Evaluation of methods of DNA extraction from Staphylococcus aureus in milk for use in real-time PCR.
The aim of this study was to evaluate the repeatability and performance of 4 methods of extracting DNA from Staphylococcus aureus (SAU) and the gene encoding bovine mitochondrial cytochrome B (BMCB) in milk samples from cows with subclinical mastitis for use in amplification by real-time polymerase chain reaction. Two milk samples were obtained from cows naturally infected with S. aureus and subjected to the following extraction methods: Qiagen DNA extraction kit; Axyprep DNA extraction kit; in silica column boil and in silica column method. After extraction in duplicate, eluates were subjected to purification and precipitation to determine purity (A₂₆₀/A₂₈₀ ratio) and concentration (μg/μL) by spectrophotometry and amplification by real-time polymerase chain reaction of target genes (SAU and BMCB). There was no effect of the DNA extraction method on DNA concentration and threshold cycle for BMCB and SAU. The purity ratio (A₂₆₀/A₂₈₀ ) was higher when using Qiagen DNA extraction (1.76 ± 0.136) compared to the other methods tested. Our results indicate that the DNA extraction kit from Qiagen produces samples of the highest purity ratio compared to other methods.
Departamento de Nutrição e Produção Animal, Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, Pirassununga, SP, Brasil.
A G
AG
Dibbern
Departamento de Nutrição e Produção Animal, Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, Pirassununga, SP, Brasil.
B G
BG
Botaro
Departamento de Nutrição e Produção Animal, Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, Pirassununga, SP, Brasil.
M P
MP
Viziack
Departamento de Nutrição e Produção Animal, Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, Pirassununga, SP, Brasil.
L F P
LF
Silva
Departamento de Nutrição e Produção Animal, Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, Pirassununga, SP, Brasil mveiga@usp.br.
M V
MV
Santos
D000818:Animals; D002417:Cattle; D004269:DNA, Bacterial / Q000302:isolation & purification*; D008892:Milk / Q000382:microbiology*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D013211:Staphylococcus aureus / Q000235:genetics*
D004269:DNA, Bacterial