• 中国科学论文统计源期刊
  • 中国科技核心期刊
  • 美国化学文摘(CA)来源期刊
  • 日本科学技术振兴机构数据库(JST)

临床输血与检验 ›› 2016, Vol. 18 ›› Issue (3): 258-261.DOI: 10.3969/j.issn.1671-2587.2016.03.019

• 实验技术与方法 • 上一篇    下一篇

新型BCA中基于实时荧光定量PCR的条形码DNA定量方法的建立*

姬长甫, 尹惠琼, 贾俊婷, 王蕊, 朱凤宣, 杨姝, 章金刚   

  1. 100850 北京,军事医学科学院野战输血研究所
  • 收稿日期:2016-01-15 出版日期:2016-06-20 发布日期:2016-09-21
  • 通讯作者: 尹惠琼(E-mail) yinyin1288@sina.com;章金刚(E-mail) zhangjg@nic.bmi.ac.cn。
  • 作者简介:姬长甫(1989-),男,河南永城人,在读硕士研究生,主要从事血液病毒免疫学检测,(Tel)13263284281 (E-mail)changfu1990@sina.com。
  • 基金资助:
    *本课题受国家自然科学基金青年基金(NO.81300448)和国家科技支撑计划子课题(NO.2015BAI07B02)资助

Establishment of Fluorescence-based Quantitative Real-time PCR for Detection of Barcode DNA in the Novel Bio-barcode Assay

JI Chang-fu, YIN Hui-qiong, JIA Jun-ting, et al   

  1. Institute of Transfusion Medicine, the Academy of Military Medical Sciences, Beijing 100850
  • Received:2016-01-15 Online:2016-06-20 Published:2016-09-21

摘要: 目的建立条形码DNA实时荧光定量PCR(qPCR)的定量方法,为人源血液病毒蛋白的新型BCA检测奠定基础。方法在新型BCA检测中,设计并合成了一段条形码DNA序列及其引物和探针序列。将条形码DNA克隆入pMD-18T载体,构建条形码DNA参考品质粒。优化条形码DNA qPCR检测体系的重要反应参数,以一系列梯度稀释的条形码DNA参考品质粒为模板,建立qPCR标准曲线,对建立的条形码DNA qPCR定量方法进行敏感性和重复性评价。结果获得了一段特异的条形码DNA序列并构建其参考品质粒,建立了条形码DNA qPCR的定量方法,检测标准曲线在(109~102) copies/μl模板浓度范围内线性关系良好,检测灵敏度达100 copies/μl,组内重复的变异系数均小于10%,组间重复的变异系数范围4.42 %~39.05%。结论建立的基于实时荧光定量PCR的条形码DNA定量方法具有较高的灵敏度和良好的重复性,可用于人源血液病毒蛋白的新型BCA检测体系。

关键词: 条形码DNA, 实时荧光定量PCR, 新型BCA检测

Abstract: ObjectiveTo establish a fluorescence-based quantitative real-time PCR (qPCR) for detection of barcode DNA that is applied to the novel bio-barcode assay of human viruses. MethodThe barcode DNA, primers and TaqMan probes were designed and synthesized for the novel bio-barcode assay. After being amplified by PCR, the barcode DNA were cloned into pMD18-T vector to construct the recombinant plasmids. The reaction conditions of qPCR were optimized, and recombinant plasmids were ten-fold serially diluted as quantitative standard to perform the calibration curves. Finally,the sensitivity and reproducibility of the qPCR for detection of barcode DNA were assessed.ResultThe specific barcode DNA was obtained to construct recombinant plasmids. The qPCR for detection of barcode DNA was established with wide linear range of 102~109copies/μl and high sensitivity (100 copies/μl). The intra-group CVs were less than 10%, and inter -group CVs were between 4.42 % and 39.05%. ConclusionThe qPCR for detection of barcode DNA is established with high sensitivity and good reproducibility, which can be used for the further study of novel bio-barcode assay.

Key words: Barcode, DNA, Fluorescence-based, quantitative, real-time, PCR, Novel, bio-barcode, assay

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