Desenvolvimento e padronização de conjuntos de primers e sondas para detecção rápida do vírus da imunodeficiência felina (FIV) e vírus da leucemia felina (FeLV)
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Data
2022-12-12
Tipo de documento
Artigo Científico
Título da Revista
ISSN da Revista
Título de Volume
Área do conhecimento
Ciências da Saúde
Modalidade de acesso
Acesso fechado
Editora
Autores
Szelag, Beatriz Souza
Nogueira, Julia
Klemes Jr., Vanderlei
Orientador
Moreira, Debora
Coorientador
Cerqueira, Anderson Romério Azevedo
Resumo
O vírus da imunodeficiência felina (FIV) e o vírus da leucemia felina
(FeLV) são retrovírus de grande importância para a medicina veterinária, já que
acometem gatos selvagens e domésticos mundialmente. Por serem doenças
que podem evoluir para quadros clínicos severos em pouco tempo, faz-se
necessário o diagnóstico preciso e rápido, para que os animais tenham
melhores opções terapêuticas. Os testes disponíveis no mercado, como o
ELISA, são bons, porém não tão práticos e sensíveis o suficiente. Portanto, a
qPCR e a RT-qPCR vieram para solucionar os problemas encontrados por
outros métodos, proporcionando um diagnóstico prático, rápido e sensível.
Levando em conta o advento da PCR em tempo real para a detecção do FIV e
FeLV, este trabalho teve como objetivo desenvolver e padronizar conjuntos de
primers e sondas para detecção singleplex e multiplex destes patógenos.
Inicialmente foi feita uma pesquisa na ferramenta BLAST da NCBI para
encontrar regiões de similaridade do genoma do RNA viral do FIV e do FeLV,
para alinhamento dos primers. As regiões foram escolhidas de modo que não
houvesse interferência com outras sequências genéticas, garantindo maior
especificidade. A sonda escolhida foi a de hidrólise, pois garante maior
especificidade e possibilidade de desenvolver sistemas de detecção multiplex.
Foram feitos testes para determinar as condições de maior eficiência da
reação, como: gradiente de temperatura, concentração dos primers e sondas e
sensibilidade analítica. Foram avaliadas também a reprodutibilidade e
repetibilidade para análise da precisão dos ensaios. Foi constatado que os
conjuntos desenvolvidos apresentaram sensibilidade semelhante a kits
disponíveis no mercado (aproximadamente 500 cópias por mL), e a variação
entre os ensaios foi igual ou inferior a 5%, indicando que foram precisos. Sendo
assim, os conjuntos de primers e sondas desenvolvidos foram validados com
sucesso e apresentam boa eficiência, estando prontos para serem validados
também com amostras biológicas.
Palavras-chave: FIV, FeLV, PCR em tempo real, diagnóstico, felinos.
Feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV) are retroviruses of great importance to veterinary medicine, as they affect wild and domestic cats worldwide. As these are diseases that can progress to severe clinical conditions in a short time, an accurate and fast diagnosis is necessary, so that the animals have better therapeutic options. Tests available on the market, such as ELISA, are good, but not practical and sensitive enough. Therefore, qPCR and RT-qPCR emerged to solve the problems encountered by other methods, providing a practical, fast and sensitive diagnosis. Taking into account the advent of real-time PCR for the detection of FIV and FeLV, this work aimed to develop and standardize a set of primers and probes for singleplex and multiplex detection of these pathogens. Initially, a search was carried out using the NCBI BLAST tool to find regions of similarity in the genome of the viral RNA and the proviral DNA of both FIV and FeLV, for alignment of the primers. The regions were chosen so that there was no interference with other genetic sequences, ensuring greater specificity. The hydrolysis probe was chosen, as it guarantees greater specificity and the possibility of developing multiplex detection systems. Tests were carried out to determine the conditions of greater efficiency of the reaction, such as: temperature gradient, concentration of primers and probes and analytical sensitivity. Reproducibility and repeatability were also evaluated to analyze the accuracy of the tests. It was found that the set developed showed sensitivity similar to kits available on the market (approximately 500 copies per mL), and the variation between assays was equal to or less than 5%, indicating that they were accurate. Thus, the set of primers and probes developed were successfully validated and showed great efficiency, being ready to be validated also with biological samples. Keywords: FIV, FeLV, real-time PCR, diagnosis, felines.
Feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV) are retroviruses of great importance to veterinary medicine, as they affect wild and domestic cats worldwide. As these are diseases that can progress to severe clinical conditions in a short time, an accurate and fast diagnosis is necessary, so that the animals have better therapeutic options. Tests available on the market, such as ELISA, are good, but not practical and sensitive enough. Therefore, qPCR and RT-qPCR emerged to solve the problems encountered by other methods, providing a practical, fast and sensitive diagnosis. Taking into account the advent of real-time PCR for the detection of FIV and FeLV, this work aimed to develop and standardize a set of primers and probes for singleplex and multiplex detection of these pathogens. Initially, a search was carried out using the NCBI BLAST tool to find regions of similarity in the genome of the viral RNA and the proviral DNA of both FIV and FeLV, for alignment of the primers. The regions were chosen so that there was no interference with other genetic sequences, ensuring greater specificity. The hydrolysis probe was chosen, as it guarantees greater specificity and the possibility of developing multiplex detection systems. Tests were carried out to determine the conditions of greater efficiency of the reaction, such as: temperature gradient, concentration of primers and probes and analytical sensitivity. Reproducibility and repeatability were also evaluated to analyze the accuracy of the tests. It was found that the set developed showed sensitivity similar to kits available on the market (approximately 500 copies per mL), and the variation between assays was equal to or less than 5%, indicating that they were accurate. Thus, the set of primers and probes developed were successfully validated and showed great efficiency, being ready to be validated also with biological samples. Keywords: FIV, FeLV, real-time PCR, diagnosis, felines.
Palavras-chave
FIV, FeLV, PCR em tempo real, Diagnóstico, Felinos