RESUMO
Newcastle disease (ND), infectious laryngotracheitis (ILT) and avian metapneumovirus (aMPV) can be similar making it critical to quickly differentiate them. Herein, we adapted pre-existing molecular-based diagnostic assays for NDV and ILTV, and developed new assays for aMPV A and B, for use under synchronized thermocycling conditions. All assays performed equivalently with linearity over a 5 log10 dynamic range, a reproducible (R² > 0.99) limit of detection of ≥ 10 target copies, and amplification efficiencies between 86.8%-98.2%. Using biological specimens for NDV and ILTV showed 100% specificity. Identical amplification conditions will simplify procedures for detection in diagnostic laboratories.
Assuntos
Metapneumovirus , Doença de Newcastle , Doenças das Aves Domésticas , Animais , Galinhas , Metapneumovirus/genética , Doença de Newcastle/diagnóstico , Vírus da Doença de Newcastle/genética , Aves Domésticas , Doenças das Aves Domésticas/diagnóstico , Reação em Cadeia da Polimerase em Tempo Real/veterináriaRESUMO
Infectious bronchitis (IB) is a highly contagious upper respiratory tract disease of chickens caused by infectious bronchitis virus (IBV), which has various serotypes that do not cross-protect. Vaccine control strategies for this virus are only effective when designed around the currently circulating serotypes. It is essential to not only rapidly detect IBV but also to identify the type of virus causing disease. Six TaqMan™-based quantitative real-time RT-PCR assays (Universal, Ark, Mass, DE/GA98, GA07, GA08) were developed and examined the sensitivity and specificity for each assay. Assays were developed targeting the hypervariable region in the S1 gene subunit. The analytical sensitivity of TaqMan™-based quantitative real-time RT-PCR assays (qRT-PCR) assays was evaluated using synthetic DNA standards that were identical with the target sequence and specificity was further validated using clinical and biological specimens. All developed assays performed equivalently when using synthetic DNA templates as standard material, as it achieved linearity over a 5 log10 dynamic range with a reproducible limit of detection of ≤10 target copies per reaction, with high calculated amplification efficiencies ranging between 90%-115%. Further validation of specificity using clinical and biological specimens was also successful.
Assuntos
Aves/virologia , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/veterinária , DNA Viral/síntese química , Vírus da Bronquite Infecciosa/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/métodos , Animais , Infecções por Coronavirus/virologia , Primers do DNA/genética , Sondas de DNA/genética , DNA Viral/genética , Vírus da Bronquite Infecciosa/classificação , Vírus da Bronquite Infecciosa/genética , Limite de Detecção , Reação em Cadeia da Polimerase em Tempo Real/normas , Estudos Retrospectivos , Sensibilidade e EspecificidadeRESUMO
Acid adaptation has previously been shown to increase the infectivity of Vibrio cholerae in the infant mouse model. To better understand this phenomenon, we monitored the spatial distribution and temporal changes in the ratios of acid-adapted cells to unadapted V. cholerae cells in the small intestine, as well as the timing of virulence factor expression. We found that the competitive advantage afforded by acid adaptation does not become manifest until greater than 3 h postinfection; thus, acid adaptation does not increase V. cholerae passage through the gastric acid barrier. Additionally, acid-adapted and unadapted V. cholerae cells colonize the same sections of the small intestine and show similar kinetics of transcriptional induction of the virulence genes tcpA and ctxA. These studies suggest that the increased infectivity of acid-adapted V. cholerae is due to a more rapid onset of multiplication and/or to an increased multiplication rate within the infant mouse intestine.
