猪血清淀粉样蛋白3分子SYBR Green Ⅰ实时荧光定量PCR检测方法的建立及初步应用  

DEVELOPMENT AND PRELIMINARY APPLICATION OF SYBR GREEN Ⅰ REAL-TIME PCR ASSAY FOR DETECTION AND QUANTITATION OF PORCINE SERUM AMYLOID PROTEIN 3(SAA3)

作　　者：郇贝丽 刘珂[1] 马改妮[1] 魏建超[1] 邵东华[1] 李蓓蓓[1] 邱亚峰[1] 石元元[1] 马志永[1] HUAN Bei-li LIU Ke MA Gai-ni WEI Jian-chao SHAO Dong-hua LI Bei-bei QIU Ya-feng SHI Yuan-yuan MA Zhi-yong(Shanghai Veterinary Research Institute, CAAS, Shanghai 200241, China)

机构地区：[1]中国农业科学院上海兽医研究所,上海200241

出　　处：《中国动物传染病学报》2017年第3期80-86,共7页Chinese Journal of Animal Infectious Diseases

基　　金：973计划(2014CB542702)

摘　　要：分别根据猪血清淀粉样蛋白3(serum amyloid A3,SAA3)基因序列以及猪三磷酸甘油醛脱氢酶(glyceraldehyde-3-phosphate dehydrogenase,GAPDH)基因全长序列设计特异性扩增引物,进行PCR扩增,并将PCR扩增片段连接至相应载体上构建重组质粒。两个重组质粒经测序鉴定和纯化后,倍比稀释作为标准曲线样品,用于实时荧光定量PCR中SAA3、GAPDH标准曲线的制备,并进行反应的灵敏性、特异性和重复性检测。结果显示,标准曲线线性关系R2均在0.98以上;特异性检测显示该引物可以特异性检测到猪SAA3、GAPDH扩增曲线;组内和组间变异系数均小于5%,说明本研究成功建立猪SAA3的荧光定量PCR检测方法。运用建立的荧光定量RT-PCR对正常以及感染猪繁殖与呼吸综合征病毒的肺泡巨噬细胞和猪组织进行检测,可检测到猪SAA3的表达,正常组与接毒组之间显示出明显的表达差异。本研究初步建立了检测猪SAA3基因的SYBR Green荧光定量RT-PCR的方法,为后续对猪传染性疾病与猪SAA3之间相互关系的研究提供了一种特异、灵敏的检测方法。The objective of the present study was to develop a SYBR Green I Real-time PCR assay for detecting and quantifying porcine serum amyloid protein 3 （SAA3）. According to the SAA3 （NM_001044552.1） and GAPDH （AF017079.1） gene sequences, specific primes were designed to their amplification. The resulting fragments were cloned into the vector to construct the recombinant plasmids. Using the recombinant plasmids as standard products, a Real-time quantitative RT-PCR was performed to construct the standard curves of porcine SAA and GAPDH and detect the sensitivity, specificity and repeatability. The results showed a precise linear relationship with a correlation coefficient of R2 〉 0.98. The amplification curves showing a single peak was detected for porcine SAA3 and GAPDH. The coefficient of variation was less than 0.5% within and between the assays. The SYBR Green I Real time PCR assay was used to detect porcine tissues and PAMs treated with PRRSV（Porcine reproductive and respiratory syndrome virus）. Porcine SAA3 was found in these samples and there was significant difference in SAA3 content between infection group and control group. The SYBR Green l Real-time PCR assay developed here was highly specific, sensitive, and reproducible and might be a tool for monitoring the relationship between pig infectious diseases and porcine SAA3.

关 键 词：猪血清淀粉样蛋白3 SYBR Green Ⅰ 实时荧光定量PCR 

分 类 号：S852.42[农业科学—基础兽医学]