Quantification of ICP4 Gene of Herpes Simplex Virus Type 2 with Real-time Fluorescent Quantitative Polymerase Chain Reaction / 医学研究杂志

ABSTRACT

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 results,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土0.137,17.988?0.162,22.081土0.259,25.957土0.345,29.565?0.203,33.269土0.287,37.737?0.698,respectively with 107~ 101 copies/?l.The coefficient of variability were 0.965%,0.902%,1.174%,1.329%,0.686%,0.862% and1.851%,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.