滚环扩增型压电石英晶体传感器检测乙型肝炎病毒

doi:10.7503/cjcu20140514

高等学校化学学报 ›› 2014, Vol. 35 ›› Issue (8): 1635.doi: 10.7503/cjcu20140514

滚环扩增型压电石英晶体传感器检测乙型肝炎病毒

姚春艳, 王云霞, 府伟灵()

第三军医大学西南医院检验科, 重庆 400038

收稿日期:2014-06-06 出版日期:2014-08-10 发布日期:2019-08-01
作者简介:联系人简介: 府伟灵, 男, 博士, 教授, 博士生导师, 主要从事生物传感器与分子生物学研究. E-mail:weilingfu@yahoo.com
基金资助:
国家自然科学基金(批准号: 30970766,81371885)资助

HBV DNA Detection by RCA-QCM Biosensor^†

YAO Chunyan, WANG Yunxia, FU Weiling^*()

Department of Laboratory Medicine, Southwest Hospital, the Third Military Medical University, Chongqing 400038, China

Received:2014-06-06 Online:2014-08-10 Published:2019-08-01
Contact: FU Weiling E-mail:weilingfu@yahoo.com
Supported by:
Supported by the National Natural Science Foundation of China(Nos.30970766, 81371885)

摘要/Abstract

摘要：

通过捕获探针与纳米金膜之间的共价连接, 保证了滚环扩增(RCA)产物始终结合于金膜表面, Phi29 DNA聚合酶的高效扩增和Escherichia coli DNA链接酶的高度精确性使检测达到单碱基识别, 检测灵敏度达到10⁴ copies/mL. 实验结果表明, 与单碱基错配序列相比, RCA可明显增强检测的灵敏度. 该RCA基因传感器操作简单, 灵敏度和特异性较高, 在乙型肝炎病毒的快速检测方面具有一定的开发潜力.

关键词: 滚环扩增, 石英晶体传感器, 乙型肝炎病毒

Abstract:

The research describes the application of RCA(rolling circle amplification)-based quartz crystal microbalance(QCM) biosensor for the detection of hepatitis B virus(HBV) DNA. RCA is an isothermal amplification technique, which creats long single-stranded products with property of product localization. After amplification, the RCA product is maintained during the assay through the covalent bonding between the capture probes and the gold electrode surface. Using high amplification efficiency of Phi29 DNA polymerase and remarkable precision of Escherichia coli DNA ligase, the detection limit can reach 10⁴ copies/mL. Experimental results show that RCA has significantly enhanced sensitivity for the target strand compared to the single-base mismatch strand. RCA has powerful amplification ability, and QCM has superb mass sensitivity. The combination of these two methods provides a high sensitive RCA-QCM biosensor method, which has the potential to become a successful clinical application.

Key words: Rolling circle amplification, Quartz crystal microbalance biosensor, Hepatitis B virus

中图分类号:

O657

TrendMD:

姚春艳, 王云霞, 府伟灵. 滚环扩增型压电石英晶体传感器检测乙型肝炎病毒. 高等学校化学学报, 2014, 35(8): 1635.

YAO Chunyan, WANG Yunxia, FU Weiling. HBV DNA Detection by RCA-QCM Biosensor^†. Chem. J. Chinese Universities, 2014, 35(8): 1635.

图/表 5

Table 1 Sequences of oligonucletides used in the assay

Oligonucleotide	Sequence(5'— 3')
Capture probe	5'-ATGATGGGTATGGGAATACAGG-3'
Circular probe	5'-TGCAGTTTCCGTCCGTAGTAGAATGAAGATAGCGCATCGTAGGAGGACGGAGGATGATGGGTATGGGAATACAGG-3'
Primer	5'-TGCAGTTTCCGTTTTTTCCTCCGTCCTCCTACGATGCG-3'
HBV target	5'-CTACGGACGGAAACTGCACCTGTATTCCCATACCCATCAT-3'
Single-base	5'-CTACGGACGGAAACTGCAACTGTATTCCCATACCCATCAT-3'
mismatch sequence

Fig.1 Real-time frequency readings along RCA reaction

Fig.2 Illustration of circular template hybridization with target strand and single mismatch strand(A) and RCA reaction on the gold electrode of crystals(B)

Fig.3 Effects of RCA reaction temperature of the QCM biosensor(A), RCA reaction time(B) and exonuclease I concentration(C) on QCM frequency shifts (C) Error bar was estimated from three replicate measurements.

Fig.4 Relationship between the frequency change and lgc of target

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滚环扩增型压电石英晶体传感器检测乙型肝炎病毒

HBV DNA Detection by RCA-QCM Biosensor^†

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滚环扩增型压电石英晶体传感器检测乙型肝炎病毒

HBV DNA Detection by RCA-QCM Biosensor†

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HBV DNA Detection by RCA-QCM Biosensor^†