Sensimix sybr low rox qpcr mastermix

Quantification of MAP genomic DNA in the DNA extracts from the tissues was performed as previously described (7 (link)). The final volume of 25 µL of reaction mixture comprised 5 µL template DNA, 250 nM final concentration of each forward [MP10-1 (5′-ATGCGCCACGACTTGCAGCCT-3′)] and reverse [MP11-1 (5′-GGCACGGCTCTTGTTGTAGTCG-3′)] primers (48 (link)) and 12.5 µL of SensiMix SYBR Low-ROX qPCR mastermix (Bioline). The amplification was performed (initial denaturation at 95°C for 8.5 min; 40 cycles of denaturation at 95°C for 15 s, annealing at 68°C for 30 s, and extension at 72°C for 1 min, and melt curve analysis from 55 to 95°C) using an Mx3000P real-time PCR instrument (Stratagene, Agilent). The quantification of MAP DNA was performed by constructing a standard curve using 10-fold serially diluted MAP genomic DNA, ranging from 10 to 0.001 pg/reaction, included in each DNA amplification plate. The acceptance criteria for a positive test result were (i) MAP-specific amplification (melt temperature in the range of 89.1 ± 1.5°C) and (ii) MAP DNA quantity of ≥0.0005 pg/reaction, which is the analytical limit of detection of the qPCR (7 (link)).

Mycobacterium avium subspecies paratuberculosis genomic DNA was quantified in both the undiluted and the diluted DNA extracts following the HT-J qPCR method (Plain et al., 2014 (link)) which targets the IS900 sequence of MAP. Briefly, the reaction mixtures comprised of 5 μl template DNA, 250 nM final concentration of each forward [MP10-1, (5′-ATGCGCCACGACTTGCAGCCT-3′)] and reverse [MP11-1, (5′-GGCACGGCTCTTGTTGTAGTCG-3′)] primers (Kawaji et al., 2007 (link)) and 12.5 μl of SensiMix SYBR Low-ROX qPCR mastermix (Bioline) in a final volume of 25 μl, with amplification performed using an Mx3000P real-time PCR instrument (Stratagene, Agilent). Quantification of MAP DNA was done with reference to the standard curve included in every experiment of 10-fold serially diluted MAP genomic DNA ranging from 10 to 0.001 pg/reaction. The detailed acceptance criteria for HT-J positive qPCR results was previously defined and included a cut-point of ≥0.001 pg target DNA quantity per reaction (Plain et al., 2014 (link)) equivalent to Ct value of 35.1 ± 1.04 (Figure 2C).
Each undiluted DNA extract was tested in a single reaction while diluted DNA extracts were tested in duplicate and a positive mean DNA test result for both replicates was considered a positive sample test result.