AS-PCR检测TLR9基因SNP方法的建立及应用

目的 建立一种快速检测Toll样受体9（toll-like receptor 9,TLR9）基因rs187084和rs352140单核苷酸多态性（single nucleotide polymorphism,SNP）的方法,以及应用该方法检测湖北地区汉族人群各基因型的分布情况.方法 根据TLR9基因rs187084和rs352140 SNP中碱基变异频率分别设计野生型、突变型以及内参引物,建立等位基因特异性聚合酶链反应（allele specific PCR,AS PCR）检测135例体检人群外周血基因组DNA,以DNA测序法检测结果作为参照标准,并统计分析不同基因型在人群中的分布.结果 135例样本SNP位点均出现相应的野生型、突变型或者杂合子个体,AS-PCR的检测结果与DNA测序符合率为100％.湖北地区体检人群TILR9基因rs187084位点基因型为：CC型占8.14％（11/135）,TT型占41.48％（56/135）,CT型占50.37％（68/135）.CC型所占比例最小（χ^2=82.21,P＜0.05）,CT型比例略高于TT型,差异无统计学意义（χ^2=2.15,P＞0.05）.TLR9基因rs352140位点基因型为：CC型占41.48％（56/135）,TT型占11.11％（15/135）,CT型占47.41％（64/135）. TT基因型所占比例最小（χ^2=46.07,P＜0.05）,CT型略高于CC型,差异无统计学意义（χ^2 =0.96,P＞0.05）.结论 AS-PCR是一种简单、快速、可靠以及低成本检测TLR9基因SNP的方法.

Establishment and Application of AS-PCR for Detecting SNPs of TLR9

Objective To establish a rapid method for detecting rs187084 and rs352140 polymorphisms of toll like receptor 9（TI.R9） gene,and then to detect the distribution of genotypes of healthy population in Hubei by this method. MethodsWild type,nmtant and internal standard primers were designed for rs187084 and rs352140 polymorphisms of TI.R9 accord-ing to the mutation frequency of bases. 135 cases healthy population＇s peripheral blood genomic DNA were detected by allelespecific PCR（APCR）. The results of DNA sequencing were set as reference standard, counting and analyzing the distribution of genotypes in healthy population. Results Wild type,mutant and heterozygous were all appeared in the 2 SNP locus ofthe 135 cases. The coincidence rate between AS-PCR and DNA sequencing Was 100%. TLR9 gene rs187084 polymorphisminCC, TT, CT genotype distribution in Hubei population frequencies were 8.14% （11/135）, 41.48G （56/135） and50.37 % （68/135）, respectively. The percentage of CC genotype was the lowest （X2 82.21, P〈 0.05 ）, CT genotype wasslightly higher than TT genotype. There was no significant difference between CT genotype and TT genotype （X2 =2. 15 ,P〈0.05）. TI.R9 gene rs352140 polymorphism in CC, TT, CT genotype distribution in Hubei population frequencies were41.48% （56/135）,11. 11%（15/135） and 47.41% （64/135）,respectively. The percentage of TT genotype was the lowest（Ze =46.07 ,P〈0.05） ,CT genotype was slightly higher than CC genotype. There was no significant difference between CTgenotypc and CC genotype （χ^2=0.96, P〉0.05）. Conclusion A＆PCR is a simple, rapid, reliable and economical method fordetecting the SNPs of TLRg.