Inventor: Assist. Prof. Dr. Marwa Hamid Mutasher, Prof. Dr. Mai Taleb Fleih Issa/Department of Biology, College of Science, University of Baghdad
Abstract
Staphylococci possess a rigid cell wall which is so difficult to be broken by boiling or using synthetic extraction kits except using costly digestive enzymes (Lysostaphin and lysozyme with proteinase K) and for that the staphylococci are the main cause of food poisoning in Iraq, so this study aimed to achieve a rapid and inexpensive method to extract Staphylococcal DNA without using irritant chemicals or costly synthetic extraction kits which require the addition of costly enzymes only when used to extract DNA from this rigid cell wall bacteria. Milk and cheese samples were collected randomly from Baghdad local markets, staphylococci were isolated and identified using biochemical and serological tests as S. aureus, S.epidermidis, and S. chromogenes.
Laboratory experiments were done to optimize DNA extraction from Staphylococci. Neither synthetic extraction kits nor lytic enzymes, only DNase free distilled water was used in DNA extraction. Temperature and time parameters were tested to breakdown the staphylococcal cell wall. A simple and inexpensive method was conducted in this study, were DNA extracted by heating the cell lysate to 85 C for 20 min. and sudden freezing by ice bath for 10 min. thereafter a centrifugation step at 10000 rpm for 10 min. was applied, the supernatant was aspirated since it represents the nucleic acids, which were tested for its purity and quality by spectrophotometer and agaros gel electrophoresis (0.8 agaros, ethedium bromide stain, 70 volt, for 1 hour). PCR technique using fem A gene to confirm S. aureus identification and seb gene responsible for SEB enterotoxin to give assurance that the extracted DNA in this study is suitable for genetic studies and this is of great importance for our country since there is no available method in our country to detect the presence of staphylococci and its enterotoxins in foods. The method described by this study is a rapid, efforts and cost effective can be used for molecular detection of the staphylococci and their enterotoxins in imported foods.