不同商品化试剂盒定量检测肝癌晚期患者乙型肝炎病毒 DNA结果的差异

Difference of quantitative detection of hepatitis B virus DNA in patients with advanced liver cancer by different commercial kits

目的探讨不同商品化试剂盒定量检测肝癌晚期患者乙型肝炎病毒(HBV)DNA结果的差异。方法选择2013年10月2日淄博市传染病医院收治的1例肝癌患者为研究对象。分别使用罗氏诊断产品(上海)有限公司和天隆科技有限公司的HBV DNA定量检测试剂盒对该例肝癌患者血清样本进行不同时间的4次HBV载量检测。取患者3 mL血清进行二代测序,并将肝癌患者基因组中HBV开放阅读框Precore/core区序列与通用参考序列比对,分析差异区域。应用MiSeqRepoter软件对二代测序结果进行分析,寻找患者染色体中嵌合的HBV基因组序列。结果罗氏诊断产品(上海)有限公司HBV DNA定量试剂盒4次检测结果均为HBV感染阴性,天隆科技有限公司HBV DNA定量试剂盒4次检测HBV载量均>1×10^(4) IU·L^(-1),均为HBV感染阳性。二代测序结果显示,患者基因组中含有病毒DNA读长509617 bp(1%),509617 bp病毒DNA中498064 bp(98%)为HBV DNA。与通用序列比对结果显示,肝癌患者基因组中HBV Precore/core区序列中未发现突变位点,患者基因组中的HBV Precore/core区中P区1700~2140 bp区间缺失。二代测序结果显示,患者多条染色体上整合了HBV基因组,其中5号染色体上HBV基因组读长3136 bp,1号染色体上HBV基因组读长2274 bp,10号染色体上HBV基因组读长51 bp,6号染色体上HBV基因组读长47 bp。结论不同商品化HBV DNA定量试剂盒检测结果存在差异,可能是HBV感染时整合到宿主染色体上导致的序列缺失,在使用不同的检测试剂盒进行临床诊断时务必慎重。

Objective To investigate the difference of quantitative detection results of hepatitis B virus(HBV)DNA in patients with advanced liver cancer by different commercial kits.Methods One HCC patient with liver cancer admitted to Zibo Infectious Diseases Hospital on October 2,2013 was selected as the research object.The HBV DNA quantitative detection kits of Roche Diagnostic Products(Shanghai)Co.,Ltd.and Tianlong Technology Co.,Ltd.were used to detect the HBV load of serum samples from the same liver cancer patient for four times at different times.A total of 3 mL serum of the patient was collected for second generation sequencing,and the HBV open reading frame Precore/core region sequence in the genome of liver cancer patients was compared with the universal reference sequence to find the difference region.The second generation sequencing result was analyzed by MiSeqRepoter software to find chimeric HBV genome sequences in patients′chromosomes.Results The results of four tests with Roche Diagnostic Products(Shanghai)Co.,Ltd.HBV DNA quantitative kit were negative for HBV infection;the HBV load of four tests with Tianlong Technology Co.,Ltd.HBV DNA quantitative kit were>1×10^(4) IU·L^(-1),all were positive for HBV infection.The second generation sequencing results showed that 509617 bp(1%)of viral DNA were read in the patient′s genome,and 498064 bp(98%)of the 509617 bp of viral DNA were HBV DNA.The results of comparison between HBV Precore/core region sequence and general sequence in liver cancer patients′genome showed that no mutation site was found,and the 1700-2140 bp range of P region in HBV Precore/core region in patients′genome was missing.The analysis of the second generation sequencing results showed that the patient′s HBV genome was integrated on multiple chromosomes,including 3136 bp of HBV genome reads on chromosome 5,2274 bp on chromosome 1,51 bp on chromosome 10,and 47 bp on chromosome 6.Conclusion The detection results of different commercial HBV DNA quantitative kits are different,which may be due to the sequence deletion caused by the integration of HBV DNA into the host chromosome during HBV infection.It is necessary to be careful when using different detection kits for clinical diagnosis.