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Identification of enterotoxins A, B and E genes of Staphylococcus aureus isolated from cheese handled in Jaboticabal

Grant number: 07/03537-4
Support type:Scholarships in Brazil - Scientific Initiation
Effective date (Start): December 01, 2007
Effective date (End): November 30, 2008
Field of knowledge:Agronomical Sciences - Veterinary Medicine - Products of Animal Origin Inspection
Principal researcher:Oswaldo Durival Rossi Junior
Grantee:Reinaldo Juan Garrido Palacios Junior
Home Institution: Faculdade de Ciências Agrárias e Veterinárias (FCAV). Universidade Estadual Paulista (UNESP). Campus de Jaboticabal. Jaboticabal , SP, Brazil

Abstract

The eruptions of alimentary poisoning are frequent provoked by enterotoxigenic Staphylococcus aureus propagated by milk and dairy products, which play an important nutritional role for the human. S. aureus produces staphylococcal enterotoxins (SEs) thermostables, being known: SEA-SEE, SEG-SEM., SEU; beyond other exotoxins: toxic shock toxin syndrome (TSST-1) and exfoliative toxins (ETA and ETB); where all the genes of these exotoxins had been published. This paper has for objective to identify A, B and E S. aureus enterotoxins isolated from caretakers Frescal Minas cheese in the Jaboticabal city. One expects with this paper to favor the diagnostic of SEs, with the use of PCR as alternative method for the rapid, sensitive e specific detection of the clinically important exotoxins, assisting in the prevention of cases of alimentary poisoning, with scientific informations and contributing with the improvement of the Public Health. For this, harvests of 30 samples of cheese in markets of the city will be made, preparing dilutions with 25 grams of each sample that will be homogenized with peptone (225 mL) and sterilized (0.1%) water. For identification 0.1 mL of each dilution will be deposited in agar plates that will be incubated 35ºC by overnight. It will be identify 20 typical colonies of each sample and it will sow in pipes with nutrient agar inclined and this incubated 35ºC by overnight. For the method of Gram will be prepared varnishes, where the Gram positives cocci in form of grape cluster will be submitted to the test of catalase for confirmation of the sort, and consecutively to the tests of coagulase it exempts, fermentation of mannitol in anaerobe, the production of acetoin (VP) and termonuclease, for identification of the species. The isolament of DNA will be used Instagene Matrix kit. The samples selected will make S. aureus DNA’s extraction strains that will be analyzed by single PCR, where each sample will be submitted the two distinct amplifications in separate pipes of PCR. The amplification for PCR will be carried through with total volume of 25µl where it is included: DNA, Taq DNA polymerase, MgCl2, dNTP mix and each primer. Magnifying will be made in an automated thermocycler, where the thermal cycle will be: 30 cycles of denaturation of 95ºC for 1 min (2 min the first cycle); annealing: 55ºC for 1 min; e polymerization: 72ºC for 2 min, where a last cycle with 5 min of duration. It will have analyzes it through the 1,5% agarose gel electrophoresis stain with ethidium bromide (5 mg/ml) and sensitivity will be on the basis of the visualization under UV ilumination at 254 nm.

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