RT-NPCRReverse Transcription and Nested Polymerase Chain Reaction
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All samples were also assayed for HEV RNA by RT-nPCR (15).
Thereafter, 100 [micro]L of the clarified supernatants was used for total viral RNA extraction, and positive-stranded and negative-stranded HEV RNA were detected by RT-nPCR as described below.
RT-nPCR to Detect Positive-stranded and Negative-stranded HEV RNA
Furthermore, all suspected cases of influenza, except 2, were confirmed by a rapid test; most were tested further and isolates were subtyped by RT-nPCR.
To further investigate the single most prominent region of difference between FECVs and FIPVs, we established an RT-nPCR method to amplify and analyze the genomic region covering nucleotides 23442-24040 for the first PCR run and nucleotides 23451-23593 for the second run, which includes deviant position 23531.
In search of those differences, we performed a phylogenetic analysis of the partial nucleotide sequences accumulated by the RT-nPCR procedure, but the results did not enable further differentiation (data not shown).
For submissions yielding WN virus from brain, positive WN virus RT-nPCR, or both, IgM was consistently present in serum.
New laboratory tests will also be incorporated, in particular RT-nPCR, which has been shown to be accurate in detecting WN virus nucleic acid in CNS tissues.
We developed a RT-nested PCR (RT-nPCR) to rapidly and accurately detect WN virus in equine brain.
RT-nPCR product was analyzed by agar gel electrophoresis followed by ethidium bromide staining and UV visualization.