Efficient, specific, compact hepatitis B diagnostic device: Optical detection of the hepatitis B virus by isothermal amplification

Abstract

Hepatitis B virus (HBV) is a common viral pathogen that infects over 370 million people worldwide. It has been proven that HBV infection is associated with other clinical problems ranging from chronic hepatitis to cirrhosis or hepatocellular carcinoma. A miniature biosensor that can diagnose infectious diseases rapidly and precisely will be invaluable and in high demand. In this study, we demonstrate the feasibility of a loop-mediated isothermal amplification (LAMP) reaction, an isothermal nucleic acid amplification method, for the amplification and optical detection of HBV viral DNA in a 25 μl miniature reactor. The micro-reactor is made from multilayer polymethyl methacrylate (PMMA), and it includes an external temperature controller and an optical detection unit that yields quantitative output of a by-product turbidity signal. Six HBV primers and working buffers were designed and are preloaded into the reactor to make the process easy to handle. Experimental results show the successful amplification of HBV DNA efficiently (in 60 min), with high specificity (12 different viral DNA, RNA and 2 internal controls) and sensitivity (50 copies/tube). Our compact device is an integrated product that can be advantageous in a wide spectrum of field applications, including pathogens detection, SNP genotyping and other gene testing.