RT/PCR
RT/PCR is used to amplify and examine a 743 base pair (bp) region of VP2.
The products of RT/PCR are identified by differential migration due to fragment size with electrophoresis. 10 ul of RT/PCR product is added to 5 ul Orange G dye and placed in a 1% agarose gel. The RT/PCR products are stained with ethidium bromide (10mg/ml). The gel is viewed under UV light. A 100 bp ladder was used as a marker.
Six different RFLP's were detected for serotype 1 vaccine viruses and previously described strains of IBDV, as well as new rflp patterns from strains of IBDV found inside and outside of the United States (Tables), . We have observed greater genetic heterogeneity in the 743 bp fragment of VP2 examined among field strains when compared to vaccine strains.
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RT/PCR - RFLP
The RT/PCR products are digested with the use of the restriction enzymes BstN I and Mbo I. The RT/PCR - RFLP assay examines the 743 bp fragment for the presence and location of multiple BstN I or Mbo I restriction sites by comparing the sizes of resulting restriction fragments.
The restriction fragments are examined on a 2.5% agarose gel followed by staining with a compound that will fluoresce under ultra-violet light.
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