探讨血红蛋白对PCR检测乙型肝炎的影响

Effect of Hemoglobin on Detecting Hepatitis B virus in PCR Technique

目的 :探讨血红蛋白对弱阳性乙型肝炎 (HBV) PCR定量检测结果造成的影响。方法 :采用煮沸法处理血清 ,分别设置阴性对照和阳性对照 (含量 3.0× 10 4 copies/ m L HBV DNA)。取浓度分别为 0 .1μg/ L、0 .5 μg/ L、1.0μg/ L、2 .5 μg/ L、5 .0 μg/ L、2 5 .0 μg/ L、5 0 .0 μg/ L、10 0 .0 μg/ L、5 0 0 .0 μg/ L 的血红蛋白 10 μL 加入管中 ,补充血清 (含3.0× 10 4 copies/ m L HBV DNA)至总体积 4 0 μL。用煮沸法提取 ,荧光定量 PCR检测 HBV DNA。结果 :血清标本中加入血红蛋白浓度 <5 .0 μg/ L 的 HBV DNA PCR结果全为阳性 ,经配对 χ2检验与阳性对照差异无显著性 (P>0 .0 5 )。加入血红蛋白浓度≥ 5 .0μg/ L时出现假阴性 ,经配对χ2 检验与阳性对照的差异有显著性 (P<0 .0 5 )。从各浓度组的变异系数 CV观察 ,血红蛋白浓度越高出现假阴性的机率就越大。结论 :在 HBV DNA PCR检测时 。

Objective To explore the reason of hemoglobin leading to false positive results when quantity polymerase chain reaction (PCR) technique was utilized to detect hepatitis B virus. Methods Negative contrast and positive standard (contains 3.0×10 4 copies/mL ,HBV DNA)were set. 10 μL boiled serum containing Hb concentration 0.1 μg/L?0.5 μg/L?1.0 μg/L?2.5 μg/L?5.0 μg/L?25.0 μg/L?50.0 μg/L?100.0 μg/L?500.0 μg/L were added to 10 testing tubes especially ,which were named samples .The total volume was supplied to 40 μL. HBV DNA was extracted in the boil way and detected by fluorescent quantity PCR. Results All results were positive when the Hb concentration in sample was lower than 0.5 μg/L, which showed the Hb concentration didn't effect HBV DNA detection. The false negative results were detected when Hb concentration in samples was higher than 5.0 μg/L or equal to 5.0 μg/L, which statistics showed significant difference between the positive standard and samples (P<0.05).Conclusion Detecting HBV DNA by quantity PCR technique, nucleic acid should be purified in order to avoid the effect of the Hb.