Evaluation of four methods of DNA extraction and cattle for bacterial quantification by real time PCR

• Grant number: 10/10584-1
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Effective date (Start): September 01, 2010
• Effective date (End): January 31, 2011
• Field of knowledge: Agronomical Sciences - Animal Husbandry - Animal Production
• Principal Investigator: Marcos Veiga dos Santos
• Grantee: Aline Gerato Dibbern
• Host Institution: Faculdade de Medicina Veterinária e Zootecnia (FMVZ). Universidade de São Paulo (USP). São Paulo , SP, Brazil

Abstract

The aim of this study is to compare the results of repeatability and yield of four techniques for extraction of bacterial DNA from S. aureus present in milk samples from cows with subclinical mastitis, with a view to amplification by polymerase chain reaction in real time. Will be used ten composite samples of milk for the species S. aureus for use in the four methodologies: A) protocol described by Cremonesi et al. (2006); B) Protocol of commercial extraction of DNA (1); C) Protocol of commercial extraction of DNA (2); D) Boil in sterile water described by Barea et al. (2004). After processing of the milk samples by four methods under test, in quadruplicate, the material extracted will be submitted to determine purity (A260/A280 ratio) and concentration (mg / mL) by spectrophotometry, and quantification of target genomes (S . aureus and cattle) by the reaction of real-time PCR (Ct). Then, the results of each treatment for each of the variables, will be submitted to descriptive statistical analysis to determine the mean, standard deviation and coefficient of variation between quadruplicate, and averages of each treatment will be submitted to analysis of variance to test the hypothesis of equality between the extraction methods for the study variables. In this study, expected to determine the method for extraction of nucleic acids more suitable for the amplification of genomic DNA by PCR in real time, with adequate repeatability for use in routine diagnosis of subclinical mastitis with the quantification of the pathogen and the cell Somatic present in bovine milk sample.

Scientific publications

(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)

DIBBERN, A. G.; BOTARO, B. G.; VIZIACK, M. P.; SILVA, L. F. P.; SANTOS, M. V.. Evaluation of methods of DNA extraction from Staphylococcus aureus in milk for use in real-time PCR. Genetics and Molecular Research, v. 14, n. 1, p. 227-233, 2015. (10/10584-1)