荧光定量聚合酶链反应检测乙型肝炎病毒DNA假阴性结果原因分析

目的探讨荧光定量聚合酶链反应(polymerase chain reaction, PCR)检测乙型肝炎病毒DNA(hepatitis B virus, HBV-DNA)产生假阴性结果的原因，以控制和减少假阴性结果。 方法依据第三版《临床检验操作规程》和2013版中国合格评定国家认可委员会(China National Accreditation Service for Conformity Assessment，CNAS)-CL36《医学实验室质量和能力认可准则在基因扩增检验领域的应用说明》，对当天送检的79份HBV DNA定量项目出现HBV-DNA假阴性的临床标本进行复查，并通过试验研究对试剂、仪器、操作过程和环境等原因分别进行确认。首先，分析2015年5月13日检测的79份临床送检的HBV-DNA定量检测血清标本和质控品的仪器原始结果，排除试剂、仪器和人员问题；其次，5次重复实验验证HBV-DNA定量结果为10⁷ IU/ml的临床血清样本是否因主要操作步骤不规范导致HBV-DNA定量检测结果下降(偏倚>7.5%)。选择第一次检测结果在6次方以上的血清标本2份，平行做5个复孔，其中1份标本做5个复孔，即用取上清液时所弃沉淀分别以不弃掉沉淀、弃掉1/4沉淀、2/4沉淀、3/4沉淀和全部沉淀分为A0、A1、A2、A3、A4组，比较弃上清液中沉淀量对HBV DNA定量检测结果的影响。另1份标本5个复孔在加模板量时，以正常加2 μl为对照组A0，其他4孔分别加1.5、1、0.5、0.1 μl模板为A1、A2、A3、A4组，分析模板加样量的不同对HBV DNA定量检测结果的影响；第三，挑选第一次HBV DNA定量检测结果为不同次方的10份血清标本，其中5份分别加入1 μl除胶剂，另5份分别加入1 μl含氯消毒液，验证是否因除胶剂或含氯消毒液导致HBV-DNA定量检测出现假阴性结果(偏倚>7.5%)。 结果操作过程中弃上清液时，A1~A4组与A0比较，各孔的偏倚均<7.5%，无差异。加入不同量的模板，A1和A2与A0比较，无差异(偏倚均<7.5%)，随着模板加入HBV DNA量的减少，A3和A4孔HBV DNA值逐渐降低，偏倚均>7.5%。加入商用除胶剂1 μl后的检测结果与初次检测结果比较，5份标本中，有3份标本检测结果偏倚>7.5%。加入含氯消毒液1 μl后的检测结果与初次检测结果比较，5份标本中有3份标本检测结果偏倚>7.5%，其中有1份标本结果由阳性(10⁷ IU/ml)变为阴性。 结论除胶剂和次氯酸气溶胶对荧光定量PCR的DNA模板扩增环节的抑制作用是产生荧光定量PCR检测HBV-DNA假阴性的主要原因，临床基因实验室应当注意使用除胶剂和次氯酸消毒液后通风与降低次氯酸在空气中浓度过高问题。

The investigation on false-negative results of hepatitis B virus DNA determination by fluorescence quantitative polymerase chain reaction

ObjectiveTo investigate the reason of false-negative results of hepatitis B virus (HBV)-DNA determination by fluorescence quantitative polymerase chain reaction (PCR), in order to control these factors in clinical trials to reduce false-negative rate. MethodsBased on the third edition of ″Clinical Laboratory Procedures″ and version 2013 China National Accreditation Service for Conformity Assessment (CNAS)-CL36 ″medical laboratory accreditation criteria for quality and competence in the field of gene amplification test application note″, reviewing 79 cases clinical specimens used in quantitative detection of HBV DNA on May 13, 2015. Experiments were conducted to evaluate the possible reasons from reagent, equipment, experiment procedure and environment, respectively. Firstly, the factors of reagents, instruments and personnel were elimiated by analyzing the above test results. Secondly, 5 repeated experiments were conducted to verify whether the lower test HBV-DNA quantitative results from clinical serum samples with the original results of 10⁷ IU/ml was due to not standard operating procedures (bias>7.5%). Two cases of serum samples with test results more than ten to the six power were selected to test in five parallel wells. Five wells of one patient specimen grouping into A0, A1, A2, A3, A4 were used to evaluate, the effect on HBV DNA quantitative test results by the discarding different amount of precipitation. The other patient specimen five wells into A0, A1, A2, A3, A4 were used to evaluate, the effect on HBV DNA quantitative test results by plus the amount of different templates. At last, 1μl addition of glue or chlorine disinfectant to the serum sample, respectively, was used to verify inhibition on the results. ResultsWhen the supernatant was discarded during the operation, compared with A0, the bias of the wells of A1~A4 group were<7.5%, no difference was showed. In the factor of different amounts of template, compared with A0, the results of A1 and A2 showed no difference(bias were<7.5%); the results of A1 and A2 showed decreased, bias were>7.5%. With the reduction in the amount of template, the quantitative value decreased. The chlorine disinfectant showed strong inhibition on the results of HBV DNA determination by fluorescence quantitative PCR, which may lead to false-negative results when preparing high concentrations of HBV DNA samples. The addition of glue also affected the PCR amplification and led to low result. ConclusionThe factor of addition of glue and hypochlorous acid aerosols inhibiting the efficacy of PCR amplification of DNA template in clinical laboratory is overlooked, so that the use of glue should be avoided, the use of hypochlorite disinfectant should be ventilated to avoid high concentration of hypochlorous acid in the air of clinical genetic laboratory.