Vetmax plus qpcr master mix

The developed ITS PCR assay was compared to the BCSP31 PCR assay for diagnostic validation because the BCSP31 gene target is one of the most commonly used targets used for the detection of Brucella spp. in animals [30 (link)] and is routinely used by OIE reference laboratories. The BCSP31 PCR can be used in a real-time PCR format, and the primers and probe for the Brucella spp. described by Probert et al. [16 (link)] was used in our study. This assay was used on a different platform (StepOne real-time PCR machine, Applied Biosystems™, Foster City, CA, USA) and the VetMAX™-Plus qPCR Master Mix (as described in 2.4, but with 300 nM forward and reverse primer concentrations and 100 nM probe concentration in the final reaction.

A commercial real time M. hyopneumoniae PCR (rt-PCR) was performed in laryngeal swabs, BALF and lung tissue samples. The assay was performed using VetMAX™-Plus qPCR Master Mix (Applied Biosystems, USA) and VetMAX™ M. hyopneumoniae Reagents (Applied Biosystems, USA), according to the manufacturer’s instructions. VetMAX™-Plus qPCR Master Mix kit includes Xeno™ DNA Control, which serves as an internal positive control for DNA purification and rt-PCR. Runs were carried out in an ABIPRISM^® 7500 machine (Applied Biosystems, Singapore). The threshold for the DNA target was set at 10% of the average maximum fluorescence value of the positive control DNA target. Cycle threshold (Ct) values equal to or lower than 40 were considered positive.

Six cross-bred pigs (Sus scrofa domesticus) of either sex, aged 6–18 months old, were used as blood donors for in vitro experiments. Pigs were housed at the Experimental Station of Istituto Zooprofilattico Sperimentale (IZS) of Sardinia (“Surigheddu”, Sassari, Italy). Animal husbandry, handling, and procedures (bleeding) were carried out according to the Italian Legislative Decree No. 26 dated 4 March 2014 and in agreement with the Guide of Use of Laboratory Animals issued by the Italian Ministry of Health (authorization No. 1232/2020-PR).
Heparinized blood samples were used for generation of monocyte-derived macrophages (moMΦ) (described in Section 4.2). Animal health was routinely monitored by trained veterinarians, and blood samples were screened for several porcine pathogens. The absence of African swine fever (ASFV), porcine parvovirus (PPV), and porcine circovirus 2 (PCV2) genome was evaluated though qualitative real-time PCR, as previously described [21 (link),60 (link)], with primers reported in the Table S1 [61 (link),62 (link),63 (link)]. The absence of the porcine reproductive and respiratory syndrome virus (PRRSV) and Mycoplasma hyopneumoniae was monitored using commercial real-time PCR kits (LSI VetMAX™ PRRSV EU/NA and VetMAX™-Plus qPCR Master Mix, both Thermo Fisher Scientific, respectively), following the manufacturer’s instructions [21 (link)].