The objective of the present study was to develop a SYBR GreenⅠ 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 amplifi cation. 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, specifi city and repeatability. The results showed a precise linear relationship with a correlation coeffi cient of R2 > 0.98. The amplifi cation curves showing a single peak was detected for porcine SAA3 and GAPDH. The coeffi cient of variation was less than 0.5％ within and between the assays. The SYBR GreenⅠ 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 signifi cant difference in SAA3 content between infection group and control group. The SYBR GreenⅠ Real-time PCR assay developed here was highly specifi c, sensitive, and reproducible and might be a tool for monitoring the relationship between pig infectious diseases and porcine SAA3.%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.
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