ABSTRACT
Two pairs of primers were designed to bind conserved genomic regions of goose parvovirus (GPV) and goose astrovirus (GAstV) to establish a simple, sensitive, and highly specific duplex quantitative PCR (qPCR) method to simultaneously detect the two viruses. The duplex qPCR can distinguish GPV (melting point: 82.1 °C) and GAstV (melting point: 79.8 °C) by the peaks of their individual melting curves. Mixed testing with other waterfowl viruses produced no nonspecific peaks. The established standard curves showed good linear relationships (R2 > 0.997) and the limits of detection (LOD) for GPV and GAstV were 5.74 × 101 and 6.58 × 101 copies/µL, respectively. Both intra- and inter-assay coefficients of variation were <2%, indicating that the method has good repeatability. Twenty tissue samples from diseased geese were examined with the duplex qPCR assay and conventional PCR. Duplex qPCR showed positive rates of 25% for GPV and 45% for GAstV, and the positive rate for GPV and GAstV coinfection was 15%, slightly higher than the results for conventional PCR. These results indicated that this duplex qPCR method is highly sensitive, specific, and reproducible, and is suitable for epidemiological studies to effectively control the transmission of GPV and GAstV.
Subject(s)
Astroviridae Infections/diagnosis , Astroviridae Infections/veterinary , Avastrovirus/isolation & purification , Benzothiazoles/metabolism , Diamines/metabolism , Parvoviridae Infections/diagnosis , Parvoviridae Infections/veterinary , Parvovirinae/isolation & purification , Quinolines/metabolism , Real-Time Polymerase Chain Reaction/methods , Animals , Geese/virology , Reference Standards , Reproducibility of Results , Sensitivity and SpecificityABSTRACT
Goose circovirus (GoCV) is a potential immunosuppressive virus that poses a great hazard to the goose industry and has been shown to be widely distributed throughout China. We have established a fast, sensitive and highly specific TaqMan real-time quantitative PCR detection method for this virus. Specific primers and probes were designed against the conserved regions of the genomic GoCV Rep gene. The results showed that the assay was highly specific and sensitive for GoCV and did not cross-react with other non-targeted waterfowl viruses. The established method will be helpful for epidemiological detection and may be effective in the prevention and control of the disease.
Subject(s)
Circovirus/genetics , Circovirus/isolation & purification , Real-Time Polymerase Chain Reaction/methods , Animals , Biological Assay , Geese/virology , Reproducibility of Results , Sensitivity and SpecificityABSTRACT
The objective of the present study was to demonstrate the effect of polyanionic copolymer mPEG-grafted-alginic acid (mPEG-g-AA)-based polyion complex (PIC) micelles on enhancing the oral absorption of salmon calcitonin (sCT) in vivo and in vitro and identify the transepithelial transport mechanism of PIC micelles across the intestinal barrier. mPEG-g-AA was first successfully synthesized and characterized in cytotoxicity. The PIC micelles were approximately of 72 nm in diameter with a narrow distribution. The extremely significant enhancement of hypocalcemia efficacy of sCT-loaded PIC micelles in rats was evidenced by intraduodenal administration in comparison with sCT solution. The presence of mPEG-grafted-chitosan in PIC micelles had no favorable effect on this action in the referred content. In the Caco-2 transport studies, PIC micelles could significantly increase the permeability of sCT across Caco-2 monolayers without significantly affecting transepithelial electrical resistance values during the transport study. No evident alterations in the F-actin cytoskeleton were detected by confocal microscope observation following treatment of the cell monolayers with PIC micelles, which further certified the incapacity of PIC micelles to open the intercellular tight junctions. In addition, TEM observations showed that the intact PIC micelles were transported across the everted gut sac. These suggested that the transport of PIC micelles across Caco-2 cell monolayers involve a predominant transcytosis mechanism via endocytosis rather than paracellular pathway. Furthermore, PIC micelles were localized in both the cytoplasm and the nuclei observed by CLSM. Therefore, PIC micelles might be a potentially applicable tool for enhancing the oral absorption of cationic peptide and protein drugs.
