Detection and differentiation of herpes simplex virus types 1 and 2 by a duplex LightCycler PCR that incorporates an internal control PCR reaction.
BACKGROUND
In recent years polymerase chain reaction (PCR) has proven to be a highly sensitive and specific method for the diagnosis of herpes simplex virus (HSV) infections. The advent of real-time HSV PCR protocols now enables rapid result turnaround times with minimal hands-on time.


OBJECTIVE
In this study, we developed a real-time duplex PCR assay (HSVgD-dPCR) comprising of HSV and internal control PCR reactions.


METHODS
Using the LightCycler, the HSVgD-dPCR targeted the HSV glycoprotein D gene and HSV typing was performed by melting curve analysis. The internal control PCR reaction targeted sequences of the DNA of the human endogenous retrovirus (ERV-3). In total, 300 swab specimens, from patients with suspected HSV infection, were tested by the HSVgD-dPCR assay. The results were then compared to the results obtained by another HSV LightCycler assay, which utilized published primer and probe sequences targeting the HSV DNA polymerase gene (Dpol-HSV-LCPCR).


RESULTS
Overall, 91 (30.3%) specimens were positive and 204 (68.0%) specimens were negative for HSV by both LightCycler assays. In addition, four (1.3%) specimens were positive by Dpol-HSV-LCPCR and negative by HSVgD-dPCR, whereas one (0.3%) specimen was positive by HSVgD-dPCR and negative by Dpol-HSV-LCPCR. The presence of HSV in these five specimens was confirmed by conventional PCR. Melting curve analysis by the HSVgD-dPCR assay enabled all HSV positive specimens to be typed, whereas sequence variation prevented three HSV positive specimens from being typed by the Dpol-HSV-LCPCR. Using the ERV-3 PCR, 5% specimens were found to contain inhibitory substances.


CONCLUSIONS
By developing the HSVgD-dPCR we have enhanced the diagnostic utility of real-time detection of HSV by incorporating an internal control reaction and by accurately typing a greater proportion of HSV positive specimens.
Whiley
David M
DM
Clinical Virology Research Unit, Sir Albert Sakzewski Virus Research Centre, Royal Children's Hospital & Health Service District, Herston Road, Herston, Queensland 4029, Australia.
Mackay
Ian M
IM
Syrmis
Melanie W
MW
Witt
Michael J
MJ
Sloots
Theo P
TP
D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D002648:Child; D002675:Child, Preschool; D004259:DNA-Directed DNA Polymerase / Q000235:genetics; D005090:Exodeoxyribonucleases / Q000235:genetics; D005260:Female; D006558:Herpes Genitalis / Q000175:diagnosis / Q000821:virology; D006561:Herpes Simplex / Q000175:diagnosis* / Q000821:virology; D018259:Herpesvirus 1, Human / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D018258:Herpesvirus 2, Human / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D007223:Infant; D008297:Male; D008875:Middle Aged; D016133:Polymerase Chain Reaction / Q000379:methods*; D012015:Reference Standards; D012680:Sensitivity and Specificity; D014759:Viral Envelope Proteins / Q000235:genetics; D014764:Viral Proteins / Q000235:genetics
D014759:Viral Envelope Proteins; D014764:Viral Proteins; C039447:glycoprotein D, Human herpesvirus 1; C040570:glycoprotein D-herpes simplex virus type 2; D004259:DNA-Directed DNA Polymerase; D005090:Exodeoxyribonucleases; C043417:DNA polymerase, Simplexvirus