用PCR-RFLP法对三带喙库蚊钠通道基因L1014F突变快速检测与分型

Rapid genotyping L1014F substitution in sodium channel of Culex tritaeniorhynchus with PCR-RFLP method

目的建立PCR-RFLP方法对三带喙库蚊钠通道基因L1014F(TTA-TTT)突变进行快速检测与分型。方法根据三带喙库蚊钠通道基因DNA序列,合成PCR引物,在上游引物3'端第二个碱基引入突变位点"C",构建酶切位点,扩增后对PCR产物酶切,在电泳结果读取分型结果并统计基因频率与基因型频率。结果建立的PCR-RFLP方法能够对三带喙库蚊钠通道基因L1014F突变进行快速检测与分型,灵敏度和特异度均达到98%以上。结论本研究建立的PCR-RFLP方法灵敏度和特异度较高,实现了对三带喙库蚊钠通道L1014F(TTA-TTT)突变的快速分型,可以推广使用。

Objective To establish a method for rapid detecting L1014 F substitution in sodium channel of Culex tritaeniorhynchus. Methods A primer was designed according to the point mutation in L1014 loci with a changed base 'C'in the second loci of 3' end and used as the sense primer so that an enzyme loci would be formed in PCR product. A common anti-sense primer was designed according to the DNA sequence of Culex tritaeniorhynchus. The point mutation could be genotyped based on the electrophoresis results. Results The optimal experiment displayed good amplification efficiency and could complete the genotyping and the mutative allele with the PCR- RFLP procedure. The sensitivity and specificity were all beyond 98%. Conclusion The PCR- RFLP method with high specificity sensitivity and reproducibility is suitable for rapid detection of L1014 F substitution in sodium channel of Culex tritaeniorhynchus.