PMQR and bla-tet resistance genes multiplex PCR

Through multiplex PCR reactions, multiple genes may be amplified simultaneously. We used a touch-down method to decrease non-specific binding. Two multiplex PCR protocols were used, one for PMQR genes (qnrA, qnrD, qnrB, qnrS, oqxAB, Aac(6′)Ib-cr, qepA, and qnrC) and one for β-lactamase (bla-TEM, bla-CTX-M, bla-OXA) and tetracycline genes (tetA and tetB) (Supplemental Table ²). We focused screening to quinolone and β-lactam resistance genes based on a previous study that observed that farm records indicating prior treatment with enrofloxacin resulted in selection of multidrug resistant isolates in calves that presented phenotypic resistance to both ciprofloxacin and ceftriaxone⁴.

For the PMQR multiplex PCR, each 25 µl reaction had 12.5 µl of EconoTaq Green Master Mix (Lucigen, Middleton, WI, USA), 1 µl of DNA template, 8 µl of primer pool (8 primer pairs at 10 µM) and 3.5 µl of nuclease free water. The thermocycler conditions were an initial denaturing cycle at 94 °C for 5 min; followed by 16 cycles of 94 °C for 45 s, touchdown from 64.5 to 60 °C, decreasing 0.5 °C every 30 sec cycle, and 72 °C for 40 s; plus 15 cycles of 94 °C for 30 s, 60 °C for 90 s and 72 °C for 40 s, and a final extension step at 72 °C for 10 min.

The β-lactamase and tetracycline genes multiplex PCR protocol was developed in the laboratory. Each 25 µl reaction also had 12.5 µl of EconoTaq Green Master Mix and 1 µl of DNA template, however, 5 µl of primer pool were used and the volume of nuclease free water was increased to 6.5 µl. The cycling conditions were 94 °C for 5 min; 16 cycles of 94 °C for 45 s, touch-down from 66 °C to 58.5 °C, decreasing 0.5 °C every 30 s cycle, and 72 °C for 60 s; more 20 cycles of 94 °C for 45 s, 54 °C for 30 s and 72 °C for 60 s, and a final extension step at 72 °C for 10 min.

Every PCR plate had a positive control (pool of DNA from resistant E.coli and Salmonella isolates owned by our research lab) and a negative control (nuclease free water). PCR products were visualized in 2.0% agarose gels. Primers used on multiplex PCR reactions are also listed on Supplemental Table ².