| Objective:To investigate the prevalence of occult hepatitis B virus(OBI)infection among voluntary blood donors in Dalian area,The molecular biological characteristics of OBI and the mutation of S gene were analyzed.To explore the safety and reliability of implementing DNA screening of hepatitis B virus in domestic blood donors,provide an important basis for prevention and control and monitoring strategies of OBI infection,improved blood screening methods,reduce the missing rate of HBV.Methods:1.Samples collection:5ml venous blood sample in the EDTA.K2 vacuum vessel collected from the blood bag bypass,saved in 2-8 ? before detecting 4 infective markers(HBsAg,-HCV,HIV,anti Ag/Ab and anti-TP),ALT and blood typingg,RhD blood typing.HBsAg,HBsAb(quantitative IU/L),HBcAb,HBeAg,HBeAb were further tested if need.2.The application of serological method(ELISA)combined with routine screening for HBsAg nucleic acid detection,from 68565 blood donors were screened in a sample of 95 cases of HBsAg negative and NAT positive samples to be tracked or backtracking,combined with hepatitis B virus serological test,5-identification test and semi nested PCR to confirm whether HBV infection;the analysis further confirm whether OBI infection.3.From manual HBV DNA in 95 cases of HBsAg-NAT+ plasma samples,the preSS and S regions were determined by nested PCR and sequence analysis of gene mutation,and compared with the standard sequence.MEGA5.2 software was used for OBI analysis of biological characteristics and amino acid mutations in S region.Results:(1)Sample were screened out of 95 cases of HBsAg-/NAT positive samples of blood donors in Dalian in 68565 cases,95 cases of HBsAg-NAT+ serological results of blood donors:the HBcAb(excluding HBsAb)accounted for 64.2%(61/95);produce protective HBsAb(not considering HBcAb)accounted for 50.5%(48/95)accounted for 16.8%of all negative;(16/95);only 29 cases were HBcAb positive,accounting for 30.5%(29/95);only 16 cases were HBsAb positive,accounting for 16.8%(16/95).(2)The number of HBsAg-()-/NAT(+)samples in plasma was determined in the presence of HBV DNA in 40 patients.Including 30 cases identified as HBV DNA reactivity,identification rate was 31.2%(30/95);unidentified HBV but the hand extracted sequences of 9 cases;another 1 cases when the blood donation did not identify HBV has not obtained sequence,but in the tracking data to identify HBV positive.(3)HBV samples from the blood samples of 40 blood donors were confirmed by OBI in,and the OBI infection rate was 1:1959(35/68565)in Dalian area of DNA.(4)95 samples in 27 samples of successful amplification and sequence determination of PCR gene by nested S,HBV genotyping results showed that 22 cases were C type,4 cases were B type,1 cases of type A2,C type,accounting for 81.5%(22/27).(5)Mutation analysis showed that the amino acid mutation rate of HBV S region was high in the MHR samples of the 27 OBI cases,especially the HBsAg antigen determinant gene and the amino acid mutation occurred in the peripheral hydrophilic region.Conclusion:In this study,95 HBsAg(-)/NAT(+)blood donors were screened from 68565 blood samples.Among them,OBI was confirmed in 35 cases,and the majority of OBI in Dalian were genotype C.The mutation of OBI(MHR)amino acids in the HBV region of S might affect the replication and protein expression of HBV,which may be related to the occurrence and development of OBI.The results of this study provide a basis for the improvement of blood screening methods and the study of OBI. |
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