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[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。
[Objective] To establish RT PCR amplification method of Keratin19 mRNA and evaluate its application prospect. [Methods] A nested RT PCR amplification system was established by nested primers of Keratin19 cDNA. The specificity of the amplified products was identified by enzyme digestion analysis and DNA dot hybridization. The sensitivity of the assay was analyzed by serial dilution method of reverse transcriptase, and 51 cases were analyzed. Clinical lymph node samples were initially tested. [Results] The amplified system had good amplification specificity and the detection sensitivity reached 1pgRNA, which was equivalent to detecting one gastric cancer cell from 105 lymphocytes. The results of clinical samples showed that this method was more sensitive than pathological examination. High sex. [Conclusion] The amplification system has good specificity, sensitivity and high reliability.