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Master's Dissertation
DOI
10.11606/D.10.2004.tde-16062005-103008
Document
Author
Full name
Nanci do Rosário Vieira
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2004
Supervisor
Committee
Soares, Rodrigo Martins (President)
Brandão, Paulo Eduardo
Richtzenhain, Leonardo Jose
Title in Portuguese
Desenvolvimento de uma Reação em Cadeia pela Polimerase (PCR) para detecção de Brucella spp. em amostras de sangue de cães naturalmente infectados
Keywords in Portuguese
Brucelose animal
Cães
Diagnóstico
Reação em cadeia por polimerase
Abstract in Portuguese
Foram desenhados três pares de primers, sendo um deles dirigido ao gene que codifica uma proteína periplasmática da Brucella (BP26) e dois outros dirigidos para a região interespaçadora entre os gene 16S e 23S do RNA ribossômico (ITS). Com os primers acima, foram padronizadas três PCRs, potencialmente capazes de detectar qualquer espécie de Brucella e denominadas PCR-ITS1, PCR-ITS2 e PCR-BP26. Soluções de DNA de sangue de cão livre de Brucella foram contaminadas com quantidades decrescentes de DNA de B. canis (RM6/66) e, então, submetidas às PCRs descritas anteriormente. Para a avaliação das PCRs quanto à capacidade de detecção de Brucella spp. em sangue de animais naturalmente infectados, foram utilizadas 75 amostras de sangue de cães provenientes de canis comerciais onde foram registrados casos clínicos sugestivos de infecção por Brucella. As 75 amostras de sangue de cães foram colhidas com anti-coagulante (citrato de sódio) por punção da veia jugular. As amostras foram separadas em duas alíquotas, uma das quais (2 ml) foi submetida ao isolamento e identificação bacteriológica e a outra alíquota (1ml), às PCRs. O DNA total das amostras de sangue foi extraído por método baseado em digestão com proteinase K, SDS e CTAB seguido de purificação com fenol e clorofórmio. Considerando os resultados da PCR de melhor desempenho (PCR-ITS1), entre as 35 amostras positivas pelo isolamento bacteriano, 30 (85,71%) foram positivas pela técnica de PCR, enquanto entre as 40 amostras negativas pelo cultivo bacteriológico, 11 (27,5%) foram positivas pela PCR. A PCR-ITS1 foi capaz de detectar um mínimo de 3,78fg de DNA bacteriano misturado a 450ng de DNA de cão, o que representa, teoricamente, a massa genômica de 1,26 bactérias. Pela aparente superior capacidade de classificar animais positivos que o método de cultura bacteriológica, a PCR-ITS1 parece ser uma técnica promissora para o diagnóstico direto da brucelose em cães.
Title in English
Development of a polymerase chain reaction (PCR) for the detection of Brucella spp. in whole-blood samples from naturally infected dogs
Keywords in English
Animal brucellosis
Diagnosis
Dogs
Polymerase chain reaction
Abstract in English
Three pair of primers were designed against Brucella genetic sequences. Two of them were directed to 16S-23S rRNA interspacer and the other was directed to a periplasmatic protein coding gene (BP26). Three PCRs assays named PCR-ITS1, PCR-ITS2 and PCR-BP26, each one putatively capable of amplifying DNA from any Brucella species were tested using the aforementioned primers. Nucleic acid extracts from whole-blood from naïve dogs were spiked with decreasing amounts of B. canis (RM6/66) DNA and the resulting solutions were tested by PCR. The capability of the PCRs to amplify Brucella spp. genetic sequences from naturally infected dogs was evaluated by using 75 whole-blood samples from dogs raised in commercial kennels were clinically affected animals have already been detected with signs suggestive of Brucella infection. The whole-blood samples were collected with sodium citrate as anti-coagulant by venal puncturing the jugular vein. The samples were split into two aliquots, one of them (2mL) being submitted to bacterial isolation and identification and the remaining sample (1mL) to PCR. The DNA from whole-blood was extracted by using proteinaseK, SDS and CTAB following phenol-chloroform purification. Considering the results from the PCR with the best performance (PCR-ITS1), within 35 isolation positive samples, 30 were positive by PCR. Within 40 isolation negative samples, 11 were positive by PCR. PCR-ITS1 was capable of detecting as few as 3.78fg of bacterial DNA mixed to 450ng of host DNA. Theoretically, 3.78fg of Brucella DNA represents the total genomic mass of 1.26 bacterial cells. The superior ability of PCR-ITS1 on classifying positive animals suggest that PCR-ITS1 is a promising tool for the direct detection of canine brucellosis.
 
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NanciVieira.pdf (1.02 Mbytes)
Publishing Date
2007-04-05
 
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