应用双向热循环消减SELEX技术筛选肺癌血清核酸适配体

Screening of Serum Nucleic Acid Aptamers for Lung Cancer by Bidirectional Thermal Circulation Subtractive SELEX

肺癌是发病率和死亡率较高的恶性肿瘤。现阶段,用于肺癌早期诊断的血清肿瘤标志物因其特异性与敏感性均较低,严重影响肺癌的临床诊断和治疗。本文用双向热循环消减指数富集的配基进化(systematic evolution of ligands by exponential enrichment,SELEX)技术,筛选肺癌和非癌血清标志物的核酸适配体,建立肺癌的检测方法,提高诊断和治疗效率。实验用环氧基琼脂磁珠为筛选介质,以非癌混合血清、肺癌混合血清作为双向靶标。应用热循环消减SELEX技术,经19轮筛选分别获得非癌和肺癌血清的特异性核酸适配体。通过高通量测序,得到40条非癌核酸适配体序列和41条肺癌核酸适配体序列。从肺癌与非癌血清特异性核酸适配体序列中分别挑选出高丰度的4条序列,合成后制成检测试剂,经临床血清验证,阳性率为90%。该检测方法检测灵敏度高,为肺癌的早期诊断和治疗提供了新的分子识别元件。

Lung cancer is a malignant tumor with high morbidity and mortality.At present,serum tumor markers used for early diagnosis of lung cancer are low in specificity and sensitivity,which seriously affect the clinical diagnosis and treatment of lung cancer.In this paper,bidirectional thermal circulation subtractive systematic evolution of ligands by exponential enrichment(SELEX)technique was used to screen aptamers for serum markers of lung cancer and non-cancer to establish a method for detection of lung cancer and to improve the efficiency of diagnosis and treatment.Epoxy agar microspheres were used as screening medium,non-cancer mixed serum and lung cancer mixed serum were used as bidirectional targets.After 15 cycles of screening,the specific aptamers of non-cancerous and lung cancer serum were obtained by the thermal circulation subtractive SELEX technique.Forty nucleic acid aptamers sequences of non-cancerous and 41 nucleic acid aptamers sequences of lung cancer were obtained by high-throughput sequencing.Four high abundance sequences were selected from serum-specific aptamers sequences of lung cancer and non-cancerous sera.These sequences were synthesized and prepared of detection reagent.Though clinical verification experiment,the positive rate was 90%.This method has high sensitivity,which provides a new molecular recognition element for early diagnosis and treatment of lung cancer.