HGF mRNA实时荧光定量PCR在淋巴瘤的应用

Establishment of real-time fluorescence quantitative polymerase chain reaction analysis system for hepatocyte growth factor mRNA expression and its application in lymphoma

目的构建肝细胞生长因子(HGF)mRNA定量标准,建立实时荧光定量PCR(FQ-PCR)检测体系,探讨在淋巴瘤的应用价值。方法提取总RNA,行RT-PCR,获HGF cDNA目标片段并构建重组质粒,作为定量标准。设计第二引物对和探针,确定扩增条件,优化组分浓度,建立HGF实时FQ-PCR检测体系,并进行方法学评价。应用该检测体系定量检测47例淋巴瘤的HGF mRNA表达,并采用受试者工作特征(ROC)曲线法,评价其在淋巴瘤诊断中的敏感度和特异度。结果成功构建了HGF定量标准,结合热启动PCR和降落PCR技术创建了HGF mRNA实时FQ-PCR检测体系。方法学评价实验表明:标准曲线的斜率为-3.513,相关系数为0.999,扩增效率达92.6%;批内、批间和日间变异系数分别为2.1%、4.0%和6.8%;灵敏度达2拷贝/μl。HGF mRNA在对照组和淋巴瘤组的表达分别为0.317±0.192和6.425±2.172,组间差异具统计学意义(P【0.001),但HD组和NHL组的表达无显著差异(P】0.05),而缓解组显著低于复发组(P【0.05)。ROC曲线法提示,当临床诊断界值为3.316时,HGF mRNA对淋巴瘤诊断敏感度和特异度分别为93.6%和100%。结论成功构建了HGF mRNA定量标准。建立的HGF mRNA FQ-PCR检测体系是理想的、可行的,可定量检测淋巴瘤HGF mRNA并具诊断价值。

Objective To construct quantitative standard for hepatocyte growth factor(HGF)mRNA expression and establish HGF mRNA real-time fluorescence quantitative PCR(FQ-PCR)analysis system.Meanwhile,to estimate its application in lymphoma.Methods Recombined plasmid was constructed with target cDNA which was obtained from isolated total RNA by RT-PCR.Having been identified and purified,recombined plasmids were quantitated and then considered as quantitative standard.A new real-time FQ-PCR analysis system was created with second pair of primers and the probe after amplification temperature and the concentrations of components were optimized.Meanwhile,its methodology evaluation was carried out.HGF mRNA expressions in 47 lymphoma cases were quantitatively analyzed by this method,and its specificity and sensitivity in lymphoma diagnosis were evaluated by receiptor operation character(ROC)curve method,too.Results HGF quantitative standard was successfully constructed,and then HGF mRNA real-time FQ-PCR analysis system was established combined with hot-start PCR and down-touch PCR technique.According to the slope of the standard curve(-3.513)and correlation coefficient(0.999),amplification efficiency of the system was 92.6%.The coefficient variation of intra-assay,inter-assay and inter-day-assay were 2.1%,4.0%and 6.8%,respectively.The sensitivity of FQ-PCR was 2 copies recombined plasmid/μl.Expressions of HGF mRNA in lymphoma group and control group were 0.317±0.192 and 6.425±2.172,respectively(P<0.001).Although it was different between remission group and relapase group(P<0.05),it was similar between HD group and NHL group(P>0.05).According to ROC analysis,its sensitivity and specificity were 93.6% and 100% with the cutoff value of 3.136 for lymphoma clinical diagnosis.Conclusion HGF mRNA real-time FQ-PCR analysis system has been successfully constructed and is proved to be an accurate and feasible method,which can quantitatively detect the expression of HGF mRNA in lymphoma,and be applied on the diagnosis of lymphoma.