The development of DNA-based quartz crystal microbalance integrated with isothermal DNA amplification system for human papillomavirus type 58 detection

Abstract

To address the effect of dramatic change in temperature and viscosity during PCR process on quartz crystal microbalance (QCM) sensor and to increase the sensitivity, isothermal amplification was employed in the system. We combined loop-mediated isothermal amplification (LAMP) technique with QCM, called as LAMP-QCM, for detection of high-risk human papillomavirus viral DNA type 58 (HPV-58) which is commonly found in Asian women. The liquid-phase LAMP-QCM prototype comprised the frequency counter, a temperature control device and housing of the quartz crystal with polished gold electrodes on both sides. QCM detection signal was monitored in real-time based on an avidin-biotin binding between avidin coated QCM surface and specific biotinylated LAMP products. Analytical performance was evaluated for precision, sensitivity and specificity. A plasmid clone containing the HPV-58 sequence was diluted from 10(6) to 1 copy and used for detection limit. Cut-off value was estimated at 28.8 Hz from negative viral template. The system could detect 100 copies with Δf at 34.0±3.6 Hz compared to 1000 copies detected by conventional LAMP. No cross-reaction was observed with other HPV types. The HPV-58 detection was compared among LAMP-QCM, conventional LAMP and nested PCR in 50 cervical cancer tissues. The positive rate of LAMP-QCM was higher than that of conventional LAMP with 100% sensitivity and 90.5% specificity. The integrated LAMP-QCM system has improved the detection limit up to ten times compared to conventional LAMP with less-time consuming.