To establish a magnetic beads based nucleic acid extraction method and apply for quantitative detection of hepatitis B virus DNA

Abstract

Objective To facilitate the establishment of fully automatic clinical diagnostic nucleic acid assay, a study was carried out for developing a fast and convenient method of extracting highly purified viral nucleic acid from human blood with magnetic beads. Method Five KHB (Shanghai Kehua Biological Engineering Company Limited) internal strong positive HBV serum samples were used in the process, for the method development, screening and selecting the magnetic beads based on magnetism, suspension power and extraction effects of HBV DNA, the optimization of the selected key components of extraction reagents were performed, which including the quantity of magnetic beads（1-1.75 μl/ml）, the buffer pH of lysis (4-9), washing conditions with or without alcohol, elution for proliferation with or without magnetic beads, and etc.The evaluation for the extraction method was carried out including linearity, precision, sensitivity, specificity, anti-interference and resistance to contamination.The magnetic beads method was compared with domestic mature boiling method in a quantitative test with 100 HBV serum samples, the magnetic beads method was also compared with the interntional “Gold standard” Roche COBAS Tagman test method （Roche method） in a quantitative test with 50 HBV serum samples and the results were statistically compared for their linear regression. Results Optimization experiment result showed superparamagnetic beads A was the best among the candidates (C,B,D) in terms of their physical properties and extraction effects of DNA.The optimum condition of the assay were as follow:magnetic beads best used at 1.25 μl/ml, buffer pH 7-8 of lysis, ethanol was eliminated from washing buffer, proliferation can be carried out with magnetic beads.The parallel comparison experiments with five KHB internal strong positive HBV serum sample and gradient diluted serum sample between beads method and boiling method were carried out.The results showed that the linear range of beads method was 4×101-4×108 IU/ml and that of boiling method was 4×102-4×108 IU/ml.The sensitivity for beads method was 30 IU/ml and that of boiling method was 100 IU/ml.The precision CV of beads method was 6.0% and boiling method was 11.9%.The false positive rate was 0 for beads method vs 3.1% for boiling method.The anti-interference ability experiment showed that when sample contained sodium heparin concentration exceed 62.5 U/ml, there was significant drop of HBV DNA detection with boiling method and no HBV DNA was detected when sodium heparin concentration in the sample reached 1000 U/ml.However, sample sodium heparin concentration had no effect on HBV DNA detection with beads method.The comparison study between beads method and boiling method was carried out by use of 100 clinical HBV positive serum samples.The result showed that the sensitivity for beads method was 86% vs boiling method 74%.The correlation between the two method was R2=0.948，P＜0.05.The comparison study between beads method and Roche method was carried use of 50 clinical HBV DNA positive serum samples and the result showed that sensitivity for beads method was 95%(49/50) vs Roche method was 100%（50/50）.The correlation was R2=0.963，P＜0.05. Conclusions The established method for automatic nucleic acid extraction with magnetic bead displayed characteristic of high yield nucleic acid extraction, broader testing range, accurate quantification and convenient in operation.This can be one choice for nucleic acid extraction and applicable for clinical automatic diagnostic system.(Chin J Lab Med,2012,35:843-850) Key words: Hepatitis B virus; DNA; viral; Magnetics