To 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.

Based 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.

When 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.

The 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.