Magmax 96 pathogen rna dna kit

Lung tissues were collected at necropsy (56 dpi) and stored at −80°C until they were processed for PCR testing. As described elsewhere (18 (link)), lung (3 by 3 cm) containing both normal and affected tissue was minced using sterile scissors and then placed in a 50-ml conical tube with 30 ml of Earle’s balanced salt solution (Sigma-Aldrich, St. Louis, MO, USA) at a concentration of 10% (wt/vol). The sample was homogenized (2 min at 1,000 rpm; Geno/Grinder; Spex SamplePrep, Metuchen, NJ, USA) and then centrifuged (10 min at 4,200 × g).
M. hyopneumoniae DNA detection for both tracheal and lung homogenates was based on commercial kits (MagMax-96 Pathogen RNA/DNA kit, PCR VetMax-Plus qPCR master mix, VetMax Mycoplasma hyopneumoniae reagents; Applied Biosystems) performed as directed by the manufacturer. DNA was extracted on the Kingfisher Flex system and amplified on Applied Biosystems 7500 real-time PCR. Each plate included a known M. hyopneumoniae-positive sample (VetMax-Plus qPCR master mix kit includes Xeno DNA Control, Applied Biosystems) and a negative control sample (RNA-free water). A test result was considered valid when the internal positive C_T value was ≤36. A sample was considered M. hyopneumoniae positive when C_T values were ≤37.

DNA from tracheal samples used in this study (50 individual tracheal samples generated from known M. hyopneumoniae-positive samples [pre-selection tests and re-tests], 70 from known M. hyopneumoniae-negative samples, and 432 pooled tracheal samples tested in pool sizes of 3, 5, and 10) was extracted using the commercial kit MagMAX™-96 Pathogen RNA/DNA kit (Applied Biosystems, Carlsbad, CA USA) and amplified using the TaqMan® Fast Virus 1-Step Master Mix with the addition of the AmpliTaq® 360DNA Polymerase (5 U/µL) enzyme (Thermo Fisher Scientific, Waltham, MA USA), and primer/probe as previously described for Mhp183^{43 (link)}. DNA amplification was performed on the automated Applied Biosystems® 7500 Real-Time PCR (ThermoFisher Scientific). A PCR result was considered valid if the internal positive control Ct was < 36, and tracheal samples were considered positive for M. hyopneumoniae DNA when Ct was < 37^{43 (link)}.

In study 1, as described elsewhere (15 (link)), oral fluid and serum samples were tested for Mycoplasma species-specific DNA and antibodies initially to confirm pigs’ negative status and later to corroborate M. flocculare, M. hyorhinis, and M. hyosynoviae infections in inoculated animals (0 to 56 dpi). Mycoplasma DNA extraction was performed using a commercial kit (MagMax-96 Pathogen RNA/DNA kit (Applied Biosystems, Carlsbad, CA USA) on the Kingfisher Flex system (Thermo Fisher Scientific, Waltham, MA, USA) and amplified on Applied Biosystems 7500 real-time PCR (Thermo Fisher Scientific). Antibody responses against M. flocculare and M. hyosynoviae were assessed using Tween 20-extracted surface protein-based ELISAs. M. hyorhinis-specific antibody testing used a chimeric VlpA to VlpG recombinant protein-based ELISA (15 (link)).