Itaq universal probes supermix

The preserved spleen and liver samples were subjected to DNA extraction using the TF lysis buffer method (Meemetta et al., 2020) . DNA quantity and quality was measured using a NanoDrop (Thermo Scientific, USA), with absorbance set at OD 260 and OD 280 . DNA samples were diluted to 100 ng/µL for quantitative PCR (qPCR) reactions. Detection of TiPV was performed using a protocol described by Liu et al. (2020) (link). PCR mixtures were comprised of 10 µL 2X SYBR Green Master Mix (Bio-Rad, USA), 0.5 μL of each forward and reverse primers (10 µM of each TiPV-Fq/TiPV-Rq) (Table 1), 2 μL DNA template and 7 μL ddH 2 O. PCR was performed at 94 ˚C for 2 min followed by 40 cycles of 94 ˚C for 10 s and 60 ˚C for 30 s. The melting curve was analyzed in the last cycle. On the other hand, probe-based qPCR targeting groEL gene was used to diagnose S. agalactiae infection, according to Leigh et al. (2019) (link). The qPCR reagents mixture contained 10 µL of 2X iTaq Universal Probes Supermix (Bio-Rad, USA), 0.5 µL of 10 µM SagroEL-probe (Table 1), 1.8 µL of each primer SagroEL-F/R (10 µM), 3 µl of DNA template and 2.9 μL ddH 2 O. The qPCR reaction was performed at 95 ˚C for 2 min followed by 40 cycles of 95 ˚C for 5 s and 60 ˚C for 30 s. DNA extracted from S. agalactiae 2809 (Linh et al., 2021) ) was used as a positive control, and ddH 2 O without DNA template was used as a negative control.