Parallel detection of five human herpes virus DNAs by a set of real-time polymerase chain reactions in a single run.
BACKGROUND
Human herpes viruses cause a spectrum of diseases that are usually self-limiting but can be reactive during immuno-suppression and may then lead to severe or even life-threatening diseases. The LightCycler technology allows rapid polymerase chain reaction (PCR) including product analysis within a closed system. This approach has been demonstrated to be suitable for routine diagnostic virus detection. Several LightCycler PCR assays have been established to the detection of human herpes viruses. The assays vary in their detection formats and PCR cycling protocols. So, they cannot be performed within a single LightCycler run.


OBJECTIVE
Development of four LightCycler PCR assays for parallel detection of DNA derived from human cytomegalovirus (CMV), Epstein-Barr virus (EBV), herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), and varicella-zoster virus (VZV) in a single run.


METHODS
Primers and hybridization probes were tailored to suit one LightCycler PCR program. LightCycler PCRs were established, detection limits were determined, and clinical samples were evaluated.


RESULTS
With quantified herpes virus type specific DNA spiked into cerebrospinal fluid, serum or EDTA plasma the detection limits were found either at 500 or 250 viral DNA copies per ml depending on the virus DNA specific PCR and on the specimen type used. The applicability of the new LightCycler assays for routine molecular testing was evaluated by testing 96 clinical samples.


CONCLUSIONS
The developed set of LightCycler PCRs permits parallel detection of CMV, EBV, HSV-1, HSV-2, and VZV in a single LightCycler run. The new molecular assays can easily be used to the rapid, simple, and convenient detection of herpes virus DNA in cerebrospinal fluid, serum and EDTA plasma in the routine diagnostic laboratory.
Stöcher
Markus
M
Institute of Laboratory Medicine, General Hospital Linz, Krankenhausstrasse 9, A-4020 Linz, Austria.
Leb
Victoria
V
Bozic
Michael
M
Kessler
Harald H
HH
Halwachs-Baumann
Gabriele
G
Landt
Olfert
O
Stekel
Herbert
H
Berg
Jörg
J
D000925:Anticoagulants; D001769:Blood / Q000821:virology*; D001800:Blood Specimen Collection; D002555:Cerebrospinal Fluid / Q000821:virology*; D003199:Computer Systems; D003587:Cytomegalovirus / Q000235:genetics; D004279:DNA, Viral / Q000032:analysis* / Q000097:blood / Q000134:cerebrospinal fluid; D004492:Edetic Acid; D006564:Herpesviridae / Q000235:genetics*; D018259:Herpesvirus 1, Human / Q000235:genetics; D014645:Herpesvirus 3, Human / Q000235:genetics; D004854:Herpesvirus 4, Human / Q000235:genetics; D006801:Humans; D016133:Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods*; D012680:Sensitivity and Specificity; D013045:Species Specificity
D000925:Anticoagulants; D004279:DNA, Viral; D004492:Edetic Acid