摘要
为了解CD44分子在雏鸡肠组织内的表达及其时空表达特性,设计1对引物,建立RT-PCR方法对CD44mRNA进行鉴定;以GAPDH基因为内参,建立检测鸡CD44基因表达水平的SYBR GreenⅠ实时荧光定量(Q-PCR)方法,对1～28日龄商品鸡肠组织不同部位中CD44mRNA表达水平进行检测。结果显示,从鸡肠组织中成功鉴定出CD44mRNA,建立的荧光定量方法能特异性检测到CD44mRNA,CD44和GAPDH基因的扩增效率分别为95%和101%,线性范围均在108～104拷贝/μL,敏感度高,最低检测限分别为45和77个拷贝。建立的CD44SYBR GreenⅠ荧光定量PCR检测方法具有特异、灵敏、快速、重复性好等优点。雏鸡不同日龄,不同肠组织中CD44表达水平有所差异,5～20日龄时的空肠组织和5～10日龄时的直肠组织中CD44表达量较高。
To understand the temporal and spatial expression characteristics of CD44 in chicken intestine,a pair of primers was designed for proving the presence of CD44 in chicken intestine.Another pair of primers for Real-time PCR was designed to establish a rapid and specific SYBR Green I Real-time PCR assay,CD44 expression levels in intestinal tissue of 1-28 day-old chickens were detected,the specificity,sensitivity and repeatability of the assay were tested.The results showed that CD44 mRNA was successfully identified in chicken intestine;the real-time PCR assay was highly specific with 95%（CD44） and 101%（GAPDH） amplification efficiency.The assay was highly sensitive,and detection thresholds were respectively 45（CD44） copies and 77（GAPDH） copies of plasmid DNA.The established SYBR Green I real-time PCR for detecting the expression level of CD44 in chickens is rapid,specific and sensitive.The expression levels of CD44 varied with different day-old chicken intestinal tissue,higher in jejunum of 5 to 20 day-old chicken and rectum of 5 to 10 day-old chicken than others.
出处
《中国兽医学报》
CAS
CSCD
北大核心
2013年第1期53-58,63,共7页
Chinese Journal of Veterinary Science
基金
国家自然科学基金资助项目(30972187)
