关键词: 血栓形成易感基因, 基因芯片, 聚合酶链式反应, 血栓性疾病

Abstract: 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.

Key words: thrombosis susceptibility gene, gene chip, polymerase chain reaction technique, thrombotic diseases