血液HBV DNA全自动检测及基因分型分析

Detection of HBV DNA with full automatic system and HBV genotyping in blood donor screening

目的建立血液HBVDNA标本汇集、核酸提取、扩增及检测的全自动筛查模式,对阳性标本进行基因分型和血清学追踪检测,为血液HBVDNA自动化筛查提供科学依据。方法在酶联免疫吸附试验(ELISA)筛查血液基础上,应用STAR2000加样仪自编程序进行全自动血样汇集(24人份),在MPLC仪上全自动提取标本核酸,应用PCR方法在COBASAMPLICOR进行扩增和检测分析结果,用国际标准核酸质控品考评检出限量,对阳性标本进行基因分型并追踪血清转换过程。结果全自动汇集、全自动核酸提取和扩增及检测HBVDNA95%检出限量为38.9IU/ml,95%CI为(21323)。通过对16512个标本共688个汇集池分析,HBVDNA阳性8例,阳性率为0.049%。其中C型3人,B型2人,D型1人,2人未能确定基因型,6例阳性标本追踪发现,3例发生了血清转换现象。结论HBVDNA全自动核酸检测方法可应用于24人份血液混样核酸筛查。

Objective To establish fully automated sample pooling, nucleic acid extraction, amplification and detection method for HBV DNA testing, and investigate the seroconversion and genotype in HBV DNA positive donors. Methods Individual donor plasma samples serologically negative for HBV were pooled by STAR2000 sampling processor with a size of 24. Nucleic acid were automatically extracted by MPLC simultaneously, amplified and detected by Roche COBAS AMPLICOR system. The sensitivity of detection was determined by international standard. HBV DNA positive donors were genotyped and followed up by serological tests. Results The 95% detection limit for this automated HBV DNA testing system was 38.9IU/ml,with 95% confidence interval (21323),eight out of 16512 specimens were PCR positive for HBV DNA,with a positive rate of 0.049%. Three of the 8 DNA positive donors were genotype C,2 genotype B, 1 genotype D,and the other 2 uncertain。Six of the eight HBV DNA positive donors were followed up, and three of them seroconverted。 Conclusion Fully automated HBV DNA detection method can be applied in blood screening,and will further increase the safety of blood supply.