Natalizumab and HSV meningitis

HSV PCR testing in our clinical lab is completed using the LightCycler® FastStart DNA Master HybProbe (Roche). Nucleic acid extracted from 200 μl of CSF fluid was tested for the presence of herpes simplex viral DNA type 1 and/or 2 using LightCycler real-time PCR on a LightCycler 1.2 instrument with FRET hybridization probes. Primers and probes are sequence-specific for the DNA polymerase gene of HSV-1/2. A 215-bp fragment of the DNA polymerase gene (Genebank accession nos. M12356 and M16321) was amplified with specific primers for HSV-1/2. The amplicon is detected by fluorescence using a specific pair of hybridization probes. The hybridization probes consist of two different short oligonucleotides that hybridize to an internal sequence of the amplified fragment during the annealing phase of the PCR cycle. One probe is labeled at the 5′-end with LightCycler® Red 640-N-hydroxy-succinimide ester (Red 640-NHS ester) or LightCycler® 705-phosphoramidite and, to avoid extension, modified at the 3′-end by phosphorylation. The other probe is labeled at the 3′-end with fluorescein. Only after hybridization to the template DNA do the two probes come in close proximity, resulting in fluorescence resonance energy transfer (FRET) between the two fluorophores. During FRET, fluorescein, the donor fluorophore, is excited by the light source of the LightCycler® 1.2 Instrument, and part of the excitation energy is transferred to LightCycler® Red 640-NHS ester or the LightCycler® 705-phosphoramidite, the acceptor fluorophore. The emitted fluorescence of LightCycler® Red 640-NHS ester is then measured by the LightCycler® 1.2 Instrument. A melting curve analysis was performed after the PCR run to differentiate positive samples in HSV-1 or HSV-2. Melting points for HSV-1 and HSV-2 are reproducible and significantly different and allow clear determination of the HSV subtype (REF no. 03315177001). An internal control is used in this process to control for substances that may interfere with DNA amplification. Detail courtesy of JS Eversly and ES Rosenberg, Massachusetts General Hospital Molecular Laboratory.