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Doctoral Thesis
DOI
10.11606/T.6.2004.tde-22032005-093557
Document
Author
Full name
Therezinha Travassos Carvalho de Almeida
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2004
Supervisor
Committee
Matte, Maria Helena (President)
Chieffi, Pedro Paulo
Chioccola, Vera Lucia Pereira
Natal, Delsio
Soares, Rodrigo Martins
Title in Portuguese
"Padronização e avaliação de métodos moleculares para detecção de oocistos de Cryptosporidium spp. (Apicomplexa: Cryptosporiidae) em amostras fecais: extração de DNA genômico e PCR (reação em cadeia pela polimerase)"
Keywords in Portuguese
Cryptosporidium
eletroforese modificada
Extração de DNA
PCR
Abstract in Portuguese
O protozoário parasita Cryptosporidium parvum tem sido reconhecido como um importante patógeno emergente. Para estudos moleculares, a maioria das técnicas para extração do DNA requer o uso de kits importados para concentrar, romper a parede muito resistente do oocisto e purificar o DNA das matrizes das amostras. O objetivo do estudo foi desenvolver um método simples e rápido, baseado na reação em cadeia pela polimerase (PCR) para detectar Cryptosporidium em fezes preservadas. Oocistos foram concentrados das amostras fecais pela flutuação em gradiente de sacarose. Dos oocistos purificados foi extraído o DNA genômico através de incubação em tampão de lise contendo 70 mM -mercaptoetanol, digerido com proteinase K e extraído com fenol-clorofórmio-álcool isoamílico. A padronização foi iniciada com a PCR única para detectar Cryptosporidium spp usando um par de primer genérico (AWA). Para identificar C. parvum foi realizada a PCR única com o par de primer especifico (LAX). Para aumentar a sensibilidade do método, foi testada a nested-PCR, usando o primer externo XIA. Foram analisadas 39 amostras de DNA do bezerro padrão, 52 amostras de 17 pacientes e 45 amostras de 14 animais. Os resultados foram: 54,28% de positividade na PCR AWA, e 71,42% na nested-PCR XIA/AWA, 67,74% na PCR LAX e 44,44% na nested-PCR XIA/LAX das amostras do bezerro. A positividade geral nas amostras de pacientes e de animais foi: 34,48% pela PCR and 54,83% pela nested-PCR para Cryptosporidium spp e 16,00% pela PCR e 50,00% pela nested-PCR para C. parvum. O emprego do corante Vistra Green melhorou significativamente a visualização do gel. Os resultados mostram que este método simples e de baixo custo pode ser melhorado para aplicação na rotina do laboratório.
Title in English
Standardization and evaluation of molecular methods to detect oocysts of Cryptosporidium spp (Apicomplexa: Cryptosporiidae) in faecal samples: extraction of genomic DNA and PCR (polymerase chain reaction).
Keywords in English
Cryptosporidium
DNA extraction
electrophoresis modification
PCR
Abstract in English
The protozoan parasite Cryptosporidium parvum has become recognised as important emerging human pathogens. For molecular studies, most of the techniques to extract genomic DNA require the use of imported kits to concentrate, rupture the very resistant oocyst wall, and purify the DNA from the samples matrix. The aim of this study was to develop a simple and rapid method based on polymerase chain reaction (PCR) to detect Cryptosporidium in preserved faeces. Oocysts were concentrated from faecal specimens by flotation on sucrose gradient. Genomic DNA was prepared from purified oocysts by adding a lysis buffer containing 70 mM -mercaptoethanol, digested with proteinase K and extracted with phenol-chlorophorm-isoamyl. The standardization was started by performing a one step PCR to detect Cryptosporidium spp using a generic set of primer (AWA). To detect C. parvum a one step PCR was assayed using the specific primer (LAX). To increase the sensitivity of the method, were tested nested-PCR assays, using an outer primer (XIA). Thirty nine DNA samples were analysed from the standard calf, 52 samples from 17 patients and 45 samples from 14 animals. The results were: 54.28% positive samples by single PCR AWA, 71.42% by nested-PCR, 67.74% by single PCR LAX and 44.44% by nested-PCR for the standard calf. The overall positivity for human and animal samples were: 34.48% by single PCR and 54.83% by nested-PCR for Cryptosporidium spp and 16.00% by single PCR and 50.00% by nested-PCR for C. parvum. Using Vistra Green for staining agarose gel, yielded the visualisation of the amplicons. These results show that this simple and cheap method could be improved to be used on the routine laboratory work.
 
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teseTDE230305.pdf (888.77 Kbytes)
Publishing Date
2005-04-04
 
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