荧光定量PCR快速检测淡色库蚊击倒抗性相关钠通道基因突变（L1014F）

Rapid Detection of kdr Point Mutation （L1014F） of Culex pipiens pallens with Real-Time Fluorescent Quantitative PCR

本研究的目的是采用荧光定量PCR扩增技术，建立快速检测淡色库蚊CulexpipienspaUens钠离子通道L1014位点击倒抗性（Knockdownresistance，kdr）相关点突变方法，该方法的原理是以突变位点为3’端设计两条特异性上游引物，和一条非特异下游引物组成两对引物平行双管法，用荧光定量PCR扩增快速检测淡色库蚊钠通道L1014F突变的SS，SR，RR3种基因型。用此方法对48个北京淡色库蚊样本进行基因分型，42个样本检测结果与等位基因特异性PCR（AS—PCR）检测结果完全一致，得到ss（TTA／TTA）基因型21个。占50．0％，SR（TTA／TTT）基因型16个，占38．1％，RR（TTT／1W）基因型5个，占11．9％，与测序结果完全一致；另外6个样本检测结果与AS—PCR检测结果不一致，测序显示：TCA（L1014S）、TTC（L1014F）突变备l株，另4株为敏感纯合子。因此，PCRSYBRGREEN扩增灵敏度（93．75％）低于AS．PCR法（100％），但特异性较高，达100％；分析原因可能是被检测样本引物结合部位多态性较高，影响了引物的结合，而AS—PCR凭经验能完成部分结果的判断。综上所述，用PCRSYBRGREEN扩增技术快速检测淡色库蚊的L1014F基因突变仍是一种值得采纳的等位基因分型方法。

To set up a real-time fluorescent quantitative PCR assay for rapid detection point mutation （TTA-TTY of L1014F） in Culex pipiens pallens sodium channel gene-related with knockdown resistance （kdr）. Two paired-primers with 3 ＇-basyl of sense-primer combined on the mutation point were contrived in parallel for each specimen. The specimens can be easily genotyped as SS, RS, RR as same as the AS-PCR did when there was no polymorphism in the target sequence complemented with primers. Of 48 specimens were detected in this assay, 42 specimens showed the same results as AS-PCR did gaining 21 SS genotypes （50. 0% ）, 16 SR genotypes （38. 1% ） and 5 RR genotypes （ 11.9% ）. There were 2 of other 6 specimens showed substitutions from TTA to TGA and TTC in the allele of LlO14, and 4 SS senotypes in other 4 specimens with polymorphism in the target sequence according to sequencing results. So in brief, the PCR assay showed lower sensitivity （93.75%） than AS-PCR did （ 100% ）, but higher specificity （ 100% ） according to direct sequencing.