单纯疱疹病毒2型ICP4基因荧光实时定量PCR检测方法的建立  

Quantification of ICP4 Gene of Herpes Simplex Virus Type 2 with Real-time Fluorescent Quantitative Polymerase Chain Reaction

作　　者：刘继峰[1] 关翠萍[1] 唐旭 徐田红[1] 许爱娥[1] 

机构地区：[1]浙江省杭州市第三人民医院皮肤科,310009

出　　处：《医学研究杂志》2008年第12期36-38,共3页Journal of Medical Research

基　　金：浙江省自然科学基金项目(Y204510)

摘　　要：目的建立实时荧光PCR定量检测单纯疱疹病毒2型(HSV2)ICP4基因的方法。方法依据HSV2ICP4基因序列设计引物,PCR纯化后克隆到pMD-18T载体上进行测序,抽提重组质粒作为标准品。根据测序结果设计荧光定量PCR的引物和探针,质粒标准品10倍稀释后进行扩增,建立标准曲线,并进行灵敏度和可靠性分析。结果测序结果显示在扩增的PCR片段中有在A-G无和T-C的无义突变。标准品101以下没有检测信号,101具有明确的检测信号,因而检测的灵敏度为10。可靠性测定结果:107～101进行10次重复Ct值的平均值分别为14.275±0.137、17.988±0.162、22.081±0.259、25.957±0.345、29.565±0.203、33.269±0.287、37.737±0.698,变异系数分别为0.965%、0.902%、1.174%、1.329%、0.686%、0.862%、1.851%。Ct值与标准品浓度的对数之间存在良好的线性关系。回归系数为0.998。结论成功建立了HSV2ICP4基因实时定量荧光PCR检测的方法,灵敏度高,重复性好,可用于HSV2ICP4基因的定量分析。Objective To establish a fluorescent quantitative polymerase chain reaction method for quantifying the ICP4 gene expression of herpes simplex virus type 2（HSV2）. Methods According to the HSV2 ICP4 gene sequence, we designed and synthesized PCR primer. The purified PCR product was sequenced after connecting with pMD - 18 T plasmid. According to the sequence assay resuits, the primer and probe of fluorescent quantitative PCR was designed and synthesized. Standard recombinant plasmid extracted from the positive bacteriumclone was used as standard substance. The plasmid as standard substance was diluted for 10 times, then PCR reaction proceeded. The sensitivity and dependability of the real - time fluorescent quantitative PCR were analyzed. Results The sequence result indicated that there was non - sense mutation of A - G and T - C. And the detection sensitivity was 101 copy. The Ct value were 14. 275 ± 0. 137,17. 988 ±0. 162,22. 081±0. 259,25. 957 3± 0. 345,29. 565± 0. 203,33. 269 ± 0. 287,37. 737 ± 0. 698 ,respectively with 10^7 - 10^1 copies/μl. The coefficient of variability were 0. 965% ,0. 902% , 1. 174% , 1. 329% ,0. 686% ,0. 862% andl. 851% , respectively. There was a good linear function in statistics between the Ct value and the concentration gradient of standard plasmid DNA specimen. The coefficient of regression was 0. 998. Conclusion The method of quantification of ICP4 gene of HSV2 with real - time fluorescent quantitative PCR is successfully established, and the method has good sensitivity and dependability, which can be used to quantitative detecting HSV2 ICP4.

关 键 词：单纯疱疹病毒2型 ICP4 实时定量荧光PCR 标准曲线 

分 类 号：R379.4[医药卫生—病原生物学] R752.11[医药卫生—基础医学]