title: The use of competitive PCR for quantitation of HSV-1 DNA.
abstract: OBJECTIVE
  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.
  
  
  CONCLUSIONS
  Competitive PCR can quantitate HSV-1 DNA.
authors.affiliation: Department of Ophthalmology, Osaka University Medical School, Osaka, Japan.
authors.forename: Ryo
authors.initials: R
authors.lastname: Kosaki
authors.forename: Takao
authors.initials: T
authors.lastname: Nakamura
authors.forename: Shirou
authors.initials: S
authors.lastname: Higaki
authors.forename: Shuji
authors.initials: S
authors.lastname: Yamamoto
authors.forename: Yoshitsugu
authors.initials: Y
authors.lastname: Inoue
authors.forename: Kozaburou
authors.initials: K
authors.lastname: Hayashi
authors.forename: Yoshikazu
authors.initials: Y
authors.lastname: Shimomura
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Tano
mesh_terms: D004279:DNA, Viral / Q000032:analysis*; D005822:Genetic Vectors; D018259:Herpesvirus 1, Human / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D016849:Keratitis, Herpetic / Q000821:virology; D016133:Polymerase Chain Reaction / Q000379:methods; D013666:Tears / Q000821:virology; D014759:Viral Envelope Proteins / Q000235:genetics
chemical_list: D004279:DNA, Viral; D014759:Viral Envelope Proteins; C039447:glycoprotein D, Human herpesvirus 1