心肌肌钙蛋白I的临床应用及检测方法进展

cTnI(心肌肌钙蛋白I)是心肌肌钙蛋白的一种亚型,正常人血液中含量很低,当心肌细胞损伤时,cTnI在血液中的含量迅速升高。近年来随着cTnI检测方法的不断改进,cTnI已成为最具有临床价值的心肌损伤标志物之一,被广泛应用于心脏疾患的临床诊断和检测。     

应用多重PCR-变性高效液相色谱技术检测大片段重复或缺失突变

目的 探讨多重PCR-变性高效液相色谱(PCR-denature high performance liquid chromatography,PCR-DHPLC)技术在假肥大型肌营养不良症和脊肌萎缩症外显子拷贝数异常检测中的应用价值.方法 应用多重PCR-DHPLC方法筛查35例假肥大型肌营养不良症患者和6例脊肌萎缩症患者.选择阳性对照和阴性对照进行方法学可靠性检验.结果 多重PCR-DHPLC可完全检测出全部阳性对照中重复或缺失片段.35例假肥大型肌营养不良症样本中检测出5例大片段重复,20例大片段缺失,突变检出率为71.4%.6例脊肌萎缩症患者均检测到第7外显子缺失.结论 多重PCR-DHPLC分析系统可作为有效筛查大片段重复或缺失突变的检测方法.     

心肌肌钙蛋白Ⅰ的临床应用及检测方法进展

cTnI(心肌肌钙蛋白Ⅰ)是心肌肌钙蛋白的一种亚型，正常人血液中含量很低，当心肌细胞损伤时，cTnI在血液中的含量迅速升高。近年来随着cTnI检测方法的不断改进，cTnI已成为最具有临床价值的心肌损伤标志物之一，被广泛应用于心脏疾患的临床诊断和检测。     

心肌肌钙蛋白Ⅰ的临床应用及检测方法进展

cTnI(心肌肌钙蛋白Ⅰ)是心肌肌钙蛋白的一种亚型,正常人血液中含量很低,当心肌细胞损伤时,cTnI在血液中的含量迅速升高。近年来随着cTnI检测方法的不断改进,cTnI已成为最具有临床价值的心肌损伤标志物之一,被广泛应用于心脏疾患的临床诊断和检测。     

酶切富集联合DHPLC检测肿瘤患者外周血EGFR和KRAS基因突变及其临床应用

Objective To establish a REDE-DHPLC method for detecting the EGFR and KRAS mutations in plasma DNA from tumor patients, and investigate its clinical significance. Methods Restriction endonucleases Mse Ⅰ , Msc Ⅰ , BstN Ⅰ and Bgl Ⅰ were used to digest the wild type fragments of exon 19,exon 21 of EGFR gene and coden 12, 13 of KRAS gene for enriching the mutation fragments, and REDE-DHPLC method was established to detect EGFR and KRAS mutations. The sensitivities of REDE-DHPLC and conventional DHPLC were analyzed by using a series of plasmids containing 50%, 10%, 5%, 1% and 0. 1% mutation genes. Then, Plasma samples and paraffin-embedded tissue samples of 120 NSCLC patients and 120 colorectal cancer patients were detected by REDE-DHPLC. Compared with conventional DHPLC and sequencing, the diagnostic efficiency of REDE-DHPLC method was evaluated by detecting the mutation status of 2 genes in plasma of NSCLC and colorectal cancer patients. Results The sensitivity values of REDE-DHPLC and conventional DHPLC for detecting mutations in 4 loci were 0. 1% and 1%respectively. Plasmid DNA containing 0.1% mutation gene was detected to be positive continually for 2 to 3 times by REDE-DHPLC. EGFR mutation rates of 120 plasma from NSCLC patients detected by REDE-DHPLC, conventional DHPLC and sequencing methods were 27. 5%, 16. 7% and 12.5% respectively, and KRAS mutation rates of 120 plasma from colorectal cancer patients were 38. 3%, 25. 8% and 16. 7%,respectively. The positive rates of EGFR and KRAS mutation detected by REDE-DHPLC were significantly higher than conventional DHPLC(x2 = 4. 092, 4. 301, all P ＜ 0. 05 ) and sequencing method (x2= 8. 438,14. 127,all P ＜ 0. 05 ). In comparison with conventional DHPLC, the sensitivities of REDE-DHPLC for detecting EGFR and KRAS mutation were 100% (20/20,31/31), the specificities were 87. 0% (87/100)and 83. 2% (74/89). In comparison with sequencing method, the sensitivities of REDE-DHPLC were 100%( 15/15,20/20), the specificities were 82.9% (87/105)and 74. 0% (74/100). The coincidence rate of the two methods for detecting EGFR and KRAS mutation were 89. 2% ( 107/120, Kappa = 0. 690, P ＜ 0. 05 ) and 87.5% ( 105/120, Kappa= 0. 718, P ＜ 0. 05 ). The Consistency of EGFR and KRAS mutation status in plasma and tissues detected by REDE-DHPLC were 91.7% (33/36, Kappa =0. 939,P ＜0. 05)and 90. 2 %(46/51, Kappa = 0. 914, P ＜ 0. 05 ), respectively. Conclusions The REDE-DHPLC method is highly sensitive and specific for detecting EGFR and KRAS mutations in plasma DNA from tumor patients. The results are easy to be interpreted without missing homozygous point mutation, which indicate that the detection of EGFR and KRAS mutations in plasma DNA by REDE-DHPLC could therefore extend to be usedin clinical laboratory.     

欧洲神经科学协会联盟关于神经遗传病的分子诊断指南:运动神经元病、周围神经病和肌肉病

一、引言自从2001年欧洲神经科学协会联盟(European Federation of Neurological Societies,EFNS)发表关于神经系统遗传性疾病的分子诊断指南以来,这一领域的发展非常迅速。依据EFNS科学委员会对于指南性文件的建议,目前已需对现有的版本进行更新。二、目标EFNS关于运动神经元病、周围神经病和肌病的分子诊断指南旨在总结分子遗传学技术的可行性和局限性,并为决策何时     

遗传性非息肉病性结直肠癌的分子病理研究

目的研究遗传性非息肉病性结直肠癌的分子病理特征,评估该病在北京地区住院人群中的发病情况。方法 94例结直肠癌组织标本均采集自2012年3~9月于北京市中西医结合医院及解放军总医院住院接受手术治疗并经病理确诊为结直肠癌的患者。使用免疫组化方法对肿瘤组织内错配修复基因MLH1、MSH2、MSH6及PMS2的蛋白表达情况进行检测。结果 94例结直肠癌患者中,至少有一种错配修复基因表达为阴性的共有13例,其中MLH1表达为阴性的有3例,MSH2表达为阴性的有11例,MSH6表达为阴性的有2例,PMS2表达为阴性的仅有1例,遗传性非息肉病性结直肠癌的发病率为13.83%,MSH2基因突变所占比例达84.62%,与遗传性非息肉病性结直肠癌国际合作组织提供的数据（40%）相比,差异有统计学意义（P〈0.05）。结论北京地区住院的结直肠癌患者中,遗传性非息肉病性结直肠癌发病率高于世界每年新发病例的水平,且MSH2基因突变致病尤为突出。     

酶切富集联合DHPLC检测肿瘤患者外周血EGFR和KRAS基因突变及其临床应用

目的 建立一种REDE-DHPLC检测外周血肿瘤游离核酸EGFR、KRAS基因突变的方法,并探讨其临床应用价值.方法 采用限制性内切酶Mse Ⅰ、Msc Ⅰ、BstN Ⅰ和BglⅠ分别切断EGFR基因第19、21号外显子和KRAS基因第12、13号密码子的野生型片段以富集突变片段,建立检测血浆EGFR和KRAS基因突变的REDE-DHPLC法,并采用50%、10%、5%、1%和0.1%稀释度的质粒标准品评价REDE-DHPLC法和传统DHPLC法的检测灵敏度.然后,采用REDE-DHPLC法检测120例NSCLC和120例结直肠癌患者血浆和石蜡组织标本中的EGFR和KRAS基因突变.同时,用传统DHPLC法和测序法进行平行检测,以评价REDE-DHPLC法检测NSCLC和结直肠癌患者血浆中2个基因突变的诊断效能.结果 REDE-DHPLC法对EGFR和KRAS基因4个突变位点检测的灵敏度均达0.1%,传统DHPLC法检测灵敏度均为1.0%,REDE-DHPLC法对含0.1%突变的质粒标准品重复2～3次检测均为阳性.REDE-DHPLC法、传统DHPLC法和测序法检测120例NSCLC患者血浆EGFR基因总突变率分别为27.5%、16.7%和12.5%,检测120例结直肠癌患者血浆KRAS基因总突变率分别为38.3%、25.8%和16.7%.REDE-DHPLC法检测EGFR和KRAS基因总突变率均高于传统DHPLC法(x2值分别为4.092、4.301,P均＜0.05)和测序法(x2值分别为8.438、14.127,P均＜0.05);将REDE-DHPLC法检测EGFR和KRAS基因突变与传统DHPLC法相比,敏感度均为100%(20/20,31/31),特异度分别为87.0%(87/100)和83.2%(74/89);与测序法相比,敏感度均为100%(15/15,20/20),特异度分别为82.9%(87/105)和74.0%(74/100).REDE-DHPLC法与传统DHPLC法检测EGFR、KRAS基因突变的符合率分别为89.2%(107/120,Kappa=0.690,P＜0.05)和87.5%(105/120,Kappa=0.718,P＜0.05).REDE-DHPLC检测血浆与组织标本中EGFR和KRAS基因总突变符合率分别为91.7%(33/36,Kappa=0.939,P＜0.05)和90.2%(46/51,Kappa=0.914,P＜0.05).结论 REDE-DHPLC法灵敏度和特异性高,结果易判读,且可有效避免纯合点突变漏检,有望成为临床实验室外周血EGFR和KRAS基因突变检测的推广方法.

Abstract：

Objective To establish a REDE-DHPLC method for detecting the EGFR and KRAS mutations in plasma DNA from tumor patients, and investigate its clinical significance. Methods Restriction endonucleases Mse Ⅰ , Msc Ⅰ , BstN Ⅰ and Bgl Ⅰ were used to digest the wild type fragments of exon 19,exon 21 of EGFR gene and coden 12, 13 of KRAS gene for enriching the mutation fragments, and REDE-DHPLC method was established to detect EGFR and KRAS mutations. The sensitivities of REDE-DHPLC and conventional DHPLC were analyzed by using a series of plasmids containing 50%, 10%, 5%, 1% and 0. 1% mutation genes. Then, Plasma samples and paraffin-embedded tissue samples of 120 NSCLC patients and 120 colorectal cancer patients were detected by REDE-DHPLC. Compared with conventional DHPLC and sequencing, the diagnostic efficiency of REDE-DHPLC method was evaluated by detecting the mutation status of 2 genes in plasma of NSCLC and colorectal cancer patients. Results The sensitivity values of REDE-DHPLC and conventional DHPLC for detecting mutations in 4 loci were 0. 1% and 1%respectively. Plasmid DNA containing 0.1% mutation gene was detected to be positive continually for 2 to 3 times by REDE-DHPLC. EGFR mutation rates of 120 plasma from NSCLC patients detected by REDE-DHPLC, conventional DHPLC and sequencing methods were 27. 5%, 16. 7% and 12.5% respectively, and KRAS mutation rates of 120 plasma from colorectal cancer patients were 38. 3%, 25. 8% and 16. 7%,respectively. The positive rates of EGFR and KRAS mutation detected by REDE-DHPLC were significantly higher than conventional DHPLC(x2 = 4. 092, 4. 301, all P ＜ 0. 05 ) and sequencing method (x2= 8. 438,14. 127,all P ＜ 0. 05 ). In comparison with conventional DHPLC, the sensitivities of REDE-DHPLC for detecting EGFR and KRAS mutation were 100% (20/20,31/31), the specificities were 87. 0% (87/100)and 83. 2% (74/89). In comparison with sequencing method, the sensitivities of REDE-DHPLC were 100%( 15/15,20/20), the specificities were 82.9% (87/105)and 74. 0% (74/100). The coincidence rate of the two methods for detecting EGFR and KRAS mutation were 89. 2% ( 107/120, Kappa = 0. 690, P ＜ 0. 05 ) and 87.5% ( 105/120, Kappa= 0. 718, P ＜ 0. 05 ). The Consistency of EGFR and KRAS mutation status in plasma and tissues detected by REDE-DHPLC were 91.7% (33/36, Kappa =0. 939,P ＜0. 05)and 90. 2 %(46/51, Kappa = 0. 914, P ＜ 0. 05 ), respectively. Conclusions The REDE-DHPLC method is highly sensitive and specific for detecting EGFR and KRAS mutations in plasma DNA from tumor patients. The results are easy to be interpreted without missing homozygous point mutation, which indicate that the detection of EGFR and KRAS mutations in plasma DNA by REDE-DHPLC could therefore extend to be usedin clinical laboratory.     

酶切富集联合DHPLC检测肿瘤患者外周血EGFR和KRAS基因突变及其临床应用

Objective To establish a REDE-DHPLC method for detecting the EGFR and KRAS mutations in plasma DNA from tumor patients, and investigate its clinical significance. Methods Restriction endonucleases Mse Ⅰ , Msc Ⅰ , BstN Ⅰ and Bgl Ⅰ were used to digest the wild type fragments of exon 19,exon 21 of EGFR gene and coden 12, 13 of KRAS gene for enriching the mutation fragments, and REDE-DHPLC method was established to detect EGFR and KRAS mutations. The sensitivities of REDE-DHPLC and conventional DHPLC were analyzed by using a series of plasmids containing 50%, 10%, 5%, 1% and 0. 1% mutation genes. Then, Plasma samples and paraffin-embedded tissue samples of 120 NSCLC patients and 120 colorectal cancer patients were detected by REDE-DHPLC. Compared with conventional DHPLC and sequencing, the diagnostic efficiency of REDE-DHPLC method was evaluated by detecting the mutation status of 2 genes in plasma of NSCLC and colorectal cancer patients. Results The sensitivity values of REDE-DHPLC and conventional DHPLC for detecting mutations in 4 loci were 0. 1% and 1%respectively. Plasmid DNA containing 0.1% mutation gene was detected to be positive continually for 2 to 3 times by REDE-DHPLC. EGFR mutation rates of 120 plasma from NSCLC patients detected by REDE-DHPLC, conventional DHPLC and sequencing methods were 27. 5%, 16. 7% and 12.5% respectively, and KRAS mutation rates of 120 plasma from colorectal cancer patients were 38. 3%, 25. 8% and 16. 7%,respectively. The positive rates of EGFR and KRAS mutation detected by REDE-DHPLC were significantly higher than conventional DHPLC(x2 = 4. 092, 4. 301, all P ＜ 0. 05 ) and sequencing method (x2= 8. 438,14. 127,all P ＜ 0. 05 ). In comparison with conventional DHPLC, the sensitivities of REDE-DHPLC for detecting EGFR and KRAS mutation were 100% (20/20,31/31), the specificities were 87. 0% (87/100)and 83. 2% (74/89). In comparison with sequencing method, the sensitivities of REDE-DHPLC were 100%( 15/15,20/20), the specificities were 82.9% (87/105)and 74. 0% (74/100). The coincidence rate of the two methods for detecting EGFR and KRAS mutation were 89. 2% ( 107/120, Kappa = 0. 690, P ＜ 0. 05 ) and 87.5% ( 105/120, Kappa= 0. 718, P ＜ 0. 05 ). The Consistency of EGFR and KRAS mutation status in plasma and tissues detected by REDE-DHPLC were 91.7% (33/36, Kappa =0. 939,P ＜0. 05)and 90. 2 %(46/51, Kappa = 0. 914, P ＜ 0. 05 ), respectively. Conclusions The REDE-DHPLC method is highly sensitive and specific for detecting EGFR and KRAS mutations in plasma DNA from tumor patients. The results are easy to be interpreted without missing homozygous point mutation, which indicate that the detection of EGFR and KRAS mutations in plasma DNA by REDE-DHPLC could therefore extend to be usedin clinical laboratory.