| Background and objective: Hepatitis B virus(HBV)is a serious hazard to human body and can be transmitted through blood,so blood screening for blood donors is very strict.After HBs Ag was detected by enzymo-linked immunoassay(HBs Ag),nucleic acid detection(NAT)was carried out,and the high sensitivity detection capability greatly reduced the risk of HBV transfusion transmission.As found in the detection of HBs Ag negative blood through NAT single Ultrio reagent inspection found to have joint inspection part of HBV,HCV,HIV-1 reactivity,but to identify the virus without reactive repeated reactive results,although at present the blood for scrap,blocking blood donors,but not in the blood test results are confirmed.In order to solve this problem,is the main purpose of the study with PEG8000 virus enrichment,with high sensitivity,real-time fluorescent quantitative PCR and nested PCR for NAT repeated reactive results are confirmed.Methods: 1.WHO standard samples were extracted HBV DNA by magnetic bead nucleic acid extraction kit,which was diluted in proportion,and the real-time fluorescence quantitative PCR was used for multiple detection,and the detection limit was obtained by calculation.The standard curve of WHO standard was used to quantify the clinical positive sample S1.Positive sample S1 extracted HBV DNA by magnetic bead nucleic acid extraction kit,diluted in proportion,and tested HBV S and Pre S/S sections with nested PCR for several times to obtain the detection limit.2.Positive samples S1 diluted in proportion,different concentration of S1 that make PEG8000 virus enrichment,using the method of magnetic beads nucleic acid extraction kit HBV DNA extracted,using real-time fluorescent quantitative PCR to sample,calculate recovery rate of the virus.On 15 NRR and 5 samples of OBI PEG8000 enrichment of nested PCR to detect HBV virus,Pre S S/S section and Elite virus detection,high-speed centrifugal enrichment reagent 12 repetitions nested PCR to detect HBV,Pre S S/S segment results compared.3.Electrochemical luminescence A-HBc,A-HBe,A-HBs,HBe Ag,HBs Ag detection of Ultrio NRR samples;Real-time fluorescence quantitative PCR detection;The detection of nested PCR HBV S section was carried out.The Ultrio reagent was performed 2 times and 1 time to identify the test,calculate the positive rate of the Ultrio NRR sample,and compare the different methods.Results: 1.Real-time fluorescence quantitative PCR detection was limited to 10IU/m L.The concentration of S1 virus in positive samples was 5 x 105 IU/m L.The detection limit of HBV S section was 2.5iu /m L.The detection limit of HBV Pre S/S section was detected by nested PCR.2.The efficiency of PEG8000 enriched HBV virus was 46.3%,and the 95% confidence interval was 33.7%-58.8%.The Elite system was able to detect all five OBI samples,and six of the 15 Ultrio Plus NRR samples were detected,with a total detection rate of 55%.PEG8000 enrichment virus(6 m L)method using the real-time fluorescent quantitative PCR and nested PCR to detect Pre S/S,S section,for five OBI also checked out entirely,15 Ultrio Plus NRR samples also checked out 6,the total detection rate of 55%.PEG8000 enrichment virus method can achieve the detection level of repeated tests.Enrichment of ultra high speed centrifugal method of virus(12 m L)using the nested PCR to test the sample Pre S/S,S section,5 OBI samples detected 4,15 Ultrio Plus NRR sample 5,check out the total detection rate was 45%.Through PEG8000 concentration virus(6 m L),the detection of Pre S/S and S sections was detected by nested PCR,and five OBI samples were also detected,15 Ultrio Plus NRR samples were also detected,and the total detection rate was 50%.PEG8000 enrichment virus method can reach the detection level of ultra-high speed centrifuge enrichment virus method.3.In the Ultrio NRR 123 test samples,the Ultrio NRR confirmed that the test positive number was 31,accounting for 25.2% of the test samples,of which the sample of a-hbc positive was 25/31,accounting for 80.6%.PEG methods and Ultrio enrichment reagent test positive for repeat number,compared with X2 test,results the two groups have significant difference(P < 0.0001),PEG enrichment method to detect the HBV virus detection rate is higher than Ultrio repeat detection reagent.The Ultrio NRR confirmed that the first-time blood donation rate was 54.8% for the positive blood donors and 45.2% for the blood donors.A total of 24 samples tested positive for the Ultrio NRR sample,which was enriched by PEG virus,with 15 samples of viral load less than 10IU/m L,accounting for 62.5%.The viral load was greater than or equal to 10 less than 20 IU/m L samples,accounting for 20.8%.The viral load was greater than or equal to 30 less than 100 IU/m L samples,accounting for 16.7%.Conclusion: 1.The detection method of PEG8000 enriched virus HBV is established to improve the detection rate of HBV. 2.Ultrio NRR blood has a high residual risk of HBV.3.The detection rate of HBV of PEG virus is significantly higher than that of the Ultrio reagent.4.Repeat blood donors have a high percentage of positive blood donors in the Ultrio NRR.5.HBV viral load is very low in Ultrio NRR positive blood. |
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