The use of competitive PCR for quantitation of HSV-1 DNA.
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.
Kosaki
Ryo
R
Department of Ophthalmology, Osaka University Medical School, Osaka, Japan.
Nakamura
Takao
T
Higaki
Shirou
S
Yamamoto
Shuji
S
Inoue
Yoshitsugu
Y
Hayashi
Kozaburou
K
Shimomura
Yoshikazu
Y
Tano
Yasuo
Y
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
D004279:DNA, Viral; D014759:Viral Envelope Proteins; C039447:glycoprotein D, Human herpesvirus 1