建立单管双向荧光PCR方法检测NQO1基因609C／T多态性

Establishing a single-tube fluorescent bidirectional PCR method to detect the 609C/T polymorphism of NQO1

目的建立单管双向荧光PCR方法快速测定人NAD（P）H：醌氧化还原酶1[NAD（P）H：quinone oxidoreductase 1，NQ01]基因609C／T多态性。方法以人NQ01基因中的609C／T位点，设计双向引物，优化反应条件，应用SYBR Green Ⅰ双向荧光PCR扩增191份人基因组DNA标本，并通过对产物进行熔解曲线分析，根据产物Tm值进行等位基因单核苷酸多态性分型。对其中62份标本用经典的PCR-限制性片段长度多态方法进行基因型分型，验证结果准确性。结果62份样本的单管双向荧光PCR方法的基因型结果与PCR-限制性片段长度多态法分型结果符合率100％。191份样本中，纯合野生型（CC）占28％，杂合型（CT）占50％，纯合突变型（TT）占22％。结论单管双向荧光PCR方法检测NQ01基因609C／T多态性，操作简便、反应过程快速，结果直观，敏感性、准确性和稳定性好，适用于临床样本检测及流行病学调查研究。

Objective To develop a single-tube fluorescent bidirectional PCR method to detect the 609C/T polymorphism of NAD（P）H： quinone oxidoreduetase 1 （NQO1） gene. Methods Two primers of NQO1 gene C609T locus were designed. Using these primers, a SYBR Green Ⅰ fluorescent bidirectional PCR, combined with melting curve analysis of the PCR products, were optimized to differentiate the 609C/T polymorphisms in 191 samples of human genomic DNA. The accuracy of the fluorescent bidirectional PCR was validated by the classical method of PCR-restriction fragment length polymorphism（RFLP） in 62 of these 191 samples. Results In the 62 samples, the genotypes determined by the fluorescent bidirectional PCR were 100% consistent with the ones by the PCR-RFLP. The frequencies of genotypes of homozygous wild-type （CC）, heterozygous （CT）, and homozygous mutant （TF） were 28%, 50%, and 22%, respectively, in the 191 samples. Conclusion The single-tube fluorescent bidirectional PCR method established here provides a simple, rapid, accurate and inexpensive assay to determine the 609C/T polymorphism of NQO1 gene. The assay is suitable to detect the single nucleotide polymorphism in large-scale samples.