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| 鸡ELAVL1基因SYBR Green Ⅰ实时荧光定量PCR检测方法的建立及应用 |
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彭慧1,2, 詹冬梅1,2, 高梦茹1,2, 陈松彪1,2, 何雷1,2, 陈建1,2, 丁轲1,2,3, 余祖华1,2
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1.河南科技大学 动物科技学院，功能微生物与畜禽健康实验室，河南 洛阳 471003;2.洛阳市活载体生物材料与动物疫病防控重点实验室，河南 洛阳 471023;3.河南科技学院 动物科技学院，河南 新乡 453003
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| 摘要: |
| 【 目的】建立鸡胚胎致死异常视觉蛋白1基因（ELAVL1）的SYBR Green Ⅰ实时荧光定量PCR检测 方法，为研究鸡ELAVL1基因在肿瘤性疾病发生发展中的作用提供技术支持。【 方法】根据鸡ELAVL1基因序列设 计特异性荧光定量PCR引物，以pMD19-T-ELAVL1质粒为模板，优化引物浓度和退火温度，进行实时荧光定量PCR扩增，建立鸡ELAVL1基因SYBR Green Ⅰ实时荧光定量PCR检测方法。以pMD19-T-ELAVL1质粒为标准品，进行实时荧光定量PCR扩增，建立标准曲线，检测该方法的特异性、敏感性和重复性。应用该方法检测鸡ELAVL1基因在不同组织中的转录水平及其在鸡淋巴瘤细胞MSB1、鸡成纤维细胞DT40、鸡肝癌细胞LMH和鸡成纤维细胞DF1中的差异表达情况。【 结果】鸡ELAVL1基因实时荧光定量PCR检测方法最佳引物浓度为0. 2 μmol/L，最佳退火温度为60 ℃；建立的标准曲线循环阈值（Ct）与模板浓度（1. 1×103-1. 1×108拷贝/μL）有良好的线性关系（R2=0.993），扩增效率为109.7%。建立的鸡ELAVL1基因实时荧光定量PCR检测方法具有良好的特异性；对重组质粒的检测下限为1.1×101拷 贝/μL，灵敏度是普通PCR的100倍 ；批内和批间变异系数分别为0.44%-1.92%和0.32%-0.57%，重复性良好。ELAVL1基因在鸡心脏、肝脏、脾脏、肺脏、肾脏、胸腺和法氏囊中均有表达，在脾脏中表达水平 最高，在肺脏中表达水平最低；在鸡淋巴瘤细胞MSB1、鸡成纤维细胞DT40和鸡肝癌细胞LMH中的表达量显著或极 显著高于鸡成纤维细胞DF-1。【 结论】建立了鸡ELAVL1基因 SYBR Green Ⅰ实时荧光定量PCR检测方法，该方法具有良好的特异性、灵敏性和重复性。 |
| 关键词: 鸡 ELAVL1基因 SYBR Green Ⅰ 实时荧光定量PCR |
| DOI：10. 13207/j. jnwafu. 2026. 08. 003 |
| 分类号: |
| 基金项目:国家自然科学基金项目（32472990） |
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| Establishment and application of SYBR Green Ⅰ real⁃time fluorescence quantitative PCR method for detection of chicken ELAVL1 gene |
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PENG Hui1,2, ZHAN Dongmei1,2, GAO Mengru1,2, CHEN Songbiao1,2, HE Lei1,2, CHEN Jian1,2, DING Ke1,2,3, YU Zuhua1,2
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1.Laboratory of Functional Microbiology and Animalhealth，College of Animal Science and Technology，Henan University of Science and Technology，Luoyang，Henan 471003，China;2.Luoyang Key Laboratory of Live Carrier Biomaterial and Animal Disease Prevention and Control，Luoyang，Henan 471023，China;3.College of Animal Science and Technology，Henan Institute of Science and Technology，Xinxiang，Henan 453003，China
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| Abstract: |
| 【 Objective】The study aims to establish a SYBR Green Ⅰ real-time quantitative PCR detection method for the detection of chicken ELAVL1 gene，thereby providing technical support for studying the role of ELAVL1 gene in the occurrence and development of tumor diseases in chickens.【Method】Specific primers for real-time quantitative PCR were designed based on the chicken ELAVL1 gene sequence. Using the pMD19-T-ELAVL1 plasmid as a template，the primer concentration and annealing temperature were optimized，and fluo?rescence quantitative PCR amplification was performed to establish a SYBR GreenⅠRT-qPCR method for detectioning chicken ELAVL1 gene. qPCR amplification was performed using the pMD19-T-ELAVL1 plasmid as the standard sample，and the standard curve was established to test the specificity，sensitivity and repeatability of the method. The method was used to measure the transcription level of ELAVL1 gene in different tissues and its differential expression in MSB1 lymphoma cells，DT40 fibroblasts，LMH hepatocellular carcinoma cells and DF1 fibroblasts.【Result】The optimal concentration of primers was 0. 2 μmol/L and the optimal annealing tem?perature was 60 ℃ . The established standard curve showed a good linear relationship between the fluorescence cycle threshold （Ct） and the template concentration in the range of 1. 1×103-1. 1×108 copy/μL （R2=0.993），with an amplification efficiency of 109.7%. The developed ELAVL1 detection method exhibited high specificity. The lower limit of detection for recombinant plasmid was 1. 1×101 copy/μL，which was 100-fold more sensitive than conventional PCR. The intra-and inter-assay coefficients of variation ranged from 0.44% to 1.92% and from 0.32% to 0.57%，respectively，demonstrating excellent repeatability. The ELAVL1 gene was ex-pressed in the heart，liver，spleen，lung，kidney，thymus and bursa of chicken，with the highest expression level in the spleen and the lowest in the lung. The expression levels of ELAVL1 were significantly or extremely signifi-cantly higher in MSB1 lymphoma cells，DT40 fibroblasts and LMH hepatocellular carcinoma cells compared to chicken DF-1 fibroblasts.【Conclusion】A SYBR Green Ⅰ real-time quantitative PCR method was established for chicken ELAVL1 gene，exhibiting high specificity，sensitivity and reproducibility. |
| Key words: chicken ELAVL1 gene SYBR Green Ⅰ real-time fluorescence quantitative PCR |
