影响内标双重PCR靶模板检测灵敏度的因素分析

Exploration on influential factors for sensitivity in duplex PCR with internal control gene

目的在内标双重PCR系统中寻找影响靶模板检测灵敏度的主要因素,并比较不同检测体系的灵敏度。方法针对HBV基因设计普通引物对和中部同序引物对,比较引物二聚体（PD）含量;控制内标基因的Ct值约为30,调整内标引物浓度为10、8、5、2、1μmol/L,比较不同体系的检测灵敏度;控制内标引物的用量不变,分别测定内标基因Ct值在15、20、25、30时的靶模板检测灵敏度。结果中部同序引物PD的Ct值在35以上,普通引物对PD的Ct值在30~33之间;10、8、5、2、1μmol/L浓度的内标引物需要内标模板的浓度分别为5×10^4、5×10^4、5×10^4、5×10^5、5×10^7IU/m L;不同内标引物用量检测灵敏度均为5×10^3IU/m L,仅使用中部同序引物的单重PCR检测灵敏度为5×10^2IU/m L;内标Ct值在15、20、25、30时靶模板检测的灵敏度分别为5×10^5、5×10^4、5×10^3、5×10^3IU/m L。结论在双重PCR检测体系中,对靶模板检测灵敏度影响最大的因素为内标的模板量,仅使用中部同序引物对的单重PCR具有最高的灵敏度。

Objective To investigate the influential factors affecting sensitivity of target template in duplex PCR detection with internal control and assess the disparity of sensitivities under different PCR reaction conditions. Methods We designed two sets of PCR primers,general primer and central homo-sequence primer,based on Hepatitis B virus genome sequence,and analyzed the amount of primer dimer formed in the both reaction systems. The sensitivities of the detection were compared under the different PCR conditions that the Ct value of internal control was kept at 30 and the concentrations of internal control primers were adjusted to 10,8,5,2 and 1μmol /L respectively. The amount of internal control primers remained unchanged. We examined the sensitivities of target template when the Ct value of internal control was at 15,20,25 and 30 respectively. Results The Ct value of primer dimer of central homo-sequence primers remained above 35,while the ones of general primers were between 30 to 33. For the internal control primer concentration of 10,8,5,2 and 1 μmol / L,the internal control template concentrations were 5 × 10^4,5 × 10^4,5 × 10^4,5 × 10^5,5 × 10^7 IU / m L respectively. The sensitivities of detection under the different conditions were all at 5 × 10^3 IU / m L using general primers,expect for the central homo-sequence primers which detecting sensitivities were raised up to 5 × 10^2 IU / m L by contrast. The detecting sensitivity of target template was 5 × 10^5,5 × 10^4,5 × 10^3,5 × 10^3 IU / m L when the Ct value of internal control was at 15,20,25 and 30 respectively. Conclusion In duplex PCR reaction with internal control,the amount of internal control template added to the reaction mix should be the major influential factor for the sensitivity of target template detection to interfer the detecting limits. The highest sensitivity may achieved by using only central homo-sequence primer in monoplex PCR.