中华检验医学杂志
中華檢驗醫學雜誌
중화검험의학잡지
CHINESE JOURNAL OF LABORATORY MEDICINE
2010年
3期
209-212
,共4页
陈志红%郭爱林%安社娟%郑有为%马冬%苏健%谢至%黄迎%陈世良%吴一龙
陳誌紅%郭愛林%安社娟%鄭有為%馬鼕%囌健%謝至%黃迎%陳世良%吳一龍
진지홍%곽애림%안사연%정유위%마동%소건%사지%황영%진세량%오일룡
高分辨率熔解法%KRAS基因%结直肠肿瘤
高分辨率鎔解法%KRAS基因%結直腸腫瘤
고분변솔용해법%KRAS기인%결직장종류
High resolution melting method%v-Ki-ras2 kirsten rat sarcoma viral oncngene homolog gene%Colorectal neoplasms
目的 建立HRM法检测大肠癌患者肿瘤组织KRAS(v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog)基因突变的方法 .方法 采用HRM法对含不同比例KRAS基因突变型质粒的系列混合样本进行检测,以评价其灵敏度.应用HRM法检测60份大肠癌患者新鲜肿瘤组织KRAS基因密码子12和13的突变状况,并与直接测序法的结果 进行比较分析.结果 HRM法只需在PCR结束后直接运行高分辨熔解,即可获得检测结果 .HRM法可检出系列混合样本中突变型质粒比例为10%的突变,其检测灵敏度达10%.HRM法从60份大肠癌患者组织标本中,检出17份KRAS基因密码子12或13突变(28.3%);直接测序法检出15份(25.0%)突变,2份未检出KRAS基因突变.HRM法检测的敏感度为100%(15/15),特异度为96%(43/45).结论 HRM法在筛选大肠癌标本的KRAS基因突变类型时,具有操作简便、快速、灵敏,单管避免污染等优点,完全符合临床个体化治疗的要求,值得推广.
目的 建立HRM法檢測大腸癌患者腫瘤組織KRAS(v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog)基因突變的方法 .方法 採用HRM法對含不同比例KRAS基因突變型質粒的繫列混閤樣本進行檢測,以評價其靈敏度.應用HRM法檢測60份大腸癌患者新鮮腫瘤組織KRAS基因密碼子12和13的突變狀況,併與直接測序法的結果 進行比較分析.結果 HRM法隻需在PCR結束後直接運行高分辨鎔解,即可穫得檢測結果 .HRM法可檢齣繫列混閤樣本中突變型質粒比例為10%的突變,其檢測靈敏度達10%.HRM法從60份大腸癌患者組織標本中,檢齣17份KRAS基因密碼子12或13突變(28.3%);直接測序法檢齣15份(25.0%)突變,2份未檢齣KRAS基因突變.HRM法檢測的敏感度為100%(15/15),特異度為96%(43/45).結論 HRM法在篩選大腸癌標本的KRAS基因突變類型時,具有操作簡便、快速、靈敏,單管避免汙染等優點,完全符閤臨床箇體化治療的要求,值得推廣.
목적 건립HRM법검측대장암환자종류조직KRAS(v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog)기인돌변적방법 .방법 채용HRM법대함불동비례KRAS기인돌변형질립적계렬혼합양본진행검측,이평개기령민도.응용HRM법검측60빈대장암환자신선종류조직KRAS기인밀마자12화13적돌변상황,병여직접측서법적결과 진행비교분석.결과 HRM법지수재PCR결속후직접운행고분변용해,즉가획득검측결과 .HRM법가검출계렬혼합양본중돌변형질립비례위10%적돌변,기검측령민도체10%.HRM법종60빈대장암환자조직표본중,검출17빈KRAS기인밀마자12혹13돌변(28.3%);직접측서법검출15빈(25.0%)돌변,2빈미검출KRAS기인돌변.HRM법검측적민감도위100%(15/15),특이도위96%(43/45).결론 HRM법재사선대장암표본적KRAS기인돌변류형시,구유조작간편、쾌속、령민,단관피면오염등우점,완전부합림상개체화치료적요구,치득추엄.
Objective To establish a HRM assay to screen for KRAS mutations in clinical colorectal cancer patients.Methods The sensitivity of HRM was analyzed by detecting somatic mutations in exon 2,notably codons 12 and 13 of the KRAS gene in the serial plasmid mixture samples which were mixed using the different proportions mutation plasmid and wide type plasmid of KRAS.HRM analysis was performed for KRAS on DNA insolated from a panel of 60 colorectal cancer samples derived from fresh tissues.The results were compared with the direct sequencing data.Results After the PCR amplification,the mutation results could be available by performing HRM analysis in the same tube on a real time PCR machine with HRM capability.HRM detection could identify KRAS mutation in a proportion of 10% of mutation plasmid DNA.All 60 samples identified the KRAS mutation by HRM and sequencing.17 samples were positive(28.3%) by HRM for KRAS exon 2 mutations,and 15 samples were confirmed the presence of codon 12 or 13 mutations(25.0%) and the other 2 samples were wild type by sequencing.The 60 samples detected by HRM were given 100% sensitivity with 96% specificity.Conclusions HRM is a sensitive intube methodology to screen for mutations in clinical samples.HRM will enable high-throughput screening to gene mutations to allow appropriate therapeutic choices for patients and accelerate research aimed at identifying novel mutations in human cancer.
