Mastermix gotaq

M. hyopneumoniae DNA detection was performed with the aid of qPCR in tracheal and lung samples, nasal swabs, and BALF. All samples were tested in duplicates, using M. hyopneumoniae specific primer pair and hydrolysis probe from a previously published protocol [29 (link)].
The reaction was composed of 1X Master mix Go taq® (Promega, Madison, USA), 0.5 μM of each primer (Invitrogen, USA), 0.3 μM of the hydrolysis probe (IDT, Iowa City- USA), ultrapure water q.s.p and 1 μL of DNA template, adding up to a final reaction volume of 10 μL. The real-time thermocycler used was a CFX-96 (Biorad - USA) model. The cycling conditions were: one cycle of initial denaturation at 95 °C for 3 min, followed by 39 cycles of 95 °C for 15 s, and annealing∕extension at 55.7 °C for 1 min. Quantification results were only used if the Cq difference was lower than 0.5 cycle [16 (link)]. In case of Cq difference higher than 0.5 cycle, the samples were tested again in triplicates.
Absolute quantification and detection limits were performed using standard curves. The curve consisted of duplicate 10-fold dilutions, starting at 10⁷ copies∕μL until 10¹ copies∕μL, of a synthetic DNA positive control (GBlock®, IDT, Iowa City, IA, USA) of the 150 bp amplified fragment. In addition, quantification data was only used if qPCR efficiency was between 90 and 105% [30 (link)].

Degenerate primers Hsp40-Fw (5′-GACCTGCGCTACAACATGGAKCT-3′) and Hsp40-Rv (5′-CCGCGYTCCAAAAGCTTCTTYGAT-3′) were visually designed and analyzed (OligoAnalyzer) according to the alignment of the dnaJ gene sequences of the type strains. The primers were used to amplify a fragment of 750 bp. The amplification reaction was performed with Master Mix GoTaq (PROMEGA, USA), with 0.5 µl of each primer (0.2 µM final PCR concentration), and 2.5 µl of DNA template in a final reaction volume of 25 µl. PCR amplification was carried out in a thermal cycler (VERITI, Applied Biosystems) as follows: 4 min denaturation step at 94°C, followed by 30 cycles at 94°C for 50 s, 60°C for 35 s, and 72°C for 1 min, with a final extension step of 5 min at 72°C. PCR products were verified by agarose gel electrophoresis, purified using WizardSV gel and PCR clean Up System (Promega), and then sequenced using the Sanger sequencing technology with Hsp40-Fw/Hsp40-Rv primers by BigDye Terminator Cycle Sequencing Ready Reaction kit (Applied Biosystems, USA) according to the manufacturer’s instructions. After sequencing, primer sequences were removed and partial sequences of the dnaJ gene (714 bp) were confirmed by at least two chromatograms (forward and reverse) for the phylogenetic analysis.

Total RNA was extracted from tissues using TriPure Isolation Reagent (Roche Diagnostics, Belgium). Complementary DNA was prepared by reverse transcription of 1 µg of total RNA using the Reverse Transcription System (Promega, Madison, WI, USA). Real-time PCR was performed with a QuantStudio 3 Real-Time PCR System (Applied Biosystems, Den Ijssel, The Netherlands) using MasterMix GoTaq (Promega, WI, USA). Data were analyzed according to the 2-ΔΔCT method. The purity of the amplified products was verified by analyzing the melting curve obtained at the end of the amplification step. The ribosomal protein L19 (Rpl19) gene was used as a reference gene. Primer sequences are given in Supplementary Table S1.