| Hcmv-miR-UL112荧光定量RT-PCR检测方法的建立 |
| 投稿时间：2015-08-31 修订日期：2015-10-12 点此下载全文 |
| 引用本文：陈琼娜,沈凯,黄燕燕,查瑶,方建玮,续力云,周吉航,陈芬,刘晓光.Hcmv-miR-UL112荧光定量RT-PCR检测方法的建立[J].医学研究杂志,2016,45(4):53-56 |
| DOI：
10.11969/j.issn.1673-548X.2016.04.015 |
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| 基金项目:浙江省自然科学基金资助项目(LY13H020001,LQ13H10001);浙江省公益性技术应用研究计划(2012C37036);舟山市科技计划项目(2011C32004,2012C13024) |
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| 中文摘要:目的 建立一种能快速准确检测hcmv-miR-UL112的方法。方法 根据miRBase网站中公布的hcmv-miR-UL112基因序列,通过设计引物、探针及优化反应条件,建立了一种实时荧光定量PCR方法对hcmv-miR-UL112进行定量检测,并进行敏感度、重复性和特异性实验,同时对PCR产物进行测序验证。结果 获得了1条特异性茎环反转录引物,一条特异性PCR正向引物和通用反向引物。PCR最佳反应条件为95℃ 10min,53℃ 2min;95℃ 15s,57℃ 40s,共40个循环。该方法的批内变异系数CV和批间CV均<5%,相关系数R2均>0.99,扩增效率均>90.0%。结论 成功建立了一种敏感度高、特异性强、重复性好并且简便快速的hcmv-miR-UL112荧光定量qRT-PCR检测方法。 |
| 中文关键词:hcmv-miR-UL112 microRNA RT-PCR 荧光定量 |
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| Development of a Fluorescent Quantitative RT-PCR assay for Hcmv-miR-UL112 |
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| Abstract:Objective To establish a rapid and accurate method for the detection of hcmv-miR-UL112. Methods The primers and optimum reaction condition were designed based on the gene sequences published in miRBase. Real-time RT-PCR was established and optimized for the detection of hcmv-miR-UL112 and carried on the sensitivity, the duplication and the specificity experiments. PCR products are validated by gene sequencing. Results The results acquired a specific stem-loop reverse transcription primer, a specific PCR forward primer and a general reverse primer. The optimum reaction condition of hcmv-miR-UL112 TaqMan fluorescence quantitative PCR is 95℃ 10min, 53℃ 2min; 40 circulations:95℃ 15s, 57℃ 40s. Both the variance inter-batch CV and the variance intra-batch CV of this method was below 5%. The correlation of association R2 reaches was as high as 0.99, and the amplification efficiency was greater than 90.0%. Conclusion We successfully established a method of hcmv-miR-UL112 fluorescence quantitative RT-PCR detection which is highly sensitive, strongly specific, well reproducible, simple and quick. |
| keywords:Hcmv-miR-UL112 miRNA RT-RCR Fluorescent quantitative |
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