应用核酸酶免疫试验中探针的熔解温度差异检测已知突变位点

Detection of Known Point Mutation by the Difference of Probe/Production Duplex Melting Temperature in Nucleotide Amplification and Enzyme Immunoassay

目的:探索建立一种利用核酸酶免疫试验中探针的熔解温度差异检测已知突变位点的方法。方法:目的靶基因经扩增后,5’末端标记洋地黄毒苷(digoxigenin,Dig)的等位基因探针与已结合在酶标板上的扩增产物杂交,其杂交的探针鄄DNA二聚体的碱基错配与未错配之间存在变性温度差异。选择适宜的熔解温度进行已知突变位点的检测,并利用酶联免疫吸附试验对标记探针进行识别、放大和结果判断。结果:实验结果表明,当熔解温度为37℃时,扩增产物的加样量为5μl/孔和检测探针加入量为10pmol/孔时所测得A450值为0.800～0.923。等位基因检测探针的熔解温度为53℃,P1、P2与靶DNA结合率分别为86%和16%。当P1、P2按不同比例杂合时,其与结合率存在着明显的线性关系(r=0.9985)。从8例人类乙型肝炎病毒(HBV)DNA阳性的血清标本检测中证实1例酪氨酸鄄缬氨酸鄄天冬氨酸鄄天冬氨酸(YVDD)突变、3例野生型,其余4例可能是杂合型突变。结论:本法具较高的检测灵敏度,且其突出的特点是能检测出标本中杂合突变所占的比例,间接反映出患者体内突变的类型和比例。这对于疾病的诊断、治疗、预后评价等都具有较高的价值。

Objective:To establish a method of detecting known point mutation by the difference of probe/production duplex melting temperature in nucleotide amplification and enzyme immunoassay(PCR-ELISA).Methods:After target DNA was amplified,the5'-digoxin-labeled probes of allelic gene was hybridized with amplified products captured in wells of mi-croplate.In the hybridization of DNA,the probe/production duplex melting temperature was different from matching alkali base and mismatching alkali base.Proper melting temperature was selected to detect known point mutation and the hy-bridized probes was enlarged and identified by the method of enzyme immunoassay.Results:When the melting tempera-ture was at37℃,the absorbance at450nm were from0.800to0.923after adding amplified products5μl per well and detective probes10pmol per well.When probe melting temperature of allelic gene was at53℃,the hybridization ratios with target DNA for probe1and probe2were86%and16%respectively.When probe1and probe2were mixed with different proportion,it had an obvious linear correlation with hybridization ratios(r=0.9985).Among the8specimens of HBV DNA positive,one was tyrosine-valine-aspartic acid-aspartic acid(YVDD)mutation,three were wild type,other four might be heterogeneous mutation.Conclusions:This method has a good sensitivity and could be used to detect the hetero-geneous mutation,indirectly reflecting the in vivo mutation type and proportion,and it will be of importance in diagno-sis,treatment and prognostic evaluation of diseases.