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


METHODS
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.
Adelson
Martin E
ME
Medical Diagnostic Laboratories, L.L.C., 2439 Kuser Road, Hamilton, NJ 08690, USA. madelson@mdlab.com
Feola
Melanie
M
Trama
Jason
J
Tilton
Richard C
RC
Mordechai
Eli
E
D000328:Adult; D002584:Cervix Uteri / Q000821:virology; D017931:DNA Primers; D004279:DNA, Viral / Q000032:analysis; D005260:Female; D006558:Herpes Genitalis / Q000175:diagnosis / Q000453:epidemiology* / Q000821:virology; D006561:Herpes Simplex / Q000175:diagnosis / Q000453:epidemiology* / Q000821:virology; D018259:Herpesvirus 1, Human / Q000235:genetics / Q000302:isolation & purification*; D018258:Herpesvirus 2, Human / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D009841:Oligonucleotides / Q000032:analysis; D016133:Polymerase Chain Reaction / Q000379:methods*; D015995:Prevalence; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000379:methods*; D012984:Software; D013048:Specimen Handling / Q000379:methods
D017931:DNA Primers; D004279:DNA, Viral; D009841:Oligonucleotides