胆红素对HBV DNA定量检测结果的影响

目的探讨高胆红素血清对实时荧光定量聚合酶链反应(PCR)检测乙型肝炎病毒核酸(HBV DNA)的影响,并选择有效减低胆红素对HBV DNA测定干扰的方法。方法将不同浓度的HBV DNA标准品(S1-S4:3.0×10~7、3.0×10~6、3.0×10~5、3.0×10~4IU/ml)按相同比例分别添加到胆红素正常血清和高胆红素血清中,按不同浓度胆红素进行分组来考察不同浓度胆红素血症对HBV DNA测定结果的影响程度大小,然后再分别测定HBV DNA的结果,每个标本重复检测三次,同时观察扩增曲线是否存在差异。对影响测定的血清标本作10、100、1000倍稀释后,进行检测,并收集5例临床高胆红素的乙肝血清标本进行验证。结果血清胆红素浓度介于401~500μmol/L、501~600μmol/L、600μmol/L三个组,HBV DNA定量检测结果高于胆红素正常血清组,差异有统计学意义(P0.05),血清胆红素浓度浓度介于20.6~100μmol/L、101~200μmol/L、201~300μmol/L和301~400μmol/L四个组,与胆红素正常血清组比较,差异无统计学意义(P0.05);血清胆红素浓度高于400μmol/L血清扩增时,扩增曲线先缓慢升高,其基线高于标准品,其阈值线处于拐点之下非S扩增区,之后升高呈现S型曲线;10、100、1000倍稀释后,扩增曲线平行且等间距,结果一致性较好。5例高胆红素血清有4例在定阈值线时,其阈值线处于拐点之下非S扩增区,定值偏高,结果高于稀释后的标本,差异有统计学意义(P0.05);有1例阈值线处于拐点处,其斜率低于标准品,结果低于稀释后的标本,差异有统计学意义(P0.05)。结论总胆红素高于400μmol/L对荧光定量PCR检测HBV DNA结果存在影响,通过稀释一般就可以降低胆红素因素干扰。

Effect of bilirubin on the detection of HBV DNA

Objective To investigate the effect of high level of serum bilirubin on detection of hepatitis B virus DNA (HBV-DNA) by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR),and select the effective method to reduce the interference of bilirubin on HBV-DNA detection. Methods The different concentrations of HBV-DNA standard products (S1-S4:3.0×107、3.0×106、3.0×105、3.0×104IU/ml) were added into the serums which contained normal and high concentration of bilirubin, according to the same proportion. The effect of different concentrations of bilirubin on the determination results of HBV-DNA were investigated. Then the detection of HBV-DNA in each serum sample were performed in triplicate ,and the difference of the ampli-fication curves was observed. The serum samples diluted by 10,100 and 1000 times were tested,respectively. The serum samples from 5 cases of clinical high bilirubin were collected and used to result validation. Results The level of HBV-DNA in the serums contained bilirubin with the concentrations in 401-500μmol/L,501-600μmol/L,>600μmol/L were significantly higher than that in normal bilirubin group (P<0.05);compared with normal bilirubin group,there is no siginificant differrence in the level detected of HBV-DNA in serum contained bilirubin with the concentrations in 20.6-100μmol/L,101-200μmol/L,201-300μmol/L and 301-400μmol/L. When the concentration of bilirubin is higher than 400μmol/L,the amplification curve raised slowly at first,and the baseline was higher than the standard. The threshold line was under the inflection point. Subsequently the amplification curve raised as S curve;for the serum samples diluted by 10,100 and 1000 times,the amplification curve was parallel and equal spaced,and the results were consistent. Among 5 clinical cases detected,4 cases presented the threshold line under the inflection point in the non-amplification region of S curve,and the detection results were significantly higher than the samples diluted (P<0. 05);1 case presented the threshold line through the inflection point in amplification curve ,the slope of which was lower than the standard curve,and the detection result was significantly lower than the sample diluted (P<0.05). Conclusion The high level of serum total bilirubin (>400μmol/L) could interfere detection of HBV DNA in fluorescence quantitative PCR,and the interference effect of bilirubin could be reduced by dilution.