3.1. Design of the MADCAT System
The principle of the multiple-probe-assisted DNA capture and amplification technology (MADCAT) is illustrated in Figure 1A. Target DNA of lysed samples are captured on a 96-well plate by a series of capture probes, each of which incorporates a target-specific sequence and an additional “tail” sequence that can interact with the oligonucleotide conjugated on the surface of each well in 96-well plate. After washing off all unbound probes and irrelevant nucleic acids, the captured targets were then amplified with the target-specific primers and probes.
Based on this multiple-probe-per-strand design principle, three sets of capture probe (CPsets A, B and C) were designed for the same region by utilizing different design schemes (Figure 1A). The CPset B and CPset C proved to be more conducive to capturing double-stranded DNA targets than CPset A (Figure 1B). The candidate three sets of primers, Primerset1 (AV-F1 and AV-R1), Primerset2 (AV-F2 and AV-R2) and OIE-recommended primers (VP-UP and VP-DP) (King et al., 2003), were computationally filtered to eliminate potential homology to the swine genome and to a panel of related viruses. Experimentally, among the three schemes, Primerset2 showed better performances (lower Ct values) than OIE-recommended primers and Primerset1 (Figure S1), and was used for the subsequent tests consequently.