HBV DNA Detection by RCA-QCM Biosensor†

doi:10.7503/cjcu20140514

Abstract

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

CLC Number:

O657

TrendMD:

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

Figures/Tables 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

References 18

[1]	Poitras C., Tufenkji N., Biosens. Bioelectron., 2009, 24, 2137—2142
[2]	Ogi H., Yanagida T., Hirao M., Nishiyama M., Biosens. Bioelectron., 2011, 26, 4819—4822
[3]	Mo Z. H., Huang H. J., Qian J. Z., Zhu L. H., Chem. J. Chinese Universities, 2007, 28(4), 649—651
	(莫志宏, 黄红稷, 钱俊臻, 朱丽华. 高等学校化学学报, 2007, 28(4), 649—651)
[4]	Song W. L., Zhu Z., Mao Y. N., Zhang S. S., Biosens. Bioelectron., 2014, 53, 288—294
[5]	Bianco M., Aloisi A., Arima V., Capello M., Ferri-Borgogno S., Novelli F., Leporatti S., Rinaldi R., Biosens. Bioelectron., 2013, 42, 646—652
[6]	Crosson C., Rossi C., Biosens. Bioelectron., 2013, 42, 453—459
[7]	Wang D. Z., Chen G. J., Wang H. M., Tang W., Pan W., Li N., Liu F., Biosens. Bioelectron., 2013, 48, 276—280
[8]	Prakrankamanant P., Leelayuwat C., Promptmas C., Limpaiboon T., Wanram S., Prasongdee P., Pientong C., Daduang J., Jea-ranaikoon P., Biosens. Bioelectron., 2013, 40, 252—257
[9]	Guo Q., Yang X., Wang K., Tan W., Li W., Tang H., Li H., Nucleic Acids Res., 2009, 37(3), e20-1—e20-6
[10]	Zhang S., Wu Z., Shen G., Yu R., Biosens. Bioelectron., 2009, 24, 3201—3207
[11]	Zuccaro L., Tesauro C., Cerroni B., Ottaviani A., Knudsen B. R., Balasubramanian K., Desideri A., Anal. Biochem., 2014, 451, 42—44
[12]	Ouyang X. Y., Li J., Liu H. J., Zhao B., Yan J., He D. N., Fan C. H., Chao J., Methods, 2014, 67, 198—204
[13]	Sato K., Ishii R., Sasaki N., Sato K., Nilsson M., Anal. Biochem., 2013, 437, 43—45
[14]	Zhao C. L., Hua M., Zhao X. H., Huang L. L., Feng Y. J., Niu S. P., Fu X. W., Yuan C., Yang Y. H., Chem. J. Chinese Universities, 2013, 34(1), 61—66
	(赵春玲, 华梅, 赵晓慧, 黄亮亮, 冯亚娟, 牛司朋, 符雪文, 袁聪, 杨云慧. 高等学校化学学报, 2013, 34(1), 61—66)
[15]	Xu L., Wei Y., Chen T., Lu J., Zhu C. L., Ni Z., Huang F., Du J., Sun Z., Qu C., Vaccine, 2010, 28, 5986—5992
[16]	Liang X., Bi S., Yang W., Wang L., Cui G., Cui F., Zhang Y., Liu J., Gong X., Chen Y., Wang F., Zheng H., Wang F., Guo J., Jia Z., Ma J., Wang H., Luo H., Li L., Jin S., Hadler S. C., Wang Y., Vaccine, 2009, 27, 6550—6557
[17]	Vincenti D., Solmone M., Garbuglia A. R., Lacomi F., Capobianchi M. R., J. Virol. Methods, 2009, 159, 53—57
[18]	Hassen W. M., Chaix C., Abdelghani A., Bessueille F., Leonard D., Jaffrezic-Renault N., Sens. Actuators B Chem., 2008, 134, 755—760