树鼩感染相关细胞因子荧光定量PCR检测方法的建立
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摘要
树鼩是一种新型的优质实验动物模型。本研究针对树鼩感染相关的细胞因子白介素6(IL-6)、IL-8、IL-10、IL-17A、γ-干扰素(IFN-γ)及管家基因磷酸甘油醛脱氢酶(GAPDH)建立荧光定量聚合酶链式反应检测方法。研究证明所建立方法具有良好的特异性。线性范围高点可达到1×10~(10) copies,低点则至10 copies(IL-6、IL-17A)、100 copies(IL-10、GAPDH)和1 000 copies(IL-8、IFN-γ)不等,在此区间各试剂线性相关系数R2均大于0.99。IL-6、IL-8、IL-10、IL-17A、IFN-γ和GAPDH最低检测值分别在8、8、4、8、128、4 copies。研究表明所建立的检测方法具有很好的特异性、灵敏度及较广的线性范围,适用于多重浓度范围的样品检测,可用于后续树鼩细胞因子的研究工作。
Tree shrew is a novel and high-quality experimental animal model. In this study, the real-time polymerase chain reaction methods were established to detect infection-related cytokines interleukin-6(IL-6), IL-8, IL-10,IL-17 A, interferon-γ(IFN-γ) and housekeeping gene glyceraldehyde-phosphate dehydrogenase(GAPDH) of tree shrew.The results indicated that the establised methods had good specificity. The high point of the linear range of these reagents reached 1 × 10~(10) copies, and the low points ranged from 10 copies(IL-6, IL-17 A), 100 copies(IL-10, GAPDH) to 1 000 copies(IL-8, IFN-γ). In this interval, the linear correlation coefficient R2 of each reagent was greater than 0.99. The lowest detectable values of IL-6, IL-8, IL-10, IL-17 A, IFN-γ and GAPDH were 8, 8, 4, 8, 128 and 4 copies, respectively. The results showed that the established detection methods had good specificity, sensitivity and wide linear range. The methods were suitable for detection of multiple concentration range samples, and could be used for the subsequent studies of tree shrew cytokines.
Tree shrew is a novel and high-quality experimental animal model. In this study, the real-time polymerase chain reaction methods were established to detect infection-related cytokines interleukin-6(IL-6), IL-8, IL-10,IL-17 A, interferon-γ(IFN-γ) and housekeeping gene glyceraldehyde-phosphate dehydrogenase(GAPDH) of tree shrew.The results indicated that the establised methods had good specificity. The high point of the linear range of these reagents reached 1 × 10~(10) copies, and the low points ranged from 10 copies(IL-6, IL-17 A), 100 copies(IL-10, GAPDH) to 1 000 copies(IL-8, IFN-γ). In this interval, the linear correlation coefficient R2 of each reagent was greater than 0.99. The lowest detectable values of IL-6, IL-8, IL-10, IL-17 A, IFN-γ and GAPDH were 8, 8, 4, 8, 128 and 4 copies, respectively. The results showed that the established detection methods had good specificity, sensitivity and wide linear range. The methods were suitable for detection of multiple concentration range samples, and could be used for the subsequent studies of tree shrew cytokines.
引文
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8 Yang Z F, Mok C K, Liu X Q, et al. Clinical, virological and immunological features from patients infected with re-emergent avian-origin human H7N9 influenza disease of varying severity in Guangdong province.PLoS One,2015,10(2):e0117846.
9 Chen Weiwei,Nie Weimin,Xu Wen,et al. Cross-sectional study of the relationship of peripheral blood cell profiles with severity of infection by adenovirus type 55.BMC Infect Dis,2014,14:147.
10 Fan Yu,Huang Zhiyong,Cao Changchang,et al. Genome of the Chinese tree shrew.Nat Commun,2013,4:1426.
2徐新平,陈红波,贲昆龙.树鞠在医学生物学中的应用.中国实验动物学报,2005,13(3):187-190.
3 Cao J, Yang E B, Su J J, et al. The tree shrews:adjuncts andalternatives to primates as models for biomedical research.J Med Primatol,2003,32(3):123-130.
4 Panda S, Kumari L, Panda S. Structural analysis of CD59 of Chinese tree shrew:A new reference molecule for human immune system specific CD59 drug discovery. Current Drug Discovery Technologies,2018,15(4):326-334.
5徐林,张云,梁斌,等.实验动物树豹和人类疾病的树韵模型研究概述.动物学研究,2013,34(2):59-69.
6 Xiao J,Liu R,Chen C S.Tree shrew(Tupaia belangeri)as a novel laboratory disease animal model.Zoological Research,2017,38(3):127-137.
7 Tsukiyama-Kohara K, Kohara M. Tupaia belangeri as an experimental animal model for viral infection.Exp Anim,2014,63(4):367-374.
8 Yang Z F, Mok C K, Liu X Q, et al. Clinical, virological and immunological features from patients infected with re-emergent avian-origin human H7N9 influenza disease of varying severity in Guangdong province.PLoS One,2015,10(2):e0117846.
9 Chen Weiwei,Nie Weimin,Xu Wen,et al. Cross-sectional study of the relationship of peripheral blood cell profiles with severity of infection by adenovirus type 55.BMC Infect Dis,2014,14:147.
10 Fan Yu,Huang Zhiyong,Cao Changchang,et al. Genome of the Chinese tree shrew.Nat Commun,2013,4:1426.