Padronização de protocolo utilizando partículas magnéticas para extração de DNA do circovírus suíno (PCV)
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Data
2022-08-26
Autores
Silva, Bruna Lindolfo da
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Universidade Estadual Paulista (Unesp)
Resumo
O Brasil ocupa o quarto lugar no ranking mundial de exportação de carne suína e para alcançar tais índices zootécnicos, os produtores necessitam do controle de várias doenças emergentes em suínos, sendo a identificação e prevenção fatores essenciais. Dentre as principais doenças, destaca-se a circovirose suína, tendo o circovírus suíno 2 (PCV-2) como agente etiológico. O diagnóstico da circovirose suína se dá por meio dos sinais clínicos associados a testes laboratoriais. A utilização da PCR quantitativa (qPCR) para a determinação da concentração do DNA viral vem sendo considerada o método de escolha neste contexto devido ao alto poder de detecção e na rapidez para obtenção dos resultados. Entretanto, a aplicabilidade desta técnica a torna problemática, devido ao alto custo para a aquisição dos insumos. Um dos principais fatores associados ao elevado custo é a utilização de kits comerciais para extração do ácido nucleico. Grande parte desses insumos são adquiridos pela importação, o que além de refletir no aumento do valor devido a logística, podem ocasionar problemas na produção devido a fatores externos como alta demanda e falta de matéria prima. Com isso, este trabalho teve como objetivo padronizar uma metodologia simples para a extração de DNA, a fim de proporcionar a otimização das análises moleculares para o diagnóstico do PCV em diferentes amostras. O protocolo desenvolvido foi aplicado em amostras de saliva, sangue total e soro de suínos infectados naturalmente. Os produtos das extrações utilizando o protocolo desenvolvido foram submetidas às análises de qPCR para a quantificação do DNA do PCV. Adicionalmente, a análise de integridade do ácido nucleico pela espectrofotometria UV-Vis foi realizada para obtenção dos dados de pureza do material extraído. Dois kits comerciais, um baseado na metodologia de extração por coluna de sílica e o outro por partículas magnéticas, foram utilizados a fim de comparar a eficiência do protocolo proposto. Os protocolos desenvolvidos neste trabalho apresentaram eficiência similar aos kits comerciais para a extração de DNA do PCV para os três tipos de amostras. Todos os protocolos de extração adotados apresentaram baixa pureza em relação às razões 260/280 e 260/230, porém tal fato não interferiu na análise por qPCR. Fatores como simplicidade de execução e possibilidade de automação favorecem a sua utilização.
Brazil occupies the fourth position in the world ranking of pork exports, and to reach such zootechnical indexes, producers need to control several emerging diseases in swine, being the identification and prevention essential factors for this. Among the main diseases, presenting Porcine circovirus 2 (PCV-2) as the etiological agent. The diagnosis of porcine circovirus diseases (PCVD) occurs through clinical signs associated with laboratory tests. The use of quantitative PCR (qPCR) to determine the concentration of viral DNA has been considered the method of choice in this context due to the high power of detection and the speed to obtain the results. However, the applicability of this technique makes it problematic, due to the high cost of acquiring the inputs. One of the main factors associated with the high cost is the use of commercial kits for nucleic acid extraction. Most of these inputs are acquired through imports, which, in addition to reflecting in the increase in value due to logistics, can cause problems in production due to external factors such as high demand and lack of raw material. Thus, this work aimed to standardize a simple methodology for DNA extraction, in order to provide the optimization of molecular analyzes for the diagnosis of PCV in different samples. The developed protocol was applied to saliva, whole blood and serum samples from naturally infected pigs. The products of the extractions using the protocol developed were submitted to qPCR analysis to quantify the PCV DNA. Additionally, nucleic acid integrity analysis by UV-Vis spectrophotometry was performed to obtain data on the purity of the extracted material. Two commercial kits, which used the extraction methodology by silica column and the other by magnetic particles, were used in order to compare the efficiency of the proposed protocol. The protocol developed in this work showed efficiency similar to commercial kits for PCV DNA extraction in serum and saliva samples. All protocols adopted showed low purity in relation to the 260/280 and 260/230 ratios, but this fact did not interfere with the qPCR analysis. Factors such as simplicity of execution and possibility of automation favor its use.
Brazil occupies the fourth position in the world ranking of pork exports, and to reach such zootechnical indexes, producers need to control several emerging diseases in swine, being the identification and prevention essential factors for this. Among the main diseases, presenting Porcine circovirus 2 (PCV-2) as the etiological agent. The diagnosis of porcine circovirus diseases (PCVD) occurs through clinical signs associated with laboratory tests. The use of quantitative PCR (qPCR) to determine the concentration of viral DNA has been considered the method of choice in this context due to the high power of detection and the speed to obtain the results. However, the applicability of this technique makes it problematic, due to the high cost of acquiring the inputs. One of the main factors associated with the high cost is the use of commercial kits for nucleic acid extraction. Most of these inputs are acquired through imports, which, in addition to reflecting in the increase in value due to logistics, can cause problems in production due to external factors such as high demand and lack of raw material. Thus, this work aimed to standardize a simple methodology for DNA extraction, in order to provide the optimization of molecular analyzes for the diagnosis of PCV in different samples. The developed protocol was applied to saliva, whole blood and serum samples from naturally infected pigs. The products of the extractions using the protocol developed were submitted to qPCR analysis to quantify the PCV DNA. Additionally, nucleic acid integrity analysis by UV-Vis spectrophotometry was performed to obtain data on the purity of the extracted material. Two commercial kits, which used the extraction methodology by silica column and the other by magnetic particles, were used in order to compare the efficiency of the proposed protocol. The protocol developed in this work showed efficiency similar to commercial kits for PCV DNA extraction in serum and saliva samples. All protocols adopted showed low purity in relation to the 260/280 and 260/230 ratios, but this fact did not interfere with the qPCR analysis. Factors such as simplicity of execution and possibility of automation favor its use.
Descrição
Palavras-chave
PCV, DNA, Extração de ácido nucleico, Partículas magnéticas, PCR quantitativa (qPCR), Nucleic acid extraction, Magnetic particles, Quantitative PCR (qPCR)