Real-Time Reverse Transcriptase–Polymerase Chain Reaction Detection and Analysis of Nucleotide Sequences Coding for a Neutralizing Epitope on Infectious Bursal Disease Viruses

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Abstract
We used real-time reverse transcriptase (RT)-polymerase chain reaction (PCR) to detect infectious bursal disease virus (IBDV) strains. The LightCycler (Roche) and hybridization probe system (Roche, Molecular Biochemicals) were used. A mutation probe labeled with fluorescein and an anchor probe labeled with Red-640 dye were prepared for each of the STC, Del E, D78, and Bursine 2 viral sequences. The mutation probes were designed to hybridize to nucleotides that encode the hydrophilic B region of VP2 for each virus. The anchor probes were designed to a relatively conserved region immediately downstream from the mutation probes. When hybridized to the RT-PCR product, a mutation and anchor probe pair produced fluorescence resonance energy transfer that was detected by the LightCycler instrument. Because they were designed to have a lower melting temperature (Tm), the mutation probes dissociated from the template before the anchor probes. The Tm values of the four mutation probes for each of their homologous viruses (exact sequence match) were STC, 69.3 +/- 1.2 C; D78, 67.8 +/- 0.9 C; Del E, 65.5 +/- 0.6 C; and Bursine 2, 71.7 +/- 0.4 C. These values were compared with the Tm values observed for a particular probe and heterologous virus. If the Tm values observed for heterologous viruses were within two standard deviations of the Tm for the probe and its homologous virus, the nucleotide sequences of the viruses were considered to be similar. If they were below two standard deviations, they were considered to have one or more nucleotide mutations. The results indicated that the STC and Variant Vax BD viruses have similar genetic sequences at the hydrophilic B region. Likewise, Bursine 2, Bursine, Bursine+, BioBurs, BioBurs W, BioBurs AB, and IBDV Blen have similar nucleotide sequences in this region. The Tm values obtained for the D78 and Del E mutation probes with heterologous viruses indicated that none of the viruses tested had nucleotide sequences that matched these probes. Because the mutation probes were designed to bind to a region that encodes a neutralizing epitope, viruses with similar sequences were expected to have antigenically similar epitopes.
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