Figure 1.
Diagram of nested PCR and bloodmeal identification assay. Primers (AvTrpF1, MaTrpF1, AvSerR1, MaSerR1) flanking the mitochondrial gene COI were used to amplify ~1900bp. This amplicon was used as the template in a PCR amplifying the 658bp ‘barcoding’ region of COI, using primers VFmix (1 VF1:1 VF1d:2 VF1i) and 5′ biotinylated VRmix (1 VR1:1 VR1d:2 VR1i). Labeled PCR products were mixed with the 15 host species-specific microsphere probes (Table 1). If probe-product hybridization was detected by the Luminex200®, a positive match was made. Unidentified PCR products were sequenced using the forward primer VF1d. Sequences were subsequently identified using the Barcode of Life Data Systems (BOLD; www.boldsystems.org).