反向荧光偏振(FP)检测血清中HBV多聚酶基因突变

Detection of mutants of HBV polymerase gene in serum by fluorescence polarization(FP)

目的:建立简便的HBV多聚酶基因序列中拉米呋啶耐药性相关的基因突变的检测方法。方法:利用pfu酶的3′-5′外切校正功能和荧光偏振光检测技术进行点突变的检测。首先PCR扩增HBV多聚酶基因,然后用3′标记荧光分子FAM的探针与扩增产物中待测核苷酸的下游序列杂交,使探针3′第一个碱基与靶序列中待测点的碱基配对。若碱基配对正确,pfu酶可以沿探针的3′端延伸;若碱基配对错误,pfu酶须将探针3′标有荧光分子的核苷酸切掉,然后再沿探针3′端延伸。由于第一种情况,荧光分子与DNA链相连,分子量增加,则荧光偏振光值增大;而第二种情况,荧光分子与探针分离,荧光分子的整体分子量减小,则荧光的偏振光值减小,这样可以通过荧光偏振光值的大小判断待测点核苷酸的改变。结果:该方法可以检测出HBV多聚酶基因序列中特定位置的核苷酸类型,可以检测1×101个拷贝的模板量。对30例经过6个月以上拉米呋啶治疗的患者血清进行检测,检测出其中有3例是甲硫氨酸(M)密码子ATG中的A→G变异;2例是ATG中的G→C变异;1例是ATG中的G→T。结论:该技术可以对血清中HBV多聚酶基因序列中点突变以及其他基因突变进行检测。

Objective：A simple method of detecting the mutants of HBV polymerase gene was established.Methods：HBV polymerase gene was amplified by PCR,the products of PCR was hybridized with the probe which is located at the upper steam behind the interesting site and the fluorescence-labeled 3＇ end of probe is matched with the interesting site,thus,Pfu polymerase allows perfectly matched probes to be extended without the fluorescence-labeled 3＇ end of it degradation but not the fluorescence-labeled 3＇ end mismatched probes unless the fluorescence-labeled 3＇ end is degraded.The fluorescence polarization of probe can be measured after reaction since the fluorescein linked to a large molecule has the character of fluorescence polarization.Results：All types of the mutants of YMDD genetic code of HBV polymerase can be detected by the method.The mutants can be detected in 1×101 copy templates.After testing 30 serum samples of patients who have been treated with lamividin for 6 months,6 samples proved to have the mutants of YMDD genetic code.Conclusion：This method can be used to detect the mutants of YMDD genetic code of HBV polymerase gene in serum and also can be used for other gene mutants.