| Background and objective:Hepatitis B virus is one of the major infectious diseases of transfusion-transmitted pathogens. Although nucleic acid detection measures carried out in our country uniformly, there are still some residual risks of HBV. Mainly because some donors’ viral load is lower than the sensitivity of the mini-pools assays, and most of the imported reagents are not designed suitable for domestic epidemic virus strains.For the current problems, the main purpose of this article is to research HBV screening or quantitative detection systems preliminary based on the Chinese epidemiological characteristicsMethod:1.For the magnetic bead based extraction reagent,the concentrations of the main components were selected and identified by thogonal design method, the main components included GuSCN, proteinase K, isopropyl alcohol and PH value.We determined the concentration of each ingredient by the optimal test value,then we chose four silica-based magnetic beads from different manufacturers,we determined the magnetic beads by the optimal extraction effect of different magnetic beads,at the same time,we also optimized the dosage in a test.After we optimized the main components of the extraction reagent,we evaluated this reagent by comparasion with commercial extraction kits including QIAamp(?) and MagPure by to evaluate the extraction efficiency.2.We searched HBV sequences for Chinese prevalence with the key words "HBV" "China", "complete genome" and others in the national center for biotechnology information (NCBI) nucleotide database,and designed primers and probe in the highly conserved regions.The PCR amplified product was prepared to make HBV fragment DNA plasmid with PMD-19 vector. Drew standard curveand calculated the limit of detection after the plamid calibrated by the third-party HBV standard material.Finally, we elavuated this method to test 100 plasma samples which had been tested by Roche cobas v2.0 system.3.The samples with low HBV viral load were made by the third-party HBV standard material dilution.wc reserched the relationship between the centrifugal force and HBV virus recovery to determined the optimal centrifugal force,then kept the optimal centrifugal force to research the relationship between centrifugal time and HBV virals recovery.Finally,we used this virals’inrichment method to test low HBV viral load and to evaluate this ultra high-speed enrichment method.Result:1.For the optimization concentration of the main components of extraction reagent were that:the concentration of GuSCN was 2.0M; concentration of proteinase K was 0.89mg/ml:isopropyl alcohol was 35%; PH value was 8. A type of magnetic beads which used with 30ul had the highest HBV viral recovery.Our extraction reagent had a significant statistical higher than the two kinds of commercial extraction kits not only tested in high, medium HBV virals load but also in low HBV virals load.2.We established a high sensitive and specific quantitative real time PCR method to detect HBV nucleotide acid;Plasmid sequence completely consistent with the HBV DNA sequence through sequencing; The plasmid standard curve was R2>0.999;Amplification efficiency was 92.75%, the linear was range from 5×10- to 2.5×109IU/mL,95%limitat of detection was 16.2 IU/mL; the intra-assay variable coefficient varied from 0.15% to 1.11%; Inter-assay variable coefficient varyied from 0.91% to 4.67%. The results of the samples were in accordance with previous test results which were detected by Roche cobas v2.0 system.3.We found that when centrifugal force between 15000g-90800g, HBV virus recoveries were increased from 41.43% to 88.29%;Then recoveries relatively stable about 80%-90%when the centrifugal force from 90800g to 138900g, when the centrifugal force continues up to 171000g, the recoveries were decreased from 88.29% to 62.94%.For the 4 groups the recoveries of different centrifugation time were 88.31%, 89.70%,88.40%,75.62%, respctively.After using enrichment technology, we found eight samples were HBV DNA positive, however, these eight samples were negative detected by cobas(?)TaqScreen MPX Test, version 2.0.4.We combined ultra-high speed centrifuge enrichment technology with HBV detection system established in this experiment, the sensitivity can be up to 2 IU/mL when a single sample amount not less than 2 ml (When using minipoolsof N blood donation samples,2ml for each sample were mixed, a total of N*2ml);Similarly, if the single sample amount not less than 4 ml (When using minipoolsof N blood donation samples,4ml for each sample were mixed, a total of N* 4ml), the sensitivity can reach 1 IU/mL.Conclusion:1.We successfully established a magnetic bead based extraction reagent for HBV nucleic acid from plasma through optimizing the concentrations of the main components.2.We established a quantitative real-time PCR method to detect HBV which based on Chinese popular characteristics.3.We established HBV screening method based on ultra high-speed centrifuge and improved virus detection rate. |
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