Genejetgenomic dna kit

Diagnosis of HP was accepted when at least the results of one of the areas were positive. The detection of HP in gastric tissue samples was carried out through the extraction of genomic DNA with the GeneJETGenomic DNA kit (Thermo Fisher Scientific, Waltham, MA), the quantification by fluorometry was conducted using the Quantus fluorometer (Promega, Madison, WI), and, subsequently, the standard detection of the colonizing genes (hspA and UreA) by quantitative polymerase chain reaction (qPCR) in the LightCycler 96 Instrument Thermal Cycler (Roche, Mannheim, Germany). The hspA gene sequence was detected with the following primers: reverse: 5′-GCT ATC TGA AAA TTT GAT TTC TTT TGC-3′ and forward: 5′-TGC GCT ATA GTT GTG TCG C-3′; and that of UreA: reverse: 5′-TTG TCT GCT TGT CTA TCA ACC-3′ and forward: 5′-GAG AAT GAG ATG AAA CTC ACC C-3′. The mixture for the qPCR was composed of PCR-grade water, Sybr GreenFastStartEssential DNA Green Master (Roche), the respective primers, and the sample of genomic DNA.^{17 ,18}

For the microbe most affected by SNPs, sample was grown on nutrient agar medium with SNPs applied using disk diffusion at MIC. Untreated group was prepared using sterilized water instead of SNP. Ultrastructure of cell surface was studied by scanning electron microscopy (JSM 6510-LV SEM, JEOL, Japan). Also, protein banding of the microbe was conducted via sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) using TriFast™ kit (Peqlab, VWR Co., UK). For DNA fragmentation, GeneJet™ genomic DNA kit (Thermo Fisher Scientific Co., USA). For protein and DNA, gel was imaged using gel documentation system (Geldoc-it, UVP, UK) and data were analyzed using Totallab® (version 1.0.1) software. Both gels were presented to clearly indicate major differences between lanes.

Extraction and purification of the DNA was performed using two extraction kits. The GeneJET Genomic DNA kit (Thermo Scientific™, Waltham, MA, USA) was used for the first 3 samples, and the PureLink™ Genomic DNA Mini Kit and Extraction Kit (Invitrogen, Waltham, MA, USA) for the last four sampling times. Three sets of primers were used for detection of the pathogens (one per pathogen). The primers were used to detect PCV2 by amplifying the gene encoding for replicasa (REP), located in the open reading frame 1 (ORF1). Primers to detect M. hyopneumoniae are modified versions of those described by Marois et al., 2010 [38 (link)] to amplify the p102 gene. Detection of M. hyorhinis was carried out by amplifying the 16S coding gene, as described by Stakenborg et al., 2006 [39 (link)]. A positive control DNA extracted from the bacteria/virus was used. Detection of PCV2 and M. hyopneumoniae via qPCR Multiplex and for the detection of M. hyorhinis, Sybr Green was used (Table 4). In addition, a melting curve from 60 °C to 95 °C, with increases of 0.5 °C, was used. After the amplification was performed, its positivity was confirmed by showing the amplified product.