实时荧光PCR结果的四维杂交的核酸技术分析

本研究采用四维杂交试剂,测定PCR产物的特征熔点温度(temperature of melting point,Tmp),对实时荧光定量PCR所获得阳性信号(特异性的或非特异性的)的结果进行分析和确认,以使检测结论更客观.将荧光探针模式的实时荧光PCR检测后的标本再进行熔解曲线温度扫描,然后在4℃冰箱冷却5min,向反应管加入1μL四维杂交液,再按照温度扫描程序做熔解曲线实验.结果显示,加四维杂交试剂之后的熔解曲线中信号峰值是收敛的,且信噪比增大.相同扩增产物的Tmp的误差是在±1℃之内.实验结果证明,四维杂交试剂对荧光探针模式实时荧光PCR结果可进行更精细的分析和确认.

Analysis of Real-time PCR of 4-Dimension HybridizationTechnology in Nucleic Acid Samples

In order to make the most objective conclusion, we recommend that 4-dimensional hybrid reagents was adopted to detect the character temperature of meltingpoint (Tmp) of PCR outcome. The results of real-time quantitative PCR obtaining the character of positive signal (specific or nonspecific) were analyzed and confirmed. The temperature of solubility curve was scanned after testing the TagMan probe mode real-time PCR. Then put it in 4 ℃ refrigerator for 5 minues and added 1μL 4-dimensional hybrid reagents in each reaction tube. Afterward according to the temperature scan process, conducted the melt curve experiment. The results showed that the signal peak value of sample was convergent with obtained melt curve and signal noise ratio (SNR) was more higher. The error of the same amplification products Tmp value was ±1 ℃. The experimental results show that the 4-dimensional hybrid reagents can perform more detailed analysis and confirm result of the TagMan probe mode real-time PCR.