Development of a sensitive,visual nucleic acid dipstick assay for rapid detection of hepatitis B virus DNA

Objective:We aimed to develop a nucleic acid dipstick assay(NADA) for rapid and visual detection of Hepatitis B virus(HBV) with a high specificity and sensitivity.Methods:PCR amplification of HBV conserved sequences was carried out by a pair of primers labeled with FITC and biotin at the 5' end respectively.The gold nanoparticles and test zones in the dipstick were modified with anti-fluorescein and strepavidin respectively.PCR products mixed with developing buffer were applied to the sample-loading area of the dipstick and the detection result can be observed 10 min afterward with naked eyes.As the buffer migrates along the strip by capillary action,the PCR products were captured at the test zones by immobilized anti-fluorescein and detected by strepavidin-funtcionalized gold nanoparticles,thus generating characteristic red lines.The excess nanoparticles were captured by immobilized biotinylated albumin at the control zone of the strip and then form another red line.We have optimized the developing buffer as well as the volume and concentration for sample-loading.The sensitivity of the optimized NADA has also been evaluated.To demonstrate the utility of NADA,a total of 48 clinical samples containing 15 negative control and 33 HBsAg positive cases were detected.The results from NADA were further com-pared with that from qPCR.Results:The sensitivity of NADA for visual detection of HBV was 250 copies/mL.In the clinical tests,the specificity of DADA and qPCR was 100%,respectively.There were no differences for the detection rates of discriminating the different serum-marker patterns by NADA and qPCR.Conclusions:Our newly developed NADA could be applied for visual detection of HBV.Compared with qPCR,NADA have the advantages of the rapidity,higher sensitivity and specificity,holding a great promise for wide ap-plications in epidemiological surveys and routine physical examinations in hospitals.