TITLE: Polymerase Chain Reaction - Comparative genomics of Staphylococcus aureus associated with subclinical and clinical bovine mastitis (Rocha et al., 2019)
AUTHORS: Lis Rocha

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Comparative genomics of </span><span style = "font-style:italic;">Staphylococcus aureus </span><span>associated with subclinical and clinical bovine mastitis (Rocha et al., 2019)</span></div><div class = "text-block"><span>Many efforts have been made to understand the pathogenesis of bovine mastitis to reduce losses and promote animal welfare. </span><span style = "font-style:italic;">Staphylococcus aureus</span><span> may cause bovine clinical mastitis, but it is mainly associated with subclinical infection, which is usually persistent and can easily reoccur. Here, we conducted a comparative genomic analysis between four strains of </span><span style = "font-style:italic;">S. aureus</span><span> causing subclinical infection (Sau170, 302, 1269, 1364), previously sequenced by our group, and two well-characterized strains causing clinical mastitis (N305 and RF122) to find differences that could be linked to mastitis outcome. A total of 146 virulence-associated genes were compared and no appreciable differences were found between the bacteria. However, several nonsynonymous single nucleotide polymorphisms (SNPs) were identified in genes present in the subclinical strains when compared to RF122, especially in genes encoding host immune evasion and surface proteins. The comparison of orthologous genes using OrthoMCL identified a membrane transporter in the genomes of the bacteria belonging to the subclinical group, but this finding was not confirmed by polymerase chain reaction (PCR) on a collection of field isolates of </span><span style = "font-style:italic;">S. aureus</span><span> associated with clinical or subclinical mastits. The secreted and surface proteins predicted by different in silico tools were compared through multidimensional scaling analysis, revealing a high degree of similarity among the six strains. However, differences were seen in the nucleotide sequences of a gene that codes for a hypothetical protein (cl3309) and a lipoprotein (cl3700). These findings were also analyzed by PCR on DNA extracted from field isolates of</span><span style = "font-style:italic;"> S. aureus</span><span>. The lipoprotein, but not the hypothetical protein, was able to separate the clinical isolates from the subclinical ones. These results show that sequence variation among bovine </span><span style = "font-style:italic;">S. aureus</span><span>, and not only the presence/absence of virulence factors, is an important aspect to consider when comparing isolates causing different mastitis outcomes</span></div></div>

[STEPS]
?. [primers cl3309sub F/R]
35 cycles of denaturation at 95.0 °C for 45 s,
?. [primers cl3309sub F/R]
Anealing: 55 °C for 45s
?. [primers cl3309sub F/R]
Extension: 72 °C for 45 s
?. [primers cl3309sub F/R]
final extension at 72.0 °C for 10 min
?. [primers cl3316F/R]
initial denaturation: 95.0 °C for 5 min;
?. [primers cl3316F/R]
35 cycles of denaturation at 95.0 °C for 45 s,
?. [primers cl3316F/R]
Annealing: 55 °C for 45 s
?. [primers cl3316F/R]
Extension: 72 °C for 45 s
?. [primers cl3316F/R]
final extension at 72.0 °C for 10 min.
?. [primers cl3700 - LipoP FCS/RC]
initial denaturation: 95.0 °C for 5 min;
?. [primers cl3700 - LipoP FCS/RC]
35 cycles of denaturation at 95.0 °C for 45 s,
?. [primers cl3700 - LipoP FCS/RC]
Annealing: 54 °C for 45 s
?. [primers cl3700 - LipoP FCS/RC]
Extension: 72 °C for 45 s
?. [primers cl3700 - LipoP FCS/RC]
final extension at 72.0 °C for 10 min.
?. [cl33009cli F/R]
initial denaturation: 95.0 °C for 5 min;
?. [cl33009cli F/R]
35 cycles of denaturation at 95.0 °C for 45 s,
?. [cl33009cli F/R]
Annealing: 45 °C for 45 s
?. [cl33009cli F/R]
Extension: 72 °C for 30 s
?. [cl33009cli F/R]
final extension at 72.0 °C for 10 min.
?. [primers cl3700 - LipoP FCS/RCS]
initial denaturation: 95.0 °C for 5 min;
?. [primers cl3700 - LipoP FCS/RCS]
35 cycles of denaturation at 95.0 °C for 45 s,
?. [primers cl3700 - LipoP FCS/RCS]
Annealing: 50 °C for 45 s
?. [primers cl3700 - LipoP FCS/RCS]
Extension: 72 °C for 1min
?. [primers cl3700 - LipoP FCS/RCS]
final extension at 72.0 °C for 10 min.
?. [Analyzing the amplified fragments]
Analyze the amplicons by electrophoresis in 1X Tris-acetate-EDTA on a 1.0% agarose gel  and visualizeimagen under UV light after staining with 2 mg.ml-1 ethidium bromide.
?. [primers cl3309sub F/R]
Initial denaturation: 95.0 °C for 5 min;