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The use of competitive PCR for quantitation of HSV-1 DNA
Ryo Kosaki1, Takao Nakamura, Shirou Higaki
1Department of Ophthalmology, Osaka University Medical School, Osaka, Japan.
Purpose:
Polymerase chain reaction (PCR) detects the genomic materials of etiological agents with high specificity and sensitivity. However, in herpes simplex virus type 1 (HSV-1) infection, the clinical significance of the results often poses controversy because of the subclinical viral shedding during latent infection. Quantitative PCR might provide additional information to help clinical evaluation of the results.
Methods:
Virus DNA was extracted from high titer stock of human HSV-1 (FK 25) by phenol/chloroform treatment. Construct (p/HSV-1) was made by inserting the glycoprotein D gene obtained from virus DNA into p(GEM-T) vector. Competitor (p/DeltaHSV-1) was made by deleting the inner 40 bp of construct (p/HSV-1) with restriction enzyme. Competitive PCR was performed using primers that amplify the glycoprotein D gene, and a template made of a 1:1 molar mixture of HSV-1 DNA and the competitor.
Results:
The PCR product reflected the initial template dose from 20 to 30 cycles. Minimum detection level of HSV-1 DNA was 0.01 ng.
Conclusion:
Competitive PCR can quantitate HSV-1 DNA.