Simultaneous detection of herpes simplex virus types 1 and 2 by real-time PCR and Pyrosequencing

J Clin Virol. 2005 May;33(1):25-34. doi: 10.1016/j.jcv.2004.09.022.


Background: Up to 80% of the US adult population has been exposed to herpes simplex virus (HSV) type 1, primarily during childhood. Also, approximately 20% of the US population has contracted genital herpes from HSV-2 infections. Clinical symptoms can present as fever, headache, malaise, myalgia, and cold sores/lesions that cause pain, itching, dysuria, and vaginal or urethral discharge. A recurrence of infection is common. HSV culturing is characterized by low sensitivity with variable success rates due to shipping conditions.

Objective: To design and validate a real-time PCR assay capable of simultaneously detecting each HSV subtype.

Study design: ATCC-purchased HSV-1 and HSV-2 positive samples and HSV-1 and HSV-2 infected clinical specimens were assayed simultaneously with shared amplification primers and subtype-specific probes against the HSV glycoprotein B gene on a Rotor-Gene 3000 platform. Separately, two PCR reactions were performed in which one primer contained a 5' biotin modification. Single-stranded DNA from the amplicon was purified and Pyrosequenced.

Results: The quantitative range of the assay extended from 10(8) through 10(0) copies of each virus (r(2) > 0.991) and specificity was determined by non-amplification of 37 different human pathogens, including other herpesviruses such as VZV, CMV, and EBV. Sensitivity and specificity values of 100% were calculated by concordance analysis between the real-time PCR and the DNA Pyrosequencing results (HSV-1: n = 119, HSV-2: n = 120). Application of this assay to 4581 cervical swab specimens collected from women visiting physicians primarily in six states provided detection rates of 3.1% for HSV-1 and 7.6% for HSV-2. The average age of women infected with HSV-1 was 29.5 versus 35.6 for HSV-2.

Conclusions: This procedure was demonstrated as both highly sensitive and specific for the detection of HSV-1 and HSV-2 in a single reaction. Also, the integration of Pyrosequencing analysis permitted an innovative and rapid verification for each subtype.

Publication types

  • Evaluation Study

MeSH terms

  • Adult
  • Cervix Uteri / virology
  • DNA Primers
  • DNA, Viral / analysis
  • Female
  • Herpes Genitalis / diagnosis
  • Herpes Genitalis / epidemiology*
  • Herpes Genitalis / virology
  • Herpes Simplex / diagnosis
  • Herpes Simplex / epidemiology*
  • Herpes Simplex / virology
  • Herpesvirus 1, Human / genetics
  • Herpesvirus 1, Human / isolation & purification*
  • Herpesvirus 2, Human / genetics
  • Herpesvirus 2, Human / isolation & purification*
  • Humans
  • Oligonucleotides / analysis
  • Polymerase Chain Reaction / methods*
  • Prevalence
  • Sensitivity and Specificity
  • Sequence Analysis, DNA / methods*
  • Software
  • Specimen Handling / methods


  • DNA Primers
  • DNA, Viral
  • Oligonucleotides