不同增强剂对荧光PCR中尿素、血红素和胆酸盐抑制作用消除效果研究

Inhibitory elimination effects of different enhancers on urea,heme and cholate in fluorescent PCR

目的探讨不同聚合酶链反应(PCR)增强剂对胆酸盐、尿素和血红素抑制作用的消除效果。方法将13种不同浓度的PCR增强剂[亚精胺、甲酰胺、PEG6000、NP40、Tween-20、甜菜碱、海藻糖、牛血清白蛋白(BSA)、明胶、硫酸铵、四甲基氯化铵(TMAC)、甘油、Triton X-100]加入含胆酸盐、尿素和血红素的扩增体系中,采用荧光PCR检测,根据循环阈值(Ct)分析不同PCR增强剂的抗抑制效果。结果6.5 mmol/L亚精胺、20%甘油可消除6.98 mmol/L胆酸盐对PCR的抑制作用,6.5 mmol/L亚精胺、100 mmol/L硫酸铵、0.2 mol/L甜菜碱可消除0.99 mol/L尿素对PCR的抑制作用。300 mmol/L BSA、6.5 mmol/L亚精胺、20%PEG6000、50%甘油可消除6.98 mmol/L血红素对PCR的抑制作用。40%甲酰胺、10%NP40、40%Tween-20、1.5 mol/L海藻糖、2%明胶、200 mmol/L TMAC和20%Triton X-100均无法降低6.98 mmol/L胆酸盐、0.99 mol/L尿素和6.98 mmol/L血红素对PCR的抑制作用。结论添加不同类型的PCR增强剂可有效提高荧光PCR的检测灵敏度和准确性。

Objective To investigate the inhibitory elimination effects of different polymerase chain reaction(PCR)enhancers on cholate,urea and heme.Methods Different concentrations of PCR enhancers[spermidine,formamide,PEG6000,NP40,Tween-20,betaine,trehalose,bovine serum albumin(BSA),gelatin,ammonium sulfate,tetramethylammonium chloride(TMAC),glycerol and Triton X-100]were added to the amplification system containing cholate,urea and heme.The anti inhibitory effects of different PCR enhancers were determined by fluorescent PCR and analyzed according to the cycle threshold(Ct)value.Results The 6.5 mmol/L spermidine and 20%glycerol could eliminate the inhibitory effect of 6.98 mmol/L cholate on PCR.The 6.5 mmol/L spermidine,100 mmol/L ammonium sulfate and 0.2 mol/L betaine could eliminate the inhibitory effect of 0.99 mol/L urea on PCR.The 300 mmol/L BSA,6.5 mmol/L spermidine,20%PEG6000 and 50%glycerol could eliminate the inhibitory effect of 6.98 mmol/L heme on PCR.The 40%formamide,10%NP40,40%Tween-20,1.5 mol/L trehalose,2%gelatin,200 mmol/L TMAC and 20%Triton X-100 could not reduce the inhibitory effects of 6.98 mmol/L cholate,0.99 mol/L urea and 6.98 mmol/L heme on PCR.Conclusions Adding different types of PCR enhancers can effectively improve the determination sensitivity and accuracy of fluorescent PCR.