Application of polymerase chain reaction to differentiate herpes simplex virus 1 and 2 serotypes in culture negative intraocular aspirates.
OBJECTIVE
To standardize and apply a polymerase chain reaction (PCR) on the glycoprotein D gene to differentiate Herpes simplex virus (HSV) 1 & 2 serotypes in culture negative intraocular specimens.


METHODS
Twenty-one intraocular fluids collected from 19 patients were subjected to cultures for HSV and uniplex PCR (uPCR) for DNA polymerase gene. To differentiate HSV serotypes, as 1 & 2, a seminested PCR (snPCR) targeting the glycoprotein D gene was standardised and applied onto 21 intraocular fluids. The specificity of the snPCR was verified by application onto ATCC strains of HSV 1 and 2, clinical isolates and DNA sequencing of the amplified products. All specimens were also tested for the presence of cytomegalovirus (CMV) and varicella zoster virus (VZV) by nucleic acid amplification methods.


RESULTS
Four of the 21 intraocular fluids were positive for HSV by uPCR. snPCR detected HSV in three additional specimens (total of seven specimens), and identified three as HSV 1 and four as HSV 2. DNA sequencing of PCR products showed 100% homology with the standard strains of HSV 1 and 2 respectively. None of the samples were positive in culture. Among the other patients, CMV DNA was detected in two and VZV DNA in five others.


CONCLUSIONS
The standardized snPCR can be applied directly onto the culture negative specimens for rapid differentiation of HSV serotypes.
Shyamal
G
G
Vision Reaserch Foundation, Sankar Nethralaya, Chennai-600 006, TN, India.
Sowmya
P
P
Sudha
B
B
Malathi
J
J
Therese
L K
LK
Madhavan
H N
HN
D004279:DNA, Viral / Q000032:analysis; D004259:DNA-Directed DNA Polymerase / Q000032:analysis / Q000378:metabolism; D003937:Diagnosis, Differential; D005090:Exodeoxyribonucleases / Q000032:analysis / Q000378:metabolism; D006566:Herpesviridae Infections / Q000175:diagnosis* / Q000821:virology; D018259:Herpesvirus 1, Human / Q000235:genetics / Q000302:isolation & purification*; D018258:Herpesvirus 2, Human / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D012173:Retinitis / Q000175:diagnosis / Q000821:virology; D014759:Viral Envelope Proteins / Q000032:analysis; D014764:Viral Proteins / Q000032:analysis / Q000378:metabolism
D004279:DNA, Viral; D014759:Viral Envelope Proteins; D014764:Viral Proteins; C040570:glycoprotein D-herpes simplex virus type 2; D004259:DNA-Directed DNA Polymerase; D005090:Exodeoxyribonucleases; C043417:DNA polymerase, Simplexvirus