title: Time-resolved fluorometry PCR assay for rapid detection of herpes simplex virus in cerebrospinal fluid.
abstract: We have introduced a time-resolved fluorometry (TRF)-based microwell hybridization assay for PCR products in detection of herpes simplex virus (HSV) in cerebrospinal fluid (CSF) specimens. TRF is a sensitive nonradioactive detection technique which involves the use of lanthanide chelates as fluorescent labels. We used PCR primers from the glycoprotein D genes of HSV type 1 (HSV-1) and HSV-2. The biotinylated PCR products were collected on streptavidin-coated microtitration wells and hybridized with short oligonucleotide probes, europium labeled for HSV-1 and samarium labeled for HSV-2. The TRF results were obtained as counts per second and as signal-to-noise (S/N) ratios. The sensitivity of the assay was 0.1 infectious units (PFU) of HSV in CSF specimens, and the S/N values increased with the virus amount, up to 68.5 for 10(3) PFU of HSV-1 and to 58.5 for 10(3) PFU of HSV-2, allowing semiquantitation of HSV in CSF. The primers and probes recognized all the studied 48 HSV wild-type samples, with S/N ratios of 12.4 to 190 (HSV-1) and 5.1 to 248 (HSV-2). We tested CSF specimens, 100 for each HSV type, which were HSV PCR negative by Southern blot and 22 CSF specimens which were HSV-1 or -2 PCR blot positive. In the TRF test, the mean S/N ratio for the HSV-1-negative CSF was 1.37 (standard deviation [SD] = 0.513) and for the HSV-2-negative CSF it was 1.03 (SD = 0.098). The HSV-1 blot-positive CSF yielded S/N ratios of 3.6 to 85.9, and the HSV-2 blot-positive CSF yielded ratios from 1.9 to 13. Using the mean S/N ratio for negative CSF specimens + 3 SD as the cutoff yielded all the previously HSV-positive specimens as TRF positive. The TRF PCR assay for HSV in CSF specimens is a rapid and sensitive method, improves interpretation of PCR results, and is well suited for automation.
authors.affiliation: Department of Virology, University of Turku, FIN-20520 Turku, Finland. Veijo.Hukkanen@utu.fi
authors.forename: V
authors.initials: V
authors.lastname: Hukkanen
authors.forename: T
authors.initials: T
authors.lastname: Rehn
authors.forename: R
authors.initials: R
authors.lastname: Kajander
authors.forename: M
authors.initials: M
authors.lastname: Sjöroos
authors.forename: M
authors.initials: M
authors.lastname: Waris
mesh_terms: D020805:Central Nervous System Viral Diseases / Q000175:diagnosis* / Q000821:virology; D002555:Cerebrospinal Fluid / Q000821:virology*; D020803:Encephalitis, Herpes Simplex / Q000175:diagnosis / Q000821:virology; D005470:Fluorometry / Q000379:methods; D006561:Herpes Simplex / Q000175:diagnosis* / Q000821:virology; D018259:Herpesvirus 1, Human / Q000235:genetics / Q000302:isolation & purification*; D018258:Herpesvirus 2, Human / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D008587:Meningitis, Viral / Q000175:diagnosis / Q000821:virology; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013997:Time Factors