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GUIDO, M. C. Detection of Brucella abortus DNA by PCR in spiked buffalo milk with B. abortus strain 1119-3. [Detecção de DNA de Brucella abortus pela PCR em leite bubalino experimentalmente contaminado pela amostra 1119-3]. 2003. 46 f. Tese (Doutorado em Reprodução Animal) - Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, São Paulo, 2003. To
improve the PCR performance for detection of Brucella spp. in buffalo milk, different DNA extraction protocols
were carried out in buffalo milk spiked with Brucella abortus strain
1119-3. These protocols were
based on utilization of guanidine isothiocyanate, boil and proteinase K, with
some variations. The analytical sensitivity was the evaluation criteria. Boiling
with initial filtration of the sample through a solution of sacarosis 40%
presented analytical sensitivity of 103 CFU/ml. The protocols based
on proteinase K presented analytical sensitivity of 104 CFU/ml. The
highest analytical sensitivity
(10 CFU/ml) and best visualization of the amplified product were verified
for the protocol using guanidine isothyocianate with
immediate precipitation in alcohol without addition of Tween 20 in the
initial sample wash. |
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