[1]马玉媛,郭逸,颜奇坡,等.猪IL-2、IL-4、IL-10和IFN-γ的TaqMan实时定量RT-PCR检测体系的建立[J].中国预防兽医学报,2011,(04):285-288.
 MA Yu-yuan#,GUO Yi#,YAN Qi-po,et al.Development of a TaqMan real-time RT-PCR assay for detecting porcine IL-2, IL-4, IL-10, and IFN gamma[J].Chinese journal of preventive veterinary medicine,2011,(04):285-288.
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猪IL-2、IL-4、IL-10和IFN-γ的TaqMan实时定量RT-PCR检测体系的建立
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《中国预防兽医学报》[ISSN:1008-0589/CN:23-1417/S]

卷:
期数:
2011年04期
页码:
285-288
栏目:
诊断和检测技术
出版日期:
2011-04-15

文章信息/Info

Title:
Development of a TaqMan real-time RT-PCR assay for detecting porcine IL-2, IL-4, IL-10, and IFN gamma
作者:
马玉媛;郭逸;颜奇坡;叶小丽;张启模;郑霖;田克恭;章金刚;
军事医学科学院野战输血研究所/国家生物医学分析中心病毒安全检测实验室;中国动物疫病预防控制中心;
Author(s):
MA Yu-yuan1# GUO Yi1# YAN Qi-po1 YE Xiao-li1 ZHANG Qi-mo1 ZHENG Lin1 TIAN Ke-gong2 ZHANG Jin-gang1*
1. Laboratory for Viral Safety of National Centre of Biomedical Analysis, Institute of Transfusion Medicine, The Academy of Military Medical Sciences, Beijing 100850, China; 2. China Animal Disease Control Center, Beijing 100094, China
关键词:
细胞因子实时定量RT-PCR细胞免疫
Keywords:
cytokines real-time RT-PCR cellular immunity
摘要:
为了解猪体内细胞免疫应答机制,本研究建立猪细胞因子IL-2、IL-4、IL-10和IFN-γ的TaqMan实时定量RT-PCR检测体系。以五指山小型猪cDNA为模板,扩增IL-2、IL-4、IL-10和IFN-γ的基因保守区,构建相应的重组质粒,并将其作为标准品构建标准曲线。结果显示,各标准曲线的相关系数r值均大于0.990,扩增效率为90%~105%;该检测方法的敏感性较高,IL-4检测下限为1copies/μL,IL-2、IL-10及IFN-γ检测下限达为102copies/μL;重复性较好,批内重复试验与批间重复试验变异系数均小于5%。本研究为病毒感染后猪体内免疫应答等研究提供方法。
Abstract:
To develope a TaqMan real-time RT-PCR assay for detecting porcine IL-2, IL-4, IL-10, and IFN-γ, the conserved regions of porcine IL-2, IL-4, IL-10, and IFN-γ genes were amplified with Wuzhishan miniature pig cDNA as the template and cloned into pMD18-T to construct the standard recombinant plasmids for establishing calibration curve, respectively. The results showed that the real-time RT-PCR assay was sensitivity and repeatability. The detection limit of IL-4 mRNA was 1 copies/μL, while the detction limit of IL-2, IL-10, and IFN-γ was 102 copies/μL, respectively. The coefficient of variation for both intra-assay and inter-assay was lower than 5%. The evaluation results further demonstrated that the established real-time RT-PCR detection was reliable. This method could be used to evaluate the immune response in pigs

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备注/Memo

备注/Memo:
基金:国家科技支撑计划资助项目(2007BAD86B01-3)
更新日期/Last Update: 2011-09-29