RESUMO
AIMS: The aim of this study was to compare the performance of four RT-qPCR assays for the detection of human and bovine group A rotaviruses and to characterize the positive samples by sequence analysis of VP4 and VP7 genes. METHODS AND RESULTS: RNA extracted from eight human rotavirus strains, and a panel of 33 human and 25 bovine faecal samples was subjected to different RT-qPCR detection systems. Among these assays, only RT-qPCR primers and probe systems B and C were able to detect all human rotavirus strains from cell culture solutions and faecal samples. However, the results showed that the system C was generally more sensitive by one or two logs than the other RT-qPCR assays tested. With the bovine faecal samples, the most efficient RT-qPCR systems were B and A with the detection in 100 and 92% of samples tested, respectively. Human group A rotavirus G1P[8] and bovine G6P[11] were the most frequently used strains identified in this study. A G3P[9] strain, closely related to a feline rotavirus isolated in the USA, was also discovered in a human rotavirus infection. CONCLUSION: The RT-qPCR system B was the only TaqMan assay evaluated in this study able to detect rotavirus RNA in all positive human and bovine faecal samples. SIGNIFICANCE AND IMPACT OF THE STUDY: Utilization of only one RT-qPCR for the detection of human and bovine group A rotaviruses and the possibility of human infection by a feline rotavirus strain.
Assuntos
Antígenos Virais/genética , Proteínas do Capsídeo/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Rotavirus/isolamento & purificação , Animais , Bovinos , Criança , Primers do DNA , Fezes/virologia , Genótipo , Humanos , Filogenia , RNA Viral/isolamento & purificação , Rotavirus/classificação , Rotavirus/genética , Infecções por Rotavirus/diagnóstico , Infecções por Rotavirus/veterinária , Infecções por Rotavirus/virologia , Sensibilidade e Especificidade , Análise de Sequência de DNARESUMO
AIMS: The goal of this study was to develop and to optimize molecular tools to detect the presence of Torque teno virus (TTV) in swine and cattle. A novel real-time polymerase chain reaction (PCR) using a TaqMan probe was developed to detect both genogroups of TTV strains. METHODS AND RESULTS: Oligonucleotide primers and hybridization probes were designed based on sequence analysis of the noncoding region, a highly conserved part of the genome. The real-time PCR assay specifically detected bovine and porcine TTV DNA without cross-amplification of other common pathogens. The assay was compared with conventional PCR and nested-PCR assays for the detection of porcine genogroups 1 and 2 and bovine TTV on plasma and faecal samples, and the assay was found faster, more reliable and reduced the risk of false positive results. CONCLUSIONS: The real-time PCR assay provided better detection results for the two TTV genogroups in both swine and cattle compared to the conventional PCR assays. SIGNIFICANCE AND IMPACT OF THE STUDY: This new TaqMan PCR assay will be a useful tool for the detection of animal TTV strains, to evaluate the viral load from animal host and finally to identify the presence of these viruses in the agri-food continuum.
Assuntos
Doenças dos Bovinos/virologia , Infecções por Vírus de DNA/veterinária , Reação em Cadeia da Polimerase/métodos , Doenças dos Suínos/virologia , Torque teno virus/isolamento & purificação , Animais , Sequência de Bases , Bovinos/virologia , Primers do DNA , Infecções por Vírus de DNA/virologia , DNA Viral/isolamento & purificação , Fezes/virologia , Dados de Sequência Molecular , Sondas de Oligonucleotídeos , Filogenia , Plasma/virologia , Reação em Cadeia da Polimerase/veterinária , Alinhamento de Sequência , Análise de Sequência de DNA , Especificidade da Espécie , Suínos/virologia , Torque teno virus/genéticaRESUMO
AIMS: The aim of this study was to compare the performance of four TaqMan RT-PCR assays with a commonly used nested RT-PCR and to include the Feline calicivirus (FCV) as an internal control. METHODS AND RESULTS: RNA extracted from 87 swine faecal samples and 103 swine blood samples was subjected to different detection systems. Faecal samples naturally contaminated with Hepatitis E virus (HEV) and negative samples were artificially inoculated with 3.2 x 10(3) PFU of FCV. Detection results obtained on faecal and plasma samples were 35.6% and 4.9% with the nested RT-PCR assay, 8.0% and 0%, 0% and 0%, 13.8% and 0% and 36.8% and 3.9% with TaqMan systems A, B, C and D respectively. The Ct means obtained with the multiplex TaqMan assay were 30.11 and 30.43 for the detection of FCV with HEV contaminated samples and negative samples. CONCLUSIONS: The TaqMan system D was more suitable for the detection of swine HEV strains than the three others and FCV was integrated successfully as an internal control. SIGNIFICANCE AND IMPACT OF THE STUDY: FCV was demonstrated as an efficient control to monitor the RNA extraction process and HEV amplification procedure in a multiplex HEV/FCV TaqMan assay. This control would be helpful in limiting false negative results.
