RESUMO
Senecavirus A (SVA) belonging to the family Picornaviridae, genus Senecavirus was incidentally isolated in 2002 from the PER.C6 (transformed foetal retinoblast) cell line. However, currently, this virus is associated with vesicular disease in swine and it has been reported in countries such as the United States of America, Canada, China, Thailand and Colombia. In Brazil, the SVA was firstly reported in 2015 in outbreaks of vesicular disease in swine, clinically indistinguishable of Foot-and-mouth disease, a contagious viral disease that generates substantial economic losses. In the present work, it was standardized a diagnostic tool for SVA based on RNA reverse transcriptase droplet digital PCR (RT-ddPCR) using one-step and two-step approaches. Analytical sensitivity and specificity were done in parallel with real-time PCR, RT-qPCR (one-step and two-step) for comparison of sensitivity and specificity of both methods. In the standardization of RT-ddPCR, the double-quenched probe and the temperature gradient were crucial to reduce background and improve amplitude between positive and negative droplets. The limit of detection and analytical specificity of techniques of one-step techniques showed superior performance than two-step methods described here. Additionally, the results showed 94.2% concordance (p < 0.001) for RT-ddPCR and RT-qPCR using the one-step assay approach and biological samples from Brazilian outbreaks of Senecavirus A. However, ddRT-PCR had a better performance than RT-PCR when swine serum pools were tested. According to the results, the one-step RT-ddPCR and RT-qPCR is highlighted to be used as an auxiliary diagnostic tool for Senecavirus A and for viral RNA absolute quantification in biological samples (RT-ddPCR), being a useful tool for vesicular diseases control programs.
Assuntos
Doenças Transmissíveis Emergentes/veterinária , Surtos de Doenças/veterinária , Infecções por Picornaviridae/veterinária , Picornaviridae/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Doenças dos Suínos/epidemiologia , Animais , Brasil/epidemiologia , Doenças Transmissíveis Emergentes/epidemiologia , Doenças Transmissíveis Emergentes/virologia , Enterovirus Humano B/genética , Enterovirus Humano B/isolamento & purificação , Picornaviridae/genética , Infecções por Picornaviridae/epidemiologia , Infecções por Picornaviridae/virologia , RNA Viral/análise , Sensibilidade e Especificidade , Suínos , Doenças dos Suínos/virologia , Doença Vesicular Suína/epidemiologia , Doença Vesicular Suína/virologiaRESUMO
The aims of this study were to standard and optimize a qPCR protocol with FAM-BHQ1 probe, and to compare its sensitivity against TaqMan qPCR and PCR methods to diagnose shrimp WSD. The FAM-BHQ1 qPCR presented higher clinical sensitivity and showed to be a robust alternative to detect WSSV in clinical samples.
Assuntos
Animais , Técnicas de Diagnóstico Molecular/métodos , Penaeidae/virologia , Reação em Cadeia da Polimerase em Tempo Real/métodos , Medicina Veterinária/métodos , Sensibilidade e EspecificidadeRESUMO
The aims of this study were to standard and optimize a qPCR protocol with FAM-BHQ1 probe, and to compare its sensitivity against TaqMan qPCR and PCR methods to diagnose shrimp WSD. The FAM-BHQ1 qPCR presented higher clinical sensitivity and showed to be a robust alternative to detect WSSV in clinical samples.(AU)
Assuntos
Reação em Cadeia da Polimerase , Reação em Cadeia da Polimerase em Tempo Real , PenaeidaeRESUMO
The aims of this study were to standard and optimize a qPCR protocol with FAM-BHQ1 probe, and to compare its sensitivity against TaqMan qPCR and PCR methods to diagnose shrimp WSD. The FAM-BHQ1 qPCR presented higher clinical sensitivity and showed to be a robust alternative to detect WSSV in clinical samples.
Assuntos
Técnicas de Diagnóstico Molecular/métodos , Penaeidae/virologia , Reação em Cadeia da Polimerase em Tempo Real/métodos , Medicina Veterinária/métodos , Animais , Sensibilidade e EspecificidadeRESUMO
This study was designed to determine the effect of cooling at a rate of - 0.13 o C/min or any slow cooling prior to freezing on stallion sperm and to determine th e effect of two thawing temperatures on the viability of sperm frozen using a lactose - EDTA - egg yolk extender (LAC) with 3.5% ethylene glycol (LAC 3.5% EG) or a LAC with the additional incorporation of methyl cellulose, trehalose and acetamide (LAC+5% AC). No differences were observed in the post - thaw parameters of sperm frozen using either one of the cryogenic agents or frozen with or without previous slow cooling. Thawing at 75 o C was better than at 37 o C (P < 0.05). An interaction was observed between the c ryoprotectant and the freezing protocol (P < 0.05). A conception rate of 23.3% was obtained after AI using equine semen frozen with LAC+5% AC.(AU)
Assuntos
Animais , Fertilidade/fisiologia , Sêmen/citologia , Espermatozoides/citologia , Equidae/classificação , CriopreservaçãoRESUMO
This study was designed to determine the effect of cooling at a rate of - 0.13 o C/min or any slow cooling prior to freezing on stallion sperm and to determine th e effect of two thawing temperatures on the viability of sperm frozen using a lactose - EDTA - egg yolk extender (LAC) with 3.5% ethylene glycol (LAC 3.5% EG) or a LAC with the additional incorporation of methyl cellulose, trehalose and acetamide (LAC+5% AC). No differences were observed in the post - thaw parameters of sperm frozen using either one of the cryogenic agents or frozen with or without previous slow cooling. Thawing at 75 o C was better than at 37 o C (P < 0.05). An interaction was observed between the c ryoprotectant and the freezing protocol (P < 0.05). A conception rate of 23.3% was obtained after AI using equine semen frozen with LAC+5% AC.