Abstract:

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.

Key words: Fluorescent polymerase chain reaction, Enhancer, Cholate, Urea, Heme