Time-resolved fluorometry PCR assay for rapid detection of herpes simplex virus in cerebrospinal fluid

We have introduced a time-resolved fluorometry (TRF)-based microwell hybrid ization assay for PCR products in detection of herpes simplex virus (HSV) i n cerebrospinal fluid (CSF) specimens. TRF is a sensitive nonradioactive de tection technique which involves the use of lanthanide chelates as fluoresc ent 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 strepta vidin-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) ra tios. 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 6 8.5 for 10(3) PFU of HSV-1 and to 58.5 for 10(3) PFU of HSV-2, allowing sem iquantitation of HSV in CSF. The primers and probes recognized all the stud ied 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 wer e HSV PCR negative by Southern blot and 22 CSF specimens which were HSV-1 o r -2 PCR blot positive. In the TRF test, the mean S/N ratio for the HSV-1-n egative CSF was 1.37 (standard deviation [SD] = 0.513) and for the HSV-2-ne gative 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 fro m 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 positiv e. The TRF PCR assay for HSV in CSF specimens is a rapid and sensitive meth od, improves interpretation of PCR results, and is well suited for automati on.