Comparison of SYBR Green and turbidimetry methods for loop mediated isothermal amplification (LAMP) product detection in diagnosis of hepatitis B virus (HBV)

Abstract

Hepatitis B virus (HBV) is one of the leading causes of hepatocellular carcinoma and liver diseases. The HBV serological and molecular detection methods have some limitations; therefore, use of these methods is not feasible in many diagnosis centers. In this study, SYBR Green was compared with spectrophotometry for turbidity assay for final detection of LAMP reaction product in diagnosis of HBV. Two hundred HBsAg positive serum samples were obtained from 3 different groups. DNA was extracted by using DNP kit and six specific primers for LAMP technique were designed. The sensitivity and specificity tests were performed and test was optimized on samples. Loop mediated isothermal amplification (LAMP) product were evaluated by absorption and SYBR Green fluorescence studies and using electrophoresis measurements. Among 200 samples with known viral load and varied titers, 172 samples (about 86%) were LAMP positive. The analysis of the reaction products by SYBR Green fluorescence, electrophoresis and spectrophotometry showed similar results. As reaction goes to completion, along with the DNA synthesis, MgP2O3 value is increased. This fact resulted in an increase of turbidity in the reaction. in comparison to conventional polymerase chain reaction (PCR) technique, LAMP technique had more sensitivity and specificity. In addition, comparison of the three final detection methods showed that the final detection of the LAMP product using SYBR Green is easier and more costeffective, also no need for electrophoresis and other post amplification methods.