鸽白细胞介素8基因实时荧光定量PCR检测方法的建立

doi:10.16431/j.cnki.1671-7236.2016.10.006

《中国畜牧兽医》 ›› 2016, Vol. 43 ›› Issue (10): 2547-2552.doi: 10.16431/j.cnki.1671-7236.2016.10.006

鸽白细胞介素8基因实时荧光定量PCR检测方法的建立

潘超, 韦天超, 农海连, 李秀凤, 蒋桂林, 苑亚东, 程二财, 磨美兰, 韦平

广西大学动物科学技术学院, 南宁 530004

收稿日期:2016-04-15 出版日期:2016-10-20 发布日期:2016-10-28
通讯作者: 韦天超 E-mail:tcwei88@126.com
作者简介:潘超(1989-),男,湖北武汉人,硕士生,研究方向:禽病防治与病原分子生物学,E-mail:1073477455@qq.com
基金资助:
国家自然科学基金项目（31360612）；广西自然科学基金项目（2013GXNSFAA019080）

Development of a Quantitative Real-time PCR Method for Detection of Pigeon IL-8 Gene

PAN Chao, WEI Tian-chao, NONG Hai-lian, LI Xiu-feng, JIANG Gui-lin, YUAN Ya-dong, CHENG Er-cai, MO Mei-lan, WEI Ping

College of Animal Science and Technology, Guangxi University, Nanning 530004, China

Received:2016-04-15 Online:2016-10-20 Published:2016-10-28

摘要/Abstract

摘要：

为建立鸽白细胞介素8（interleukin-8，IL-8）基因实时荧光定量PCR检测方法，本研究根据GenBank上公布的鸽IL-8基因序列，在保守区域设计1对特异性引物，以鸽子淋巴细胞提取的核酸为模板扩增鸽IL-8基因部分片段并克隆到pMD18-T载体上。提取重组质粒通过系列制备标准品，建立鸽IL-8基因SYBR GreenⅠ染料法实时荧光定量PCR标准曲线，并进行特异性、敏感性和重复性试验。结果显示，实时荧光定量PCR熔解曲线呈单一熔解峰，试验线性相关系数为1.0，IL-8基因的扩增效率为103%。敏感性结果显示最低可检测到16个拷贝数；用该方法检测其他鸽白细胞介素细胞因子（IL-1β、IL-6、IL-18）和双蒸水的结果均为阴性。批间、批内变异系数均≤1.52%。因此，本研究建立的实时荧光定量PCR检测方法可用于鸽IL-8 mRNA的检测，为病毒感染宿主细胞后细胞因子表达的定量分析奠定基础。

关键词: 鸽; 白细胞介素8; 实时荧光定量PCR

Abstract:

To develop a quantitative Real-time PCR method for detection of pigeon interleukin-8 (IL-8),a pair of specific primers was designed based on the conserved region of IL-8 gene sequence published on GenBank.A fragment of IL-8 gene was amplified from the pigeon lymphocytes template and cloned into pMD18-T vector.Plasmid DNA was extracted from the bacteria and was serially diluted to serve as a standard.A standard curve for the SYBR Green Ⅰ of quantitative Real-time PCR was established and the specificity,sensitivity and reproducibility of this assay were investigated.The results showed that the quantitative Real-time PCR melting curve only had one single melting peak.The assay was linear with R² values was 1.0;The reaction efficiency for the pigeon IL-8 gene was 103%.The detection limit of this assay was 16 copies per reaction.Other interleukin including IL-1β,IL-6 and IL-18 and double distilled water control were tested by this assay and the results were all negative.The CV values of intra- and inter-assay were less than 1.52%.The established quantitative Real-time PCR assay of this study was suitable for the detection of pigeon IL-8.It would provide basis for analyzing cytokine expression quantitatively after the host cell was infected by the virus.

Key words: pigeon; interleukin-8; quantitative Real-time PCR

中图分类号:

S854.4

潘超, 韦天超, 农海连, 李秀凤, 蒋桂林, 苑亚东, 程二财, 磨美兰, 韦平. 鸽白细胞介素8基因实时荧光定量PCR检测方法的建立[J]. 《中国畜牧兽医》, 2016, 43(10): 2547-2552.

PAN Chao, WEI Tian-chao, NONG Hai-lian, LI Xiu-feng, JIANG Gui-lin, YUAN Ya-dong, CHENG Er-cai, MO Mei-lan, WEI Ping. Development of a Quantitative Real-time PCR Method for Detection of Pigeon IL-8 Gene[J]. , 2016, 43(10): 2547-2552.

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鸽白细胞介素8基因实时荧光定量PCR检测方法的建立

Development of a Quantitative Real-time PCR Method for Detection of Pigeon IL-8 Gene

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