Design and properties of myxoma virus specific qPCR assay. Panel (a). The sequence of the myxoma virus M029L gene [GenBank:NC_001132] was aligned with the homologous E3L genes from vaccinia virus (WR strain, [GenBank:AY243312], swinepox [GenBank:NC_003389] and Orf virus [GenBank:NC_005336]) using ClustalW2. The positions of the myxoma virus specific primers (myxE3Lfor and myxE3Lrev) (in bold type and underlined) and the MGB-probe (within the rectangle) which recognizes a sequence that is identical in myxoma virus and swinepox virus are indicated. Panel (b). Viral DNA was prepared from cell culture grown samples of vaccinia virus (WR strain, blue diamond), myxoma virus (red triangle), swinepox virus (black square) and Orf virus (black circle) and each was assayed (in parallel and with negative (water) controls (yellow and green symbols)) in the qPCR assay for myxoma virus as described in Materials and Methods. Panel (c) Serial 10-fold dilutions of a plasmid, including the myxoma M029L gene, containing between 101 and 107 copies were tested in the myxoma virus assay. The gradient of the line is -3.293 and the efficiency of the PCR reaction was calculated as 101.2%.