| Hepatitis B, caused by HBV, is a widespread blood-borne infectious disease. The number of incidence and morbidity of Hepatitis B remain at front row in statutory infectious diseases in China for several years.At present, screening of transfusion-transmitted virus with NAT has been extensively carried out in various countries. However, representative qPCR is difficult to be adapted in many cases because of higher technical requirements. In addition, expensive reagents, instruments as well as time-consuming analyzing procedure also limit its wide applications. Meanwhile, false negative signals resulted from NAT constantly troubled researchers all around world.GICA is a rapid immunological assay originating from late 1980s. It is a combination of colloid gold labeling and Immune chromatography technology. Through filtration enrichment and capillary action of microporous membrane, antigen-antibody reaction occurs in the solid phase membrane quickly. Colloidal gold will be gathered in the test zone only in the presence of substance to be detected, thus generating a red signal. Otherwise, there is no red band in the corresponding region. It takes only 10 minutes for the whole process.We combined the advantages of PCR and GICA to develop NADA and applied it to the detection of HBV. In brief, 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 could be observed 10 min afterward with naked eyes. The sensitivity of NADA for visual detection of HBV was 250 copies/mL at the optimized condition. In the clinical tests of 48 clinical samples containing 15 negative control and 33 HBsAg positive cases, the specificity of DADA and qPCR was 100%, respectively. There were no differences for the positive detection rates of discriminating the different serum-marker patterns by NADA and qPCR.Based on our previous work, we further developed NADA with an internal control to avoid possible false negative results. The key to this approach was adding a pre-prepared internal control to serum samples to be extracted, then duplex PCR amplification was used to obtain virus and internal control products in a single tube. Meanwhile, an internal control zone was added to traditional nucleic acid dipstick to analyse two PCR products mentioned above simultaneously. Due to parallel extraction, amplification and detection between pathogen and corresponding internal control, monitor of the testing process was achieved, which makes test results more accurate and reliable. We demonstrated that the specificity of prepared internal control and corresponding nucleic acid dipstick was high. Furthermore, monitoring the whole testing process was achieved for serum samples cotaining 103 copies/mL or more when the addition of the internal control was 105 copies.Our newly developed NADA can be applied to visual detection of pathogen. The preliminary results from clinical applications showed that NADA has the advantages of rapidity, higher sensitivity and specificity. Moreover, the addition of internal control can avoid possible false negative results, holding a great promise for wide applications in epidemiological surveys and routine physical examinations in hospitals.
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