›› 2012, Vol. 32 ›› Issue (6): 693-.doi: 10.3969/j.issn.1674-8115.2012.06.002

• Original article (Basic research) • Previous Articles     Next Articles

Surface plasmon resonance technology combined with rolling circle amplification for detection of hepatitis C virus

JI Ming-hui1,2, HU Gui-fang1, ZHENG Yi3, GU Da-yong2, LONG Jun4, LU Wei-ping5, HE Jian-an2, TAN Shu-qin1, SHI Lei2, LIU Chun-xiao2, ZHAO Chun-zhong2, XU Yun-qing2, XU Hua6, OU Qing-ye6, SUN Qiu-xiang7, TENG Juan8   

  1. 1.School of Public Health and Tropical Medicine, Southern Medical University, Guangzhou 515000, China；2.Institute of Disease Control and Prevention, Shenzhen International Travel Health Care Center, Shenzhen Entry-exit Inspection and Quarantine Bureau, Shenzhen 518045, China；3.Center of Bio-technique &|Bio-medicine, Shenzhen Key Laboratory of Antibody &|Gene Therapy, Graduate School at Shenzhen, Tsinghua University, Shenzhen 518000, China；4.Zhujiang Hospital, Southern Medical University, Guangzhou 515000, China；5.Daping Hospital, Third Military Medical University, Chongqing 400042, China；6.Department of Occupational Medicine and Environmental Toxicology, School of Public Health, Nantong University, Nantong 226019, China；7.School of Public Health, Sun Yat-sen University, Guangzhou 515000, China；8.International Travel Health Care Center, Hainan Entry-exit Inspection and Quarantine Bureau, Haikou 570311, China
  • Online:2012-06-28 Published:2012-07-02
  • Supported by:

    National Natural Science Foundation of China, 30972827, 81171667； Program of General Administration of Quality Supervision, Inspection and Quarantine of the People's Republic of China, 2010IK212；Hong Kong Innovation Circle Program (joint funding) Project, ZYB200907090128A；Shenzhen Science & Technology Planning Program, HZ0907004；Foundation for Science & Technology Research Project of Chongqing, 2011AC5033

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Abstract:

Objective To develop rolling circle amplification (RCA) method combined with specific surface plasmon resonance (SPR) nucleic acid gold-chip for the detection of hepatitis C virus (HCV). Methods According to the specific test sequence of HCV x-tail region, probes and primers for detecting HCV with RCA method were designed and synthesized, and were divided into test group, negative sample group and positive sample group for RCA experiments to detect HCV. The template concentration was diluted into ten gradients, and the detection limit of SPR combined with RCA method was evaluated. Based on the ordinary gold chip, through the surface chemical processing, the nucleic acid chip with high specificity was obtained, and the anti-protein capacity of protein chip was verified by anti-protein experiment. Real-time detection of RCA reaction and signal magnification reaction was conducted with double channel SPR apparatus. Sixty-three blood samples collected from clinics were detected by SPR combined with RCA method, comparisons were made with Real-Time PCR, and the sensitivity and specificity were evaluated. Results The minimum detection concentration of SPR combined with RCA method in HCV testing was 1 pmol/L, which was lower than the detection limit of Real-Time PCR (0.1 nmol/L). SPR chip had the favorable anti-protein absorptive capacity. The signal-to-noise ratio of double channel SPR apparatus in detection of RCA chip system was 100, which achieved the detection of HCV. The sensitivity of SPR combined with RCA method in detection of clinical samples was 90.0%（27/30）, and the specificity was 84.8% （28/33）（χ2=8.10，P=0.004）. Conclusion SPR combined with RCA method combines biological sensing technology with in situ amplification technology, which may detect HCV in a fast, label-free and real-time way.

Key words: surface plasmon resonance, hepatitis C, rolling circle amplification