微型化、恆溫增幅核酸之光學檢測器-應用於B型肝炎病毒核酸之快速偵測

In the post-genomic era, the field of genomics now faces the challenge of cataloging gene function. After prevalence of SARS, the demands of fast screening of pathogens grow dramatically. Therefore, it is urgent that develop the biochip for such requirement. As a gene testing platform, the integrated isothermal device which is combined microfabricated technology and isothermal amplification. We adapt loop-mediated amplification method to amplify Hepatitis B Virus (HBV) polymerase gene under 65℃ within one hour. Our device can be partitioned into two components, one is the disposable micro-reactor chip (part A component) and the other one is the control and measurement system (part B component). To provide a reaction chamber with optical path, our design of the part A is made with polymethyl methacrylate (PMMA) and a cover glass slide. To create an appropriate condition for the LAMP reaction, part B includes an external temperature controller and an optical detection unit that yields real time output of by-product, magnesium pyrophosphate, turbidity signal. In results, we summarize the optimal rule of primer design for LAMP reaction and use LAMP reaction to amplify HBV DNA efficiently (in 60 minutes) with high specificity (12 different viral DNA and RNA) and sensitivity (50 copies/25μl). In addition, we adapt simulated reaction to mimic the precipitate of magnesium pyrophosphate. For quantitative analysis, the standard curve (R2 = 0.9605) is established and seven clinical serum specimens with different amounts of HBV DNA can be successfully detected by using this integrated isothermal device in 30 minutes with threshold level of 10,000 copies/ml. We have successfully demonstrated the feasibility of the LAMP reaction for HBV DNA amplification and detection in this novel integrated isothermal device within one hour. The compact device can amplify HBV DNA with high specificity and efficiency under isothermal conditions by using LAMP reaction. It also provides appropriate reaction conditions and a steady optical detection system for detecting turbidity derived from magnesium pyrophosphate formation without fluorescence labeling. Thus, using this integrated isothermal device has great assistance for early diagnosing and monitoring the progress of chronic hepatitis B. It can improve the prognosis of patients and save medical expense enormously. In the future, we hope to provide a multi-channel, portable, label-free, real-time monitoring personalized medical device for rapid identification and quantification of pathogenic organisms and point-of-care applications.