巢式RT-PCR扩增Keration19诊断胃癌淋巴结微转移方法及应用

Method of Detecting Gastric Cancer Micrometastases in Lymph Nodes by Nest RT-PCR Amplification of Keration 19 and its Application

[目的]建立RT PCR扩增Keratin19mRNA的方法 ,评价其应用前景。[方法]采用Keratin19cDNA的套式引物建立巢式RT PCR扩增体系 ,以酶切分析及DNA点杂交法鉴定扩增产物的特异性 ,逆转录产物系列稀释法分析检测敏感性 ,并对51例临床胃周淋巴结样品作初步检测。[结果]该扩增体系具有较好的扩增特异性 ,检测敏感性达1pgRNA ,相当于从105 个淋巴细胞中检出1个胃癌细胞 ;对临床样品检测结果显示该法较病理检查法敏感性高。[结论]该扩增体系具有较好的特异性、敏感性和较高的可靠性。

To develop a RT-PCR method to amplify Keratin 19 mRNA for detecting gastric cancer cells metastases in gastric lymph nodes and to evaluate its specificity and sensitivity.The RT-PCR system was set up by using nest primer.Its amplification products wasidentified by restriction enzyme digestion and DNA dot blot.The sensitivity was estimated with serial diluted reverse transcript templates.Moreover,51 lymph nodes were analyzed by this RT-PCR method and pathological examination,respectively.This system possessed a good specificity,its detection sensitivity was 1pg RNA from 100ng control RNA which means detecting out one gastric cancer cell from 105 lymph cells.The results performed on clinical gastric lymph nodes showed that the sensitivity of RT-PCR was superior to pathological examination.The dot hybridization test further proved the high specificity of RT-PCR.〔Conclusion〕 The amplification system presents good reliability,high sensitivity and specificity.