聚羟基丙烯酸和Van-clear在qRT-PCR法检测非小细胞肺癌EGFR基因突变中的应用研究
Application of polyhydroxy acrylic acid and Van-clear in the detection of EGFR gene mutations in non small-cell lung cancer by qRT-PCR method

摘要：目的 比较环保固定液聚羟基丙烯酸、环保透明脱蜡液Van-clear单独或联合替代传统固定液4%中性缓冲甲醛、传统透明脱蜡液二甲苯应用于实时荧光定量PCR(qRT-PCR)法，检测非小细胞肺癌(NSCLC)表皮生长因子受体(EGFR)基因突变率的敏感性、特异性及符合率的差异，评估qRT-PCR法及其环保技术平台的临床应用价值。方法 选取中山市博爱医院、中山市中医院2013年5月至2016年3月期间手术切除的原发性NSCLC标本91例，同一肿瘤病变部位切取5个样本，采用随机数字表法分为A、B、C、D、E。A组使用4%中性缓冲甲醛固定、二甲苯透明脱蜡制作切片、直接测序法检测EGFR基因突变；B组使用4%中性缓冲甲醛固定、二甲苯透明脱蜡制作切片、qRT-PCR法检测EGFR基因突变；C组使用聚羟基丙烯酸固定、二甲苯透明脱蜡制作切片、qRT-PCR法检测EGFR基因突变；D组使用4%中性缓冲甲醛固定、Van-clear透明脱蜡制作切片、qRT-PCR法检测EGFR基因突变；E组使用聚羟基丙烯酸固定、Van-clear透明脱蜡制作切片、qRT-PCR法检测EGFR基因突变。分别测定5组NSCLC中EGFR基因第18、19、20、21号外显子的突变情况。 结果 ①B、C、D、E组与A组比较，EGFR基因发生突变人数百分率、基因靶位点突变率的差异均无统计学意义(P>0.05)；②B、C、D、E组与A组比较，EGFR靶位点突变检测结果的灵敏度好、特异度强、符合率高。结论 聚羟基丙烯酸、环保透明脱蜡液Van-clear单独或联合替代传统试剂，应用于qRT-PCR法检测NSCLC中EGFR基因突变，与以传统试剂应用于直接测序法的检测结果一致性好，有在临床推广应用的意义和价值。
ABSTRACT: Objective To compare two different methods to detect the differences of gene mutation rate, sensitivity, specificity and coincidence rate of epidermal growth factor receptor (EGFR) in non-small-cell lung cancer (NSCLC) so as to assess the clinical value of qRT-PCR method and its environmental-friendly technology platforms. One uses environmental fixative poly hydroxyl acrylic acid and green transparent liquid dewaxing Van-clear alone or in combination to replace the traditional fixative 4% (volume fraction) neutral buffered formalin and the traditional transparent dewaxing liquid xylene in application of quantitative real-time polymerase chain reaction (qRT-PCR). The other uses traditional reagents in direct sequencing. Methods We selected 91 cases of primary NSCLC specimens resected between May 2013 and March 2016 in Zhongshan Bo’ai Hospital and Zhongshan Hospital of Traditional Chinese Medicine. Five samples were taken from the same tumor lesion. We used a random number table to randomly divide these samples into Groups A, B , C, D, and E. Group A received direct sequencing method in detection of EGFR gene mutations. Besides, during the experiment, 4% neutral buffered formalin was used for fixing, and xylene transparent dewaxing was used to make slices for DNA extraction dewaxing. Group B received qRT-PCR method to detect EGFR gene mutations. Meanwhile, during the experiment, 4% neutral buffered formalin was used for fixing, and xylene transparent dewaxing was used to make slices for DNA extraction dewaxing. Group C received qRT-PCR method in detection of EGFR gene mutations. At the same time, during the experiment, polyhydroxy acrylic acid was used for fixing, and xylene transparent dewaxing was used to make slices for DNA extraction dewaxing. Group D received qRT-PCR method to detect EGFR gene