. Introduction Mastitis is recognized as the most important dairy herd problems worldwide [1] Economic losses of mastitis include decrease in milk quantity & quality and high cost of treatment [2]. S. aureus is one of the most common etiological pathogens, causing intramammary infection in dairy herds leading to severe economic losses in dairy industry [3] which colonize in the mammary gland [4]. S. aureus produce a large number of potential virulence factors which have an important role in the pathogenesis of mastitis [5]. These include, Coagulase which is considered the most important virulence factors that clot plasma and coats the bacterial cell, so prevent the phagocytosis [6] via enables staphylococci to captured within a fibrin meshwork, …show more content…
After incubation, 200 µl of 100% ethanol was added to the lysate. The sample was then washed and centrifuged following the manufacturer’s recommendations. Nucleic acid was eluted with 100 µl of elution buffer provided in the kit. Oligonucleotide Primer. Primers used were supplied from Metabion (Germany) are listed in table (1). PCR amplification. Primers were utilized in a 25- µl reaction containing 12.5 µl of Emerald Amp Max PCR Master Mix (Takara, Japan), 1 µl of each primer of 20 pmol concentrations, 4.5 µl of water, and 6 µl of DNA template. The reaction was performed in an applied biosystem 2720 thermal cycler. 3. Analysis of the PCR products The products of PCR were separated by electrophoresis on 1.5% agarose gel (Applichem, Germany, GmbH) in 1x TBE buffer at room temperature using gradients of 5V/cm. For gel analysis, 15 µl of the products was loaded in each gel slot. A 100 bp and 100DNA Ladders (Qiagen, Germany, GmbH) was used to determine the fragment sizes. The gel was photographed by a gel documentation system (Alpha Innotech, Biometra), and the data was analyzed through computer software. Table 1: Primers Sequences, Target Genes, Amplicon Sizes and Cycling Conditions. Target gene Primers sequences Amplified segment (bp) Primary