SYBR GreenⅠ荧光定量PCR扩增并制作标准曲线,建立了猪伪狂犬病毒的荧光定量PCR检测方法.该方法检测灵敏度可达1.26×102拷贝.μL-1,与猪圆环病毒,猪细小病毒,猪繁殖与呼吸综合征病毒,猪瘟病毒不发生交叉反应,具有良好的特异性和重复性;对15份疑似病料进行了检测,发现均为荧光定量PCR阳性,而常规PCR只能检测出6份阳性.结果表明,建立的实时荧光定量PCR具有特异、敏感、快速、定量、重复性好等优点,可用于临床PRV感染的检测.[著者文摘]
Mostrar más [+] Menos [-]Inglés. Using PCR assay, a 187 bp region of the Pseudorabies virus gH gene was cloned to pGEM T vector, and the constructed plasmid was named pGEM - gH. Serial dilutions of plasmid pGEM - gH were used to quantify the virus genomic copy number, and served as standards curve. We developed a SYBR Green I real-time PCR to detect the pseudorabies virus. Pseudorabies virus sensitivity analysis showed that the developed SYBR Green I real-time PCR could detect 1.26 × 10. template ・ μL. 1. The assay exhibited specificity as all negative controls and other porcine pathogens, such as PRRSV, PPV, PCV, CSFV showed negative results. It is shown the real-time PCR results of 15 suspicious positive samples are positive. However, it was 6 positive by normal PCR. The results suggested that as a result of the sensitivity and specificity of the assay with a rapid and simple procedure, the real-time PCR would be useful for the clinical diagnosis of pseudorabies virus infection.
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Este registro bibliográfico ha sido proporcionado por Institute of Agricultural Information, Chinese Academy of Agricultural Sciences