血栓形成易感基因芯片的研制及效果评价

目的：探讨血栓形成易感基因芯片的研制方法及初步临床验证，建立一种快速且高通量检测血栓形成易感基因突变的方法。方法：根据GenBank上已发表的血栓形成易感基因序列，将设计好的参照探针和特异性探针点置于醛基化处理的玻片上，经紫外交联后固定，制成血栓形成易感基因芯片。以阳性参考品（各检测位点突变型和正常型基因）和阴性参考品（双蒸水）为模板，经PCR反应后与芯片进行杂交，对基因芯片的有效性进行检测。以靶序列经过测序验证的人类基因组DNA为模板，经PCR反应后芯片进行杂交，检测基因芯片的特异度和灵敏度，并对来自吉林、河南和云南3个地区的健康受试者150人和有不明原因血栓性疾病家族史的血栓患者24例进行临床验证。分析指标为检测相应位点的杂交信号强度。结果：以阳性参考品和阴性参考品为模板进行杂交，相应位点出现特异性的杂交信号，说明基因芯片检测位点有效。用于检测选定突变位点的基因芯片均有特异性的杂交信号，说明基因芯片的特异性良好。标准基因组DNA逐级稀释后检测基因芯片的灵敏度为50~100 mg·L^-1。临床验证结果，在健康受试者150人中8人检出血栓形成易感基因突变，在有不明原因血栓性疾病家族史的血栓患者24例中20例检出血栓形成易感基因突变，该芯片对不明原因血栓性疾病家族史的血栓患者血栓形成易感基因突变检出率明显高于健康受试者（P<0.05）。结论：研制的血栓形成易感基因芯片具有良好的特异度和灵敏度，对血栓形成易感基因有较高的检出率，在血栓性疾病的早期诊断和易感风险评估中具有潜在价值。

Development and evaluation of thrombosis susceptibility gene chip

Objective: To explore the development methods and preliminary clinical validation of thrombosis susceptibility gene chip, and to illustrate a rapid and high-throughput method for detecting the thrombosis susceptibility gene mutation. Methods: According to the published gene sequences of thrombotic susceptibility genes in GenBank, the reference probes and special probes were pointed on the glass slide. After ultraviolet crosslinking, thrombosis susceptibility gene chip was eatablished. The validity of gene chip was tested by positive reference (mutant gene and normal gene at each detection site) and negative reference (distilled water) as templates, thereby performing PCR reaction. The specificity and sensitivity of gene chip were detected by using the human genome DNA of target sequence proven by sequencing as templates, thereby performing PCR reaction. Meanwhile 150 healthy subjects and 24 thrombosis patients with family history of unexplained thrombotic disease from Jilin province, Henan province and Yunnan province were carried out the clinical verification of gene chip. The analysis index was the hybridization signal strength of the corresponding site. Results: The hybridization results of positive reference and negative reference as templates showed that the specific hybridization signals appeared at the corresponding sites, which indicated that the detection sites of gene chip are effective. The gene chip used to detect the selected mutation sites had specific hybridization signals, suggesting there were good specificity of gene chip. The sensitivity of gene chip was 50-100 mg·L^-1 by testing genomic DNA with stepwise dilution. Eight individuals with thrombosis susceptibility gene mutations were found in 150 healthy subjects. Twenty individuals with thrombosis susceptibility gene mutations were found in 24 thrombosis patients with family history of unexplained thrombotic disease. The statistcs results showed that the detection rate of thrombosis susceptibility gene mutations in the thrombosis patients with unexplained thrombotic disease family history was significantly higher than that of the healthy subjects (P<0.05). Conclusion: The developed thrombosis susceptibility gene chip has great specificity, sensitivity, and high detection rate of thrombosis susceptibility genes. It has potential values in the early diagnosis and susceptibility risk assessment of thrombotic diseases.