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Master's Dissertation
DOI
10.11606/D.10.2009.tde-13072009-111831
Document
Author
Full name
Maria Del Pilar Vejarano Ruibal
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2009
Supervisor
Committee
Ferreira Neto, José Soares (President)
Ferreira, Fernando
Homem, Valéria Stacchini Ferreira
Title in Portuguese
Avaliação de diferentes protocolos de extração de DNA para detecção de Brucella abortus a partir de diferentes tecidos de vacas infectadas experimentalmente com a cepa 2308
Keywords in Portuguese
B. abortus
Extração de DNA
PCR
Vacas
Abstract in Portuguese
Este estudo comparou o desempenho de quatro protocolos de extração de DNA a partir de homogeneizados de diferentes órgãos provenientes de vacas infectadas experimentalmente com a B. abortus 2308. Os protocolos de extração comparados foram o método de GT (lise com isotiocianato de guanidina), Boom (lise com GT e tratamento com suspensão carreadora Diatomaceous earth), PK (lise com proteinase K) e Santos (lise por fervura e congelamento com nitrogênio líquido). Foram constituídos os grupos padrão ouro positivo e negativo baseados na bacteriologia clássica e compostos por: 54 cotilédones (27 pos. e 27 neg.), 39 linfonodos supra mamários (12 pos. e 27 neg.), 44 pré-escapulares (17 pos. e 27 neg.), 33 fígados (6 pos. e 27 neg.), 37 baços (10 pos. e 27 neg.), e 34 úberes (7 pos. e 27 neg.). Todas as amostras foram submetidas aos quatro protocolos de extração e a um mesmo processo de amplificação com os primers B4 e B5. Nos resultados consolidados por órgãos, a proporção de positivos no cotilédone foi maior do que a encontrada no linfonodo supramamário (p=0,0001), linfonodo pré-escapular (p<0,0001), fígado (p=0,0006), baço (p<0,0001) ou úbere (p=0,0019). Os resultados acumulados para os métodos de extração mostraram que o protocolo de Santos teve maior sensibilidade relativa do que o método de Boom (p=0,003) e GT (p=0,0506), e foi igual ao PK (p=0,2969). As demais comparações de proporções não resultaram em diferenças estatisticamente significantes. No estudo verificou-se amostas positivas a PCR e negativas ao isolamento e viceversa. Assim, apesar das desvantagens do método bacteriológico clássico, a melhor estratégia para o diagnóstico direto da infecção de vacas por B. abortus em homogeneizado de órgãos é a utilização conjunta do isolamento e da PCR, examinando os cotilédones e utilizando os métodos de extração de DNA Santos ou PK.
Title in English
Evaluation of different protocols of DNA extraction for Brucella abortus detection from different tissues from experimentally infected cows with 2308 strain
Keywords in English
B. abortus
Cows
DNA extraction
PCR
Abstract in English
This study compared the performance of four protocols of DNA extraction from suspensions of different tissues from experimentally infected cows with 2308 strain. The compared extraction protocols were GT protocol (lyse with guanidine isotiocianate), Boom (lyse with GT and treated with the carrying suspension Diatomaceous earth), PK (lise with proteinase K) and Santos (lyse by boiling and freezing with liquid nitrogen). Based on classical bacteriology, positive and negative gold standard groups were built and consisted of 54 cotyledons (27 pos. and 27 neg.), 39 supramammary lymph nodes (12 pos. and 27 neg.), 44 prescapulars (17 pos. and 27 neg.), 33 livers (6 pos. and 27 neg.), 37 spleens (10 pos. and 27 neg.), and 34 udders (7 pos. and 27 neg.). All the samples were submitted to the four DNA extraction protocols and the same amplification process using the primers B4 and B5. According to consolidated results by tissue, the proportion of positives in cotyledon was bigger than supramammary lymph node (p=0,0001), prescapular lymph node (p<0,0001), liver (p=0,0006), spleen (p<0,0001) and udder (p=0,0019). The consolidated results for the extraction methods show that Santos protocol had bigger relative sensitivity than Boom method (p=0,003) and GT (p=0,0506), and was equal than PK (p=0,2969). There were not significant statistical differences in the others comparisons of proportions. In the study, PCR-positive and isolation-negative samples and vice-versa were verified. However, the disadvantages of the classic bacteriological method, the best strategy for direct diagnosis of the infection of cows for B. abortus in homogenized of tissues is combined use of isolation and PCR, examining the cotyledons and using the methods of DNA extraction from Santos or PK.
 
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Publishing Date
2009-10-13
 
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