鸭源NF-κB1基因荧光定量PCR方法建立及其初步应用

doi:10.3969/j.issn.1004-1524.2021.02.04

浙江农业学报 ›› 2021, Vol. 33 ›› Issue (2): 215-222.DOI: 10.3969/j.issn.1004-1524.2021.02.04

鸭源NF-κB1基因荧光定量PCR方法建立及其初步应用

张飘¹(), 贺欣薇¹, 杨霞¹, 曾茂芹¹, 刘妍罕¹, 张扬子², 杨颖¹^,³, 温贵兰¹^,³, 程振涛¹^,³, 文明¹^,³^,^*()

1.贵州大学动物科学学院,贵州贵阳 550025
2.贵州省畜牧兽医研究所,贵州贵阳 550025
3.贵州省动物生物制品工程技术研究中心,贵州贵阳 550025

收稿日期:2020-07-06 出版日期:2021-02-25 发布日期:2021-02-25
通讯作者: 文明
作者简介:*文明,E-mail: as.mwen@gzu.edu.cn
张飘(1995—),男,贵州遵义人,硕士研究生,主要从事动物疾病防控研究。E-mail: 18586723070@163.com
基金资助:
国家自然科学基金(31260607);国家自然科学基金(31560703);贵州省百层次创新型人才项目(黔科合人才〔2016〕4009号);贵州省科技平台及人才团队计划(黔科合平台人才〔2018〕5253号)

Establishment and preliminary application of fluorescence quantitative PCR method for duck NF-κB1 gene

ZHANG Piao¹(), HE Xinwei¹, YANG Xia¹, ZENG Maoqin¹, LIU Yanhan¹, ZHANG Yangzi², YANG Ying¹^,³, WEN Guilan¹^,³, CHENG Zhentao¹^,³, WEN Ming¹^,³^,^*()

1. College of Animal Science, Guizhou University, Guiyang 550025, China
2. Guizhou Institute of Animal Husbandry and Veterinary Medicince, Guiyang 550025, China
3. Research Center of Engineering Technology for Animal Biological Products of Guizhou, Guiyang 550025, China

Received:2020-07-06 Online:2021-02-25 Published:2021-02-25
Contact: WEN Ming

摘要/Abstract

摘要：

为建立鸭NF-κB1(nuclear factor-kappa B, NF-κB)基因的荧光定量PCR检测方法,并以建立的方法检测鸭胚成纤维细胞(duck embryo fibroblasts , DEF)在感染鸭肠炎病毒(DEV)后NF-κB1基因随时间变化转录表达量的变化情况。根据GenBank 上NF-κB1基因保守序列设计特异性引物,以鸭胚成纤维细胞 mRNA提取样本反转录为cDNA,进行NF-κB1基因克隆质粒构建,以此为模板建立鸭NF-κB1基因荧光定量PCR检测方法并进行特异性、重复性和敏感性试验,并利用建立的方法DEF感染DEV后NF-κB1基因随时间变化的转录变化进行检测。结果显示:建立的鸭NF-κB1基因荧光定量PCR方法标准曲线呈现典型的S型,方程为y=-3.12x+44.086(R²=1),扩增效率为109.2%;其熔解温度Tm值为(83.5±0)℃,曲线呈特异性单峰,批内变异系数小于0.3%,批间变异系数小于0.2%;检测灵敏度可达到1.79个拷贝。DEF细胞在感染DEV后NF-κB1基因随时间改变转录水平变化无规律,但整体表达水平高于正常细胞,差异显著(P<0.05),36~84 h NF-κB1基因的转录水平与正常细胞中差异极显著(P<0.01)。本研究成功建立了鸭NF-κB1基因荧光定量PCR检测方法,并对DEF细胞感染DEV后NF-κB1基因的转录水平进行了研究,为后续实验研究提供了技术和数据支撑。

关键词: 鸭, NF-kB1基因, 荧光定量PCR, 建立, 应用

Abstract:

In order to establish a fluorescent quantitative PCR method for the detection of nuclear factor-kappa B (NF-κB) gene in ducks, the expression of NF-κB 1 gene in duck embryo fibroblasts (DEF) was detected with the method established in this study. Based on the conserved sequence of NF-κB1 gene in GenBank, a specific primer was designed, and the reverse transcription of NF-κB 1 mRNA from duck embryo fibroblast was used to construct the NF-κB 1 gene cloning plasmid, the qPCR assay was used to detect NF-κB1 gene in ducks, and the specific, repeatable and sensitive tests were carried out. The transcription changes of NF-κB1 gene with time after DEV infection were detected by the established method. The results showed that the standard curve of NF-κB1 gene was typical s-type, the equation was y=-3.12x+44.086(R²=1), the amplification efficiency was 109.2%, the Tm value of the fusion temperature was (83.5±0) ℃, and the curve showed specific single peak, the intra- and inter-assay coefficients of variation were less than 0.3% and 0.2%, respectively, and the detection sensitivity was 1.79 copies. The transcription level of NF-κB 1 gene in DEF cells was not regular with time after being infected with DEV, but the expression level of NF-κB 1 gene in DEF cells was higher than that in normal cells (P<0.05), 36-84 h NF-κB1 gene transcription level was significantly different from that in normal cells (P<0.01). In this study, a fluorescent quantitative PCR method for the detection of duck NF-κB1 gene was successfully established, and the transcription level of NF-κB1 gene in DEV infected DEF cells was studied.

Key words: duck, NF-κB1 gene, fluorescence quantitative PCR, establishment, application

中图分类号:

S855.3

张飘, 贺欣薇, 杨霞, 曾茂芹, 刘妍罕, 张扬子, 杨颖, 温贵兰, 程振涛, 文明. 鸭源NF-κB1基因荧光定量PCR方法建立及其初步应用[J]. 浙江农业学报, 2021, 33(2): 215-222.

ZHANG Piao, HE Xinwei, YANG Xia, ZENG Maoqin, LIU Yanhan, ZHANG Yangzi, YANG Ying, WEN Guilan, CHENG Zhentao, WEN Ming. Establishment and preliminary application of fluorescence quantitative PCR method for duck NF-κB1 gene[J]. Acta Agriculturae Zhejiangensis, 2021, 33(2): 215-222.

图/表 7

图1 NF-κB1基因PCR扩增结果 M,DNA分子质量标准DL2000;1~12,NF-κB1基因;-,阴性对照。

Fig.1 The PCR amplification results of NF-κB1 gene M, DNA marker DL2000;1~12, NF-κB 1gene;-, Negative control.

图2 NF-κB1基因重组质粒PCR鉴定结果 M,DNA分子质量标准DL2000;1~12,NF-κB1基因重组质粒;-,阴性对照。

Fig.2 Identification of NF-κB1 gene recombinant plasmid by PCR M, DNA marker DL2000;1-12, Recombinant plasmid of NF-κB1 gene;-, Negative control.

图3 NF-κB1基因标准曲线建立

Fig.3 Establishment of NF-κB1 gene standard curve

图4 NF-κB1基因标准曲线特异性(A、B)和敏感性检测(C、D) C:1~10,1.79×109~1.79×100 copies·mL-1。D:M,DNA marker DL2000;1~7,1.79×108~1.79×102 copies·mL-1;8,阴性对照。

Fig.4 NF-κB1 gene standard curve specificity (A, B) and sensitivity detection (C,D) C:1-10, 1.79×109-1.79×100 copies·mL-1. D: M, DNA Marker DL2000; 1-7, 1.79×108-1.79×102 copies·mL-1; 8, Negative control.

表1 重复性检验

Table 1 Repeatability test

模板浓度 Concentration of template/ ( copies·mL^-1)	批内试验(Ct值) Intra-assay variability(Ct)			批间试验(Ct值) Inter-assay variability(Ct)
模板浓度 Concentration of template/ ( copies·mL^-1)	标准偏差s	平均值 $x ?$	CV/%	标准偏差s	平均值 $x ?$	CV/%
1.79×10⁹	0.09	15.10	0.06	0.22	15.28	0.17
	0.29	15.53	0.20
	0.07	15.21	0.04
1.79×10⁸	0.20	18.33	0.15	0.14	18.46	0.10
	0.38	18.61	0.28
	0.09	18.43	0.07
1.79×10⁷	0.26	21.69	0.19	0.21	21.55	0.16
	0.19	21.31	0.14
	0.20	21.64	0.15

表1 重复性检验

Table 1 Repeatability test

模板浓度 Concentration of template/ ( copies·mL^-1)	批内试验(Ct值) Intra-assay variability(Ct)			批间试验(Ct值) Inter-assay variability(Ct)
模板浓度 Concentration of template/ ( copies·mL^-1)	标准偏差s	平均值 $x ?$	CV/%	标准偏差s	平均值 $x ?$	CV/%
1.79×10⁹	0.09	15.10	0.06	0.22	15.28	0.17
	0.29	15.53	0.20
	0.07	15.21	0.04
1.79×10⁸	0.20	18.33	0.15	0.14	18.46	0.10
	0.38	18.61	0.28
	0.09	18.43	0.07
1.79×10⁷	0.26	21.69	0.19	0.21	21.55	0.16
	0.19	21.31	0.14
	0.20	21.64	0.15

表2 NF-κB1基因在感染DEV前后在DEF细胞中转录水平

Table 2 Repeated field value NF-κB1 gene transcription level in DEF cells before and after infection with DEV

处理 Treatment	时间Time/h
处理 Treatment	12	24	36	48	60	72	84	96	108	120
正常 Control group	25.33 ±0.15 Aa	27.90 ±0.19 Aa	23.78 ±0.20 Cc	28.68 ±0.31 Aa	26.20 ±0.50 Aa	25.36 ±0.22 Aa	25.25 ±0.12 Bb	25.27 ±0.08 Aa	24.71 ±0.29 Aa	28.62 ±0.16 Bb
感染 Infected group	25.67 ±0.55 Aa	25.39 ±0.23 Aa	24.92 ±0.24 Bb	24.77 ±0.28 Dc	22.69 ±0.20 Cc	23.70 ±0.17 Bb	22.73 ±0.27 Aa	25.04 ±0.22 Aa	27.23 ±1.14 Ca	24.93 ±0.27 Aa

图5 感染DEV后 DEF细胞NF-κB1基因转录水平图谱

Fig.5 Level map of NF-κB1 gene expression in DEF cells infected with DEV

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鸭源NF-κB1基因荧光定量PCR方法建立及其初步应用

Establishment and preliminary application of fluorescence quantitative PCR method for duck NF-κB1 gene

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