Detection of HbsAg and hATIII genetically modified goats (Caprahircus) by loop-mediated isothermal amplification.

In this study, sensitive and rapid detection systems were designed using a loop-mediated isothermal amplification (LAMP) method to detect the genetically modified goats. A set of 4 primers were designed for each exogenous nucleic acids HBsAg and hATIII. The DNA samples were first amplified with the outer and inner primers and released a single-stranded DNA,of which both ends were stem-loop structure. Then one inner primer hybridized with the loop, and initiated displacement synthesis in less than 1 h. The result could be visualized by both agarose gel electrophoresis and unaided eyes directly after adding SYBR GREEN 1. The detection limit of LAMP was ten copies of target molecules, indicating that LAMP was tenfold more sensitive than the classical PCR. Furthermore, all the samples of genetically modified goats were tested positively by LAMP, and the results demonstrated that the LAMP was a rapid and sensitive method for detecting the genetically modified organism.

Immunochromatography detection of human lactoferrin protein in milk from transgenic cattle.

Transgenic technologies have opened a new era of transgenesis, characterized by manipulating intraspecies or interspecies genes in microorganisms, plants, or animals to change them in desired directions. The advent of genetically modified animals and related products has raised the need for analytical methods, nucleotide- or protein-based, to qualitatively and quantitatively determine the biotechnology ingredients. In this study, we collected milk samples containing human lactoferrin (hLF) protein, to exploit appropriate detection means for exogenous hLF protein. We preliminarily developed two types of competitive immunochromatography strips for quick detection, based on gold-conjugated hLF protein or gold-conjugated polyclonal antibody. As control methods, sodium dodecyl sulfate polyacrylamide gel electrophoresis, dot blot, and Western blot methods were used to check the accuracy of strips, and highly sensitive ELISA with chemiluminescent substrates was developed to determine the concentration of hLF in milk. Comparing the test results of lateral flow strips with qualitative assays, we found our strips gave the same results in a few minutes, showing great advantages with no need of professional technicians or any equipment. Our results demonstrated that all the applied methods were effective to detect hLF, suggesting that they could be used to monitor the production of transgenic milk.

Detection of two exogenous genes in transgenic cattle by loop-mediated isothermal amplification.

Nucleotide-based analytical approaches are indispensable and effective, targeting for the transgenic ingredients in biotechnical products in terms of safety assessment. In this study, a loop-mediated isothermal amplification method was developed for the specific detection of exogenous nucleic acids of hLTF/hLALBA-induced transgenic cattle. The detection limit of the LAMP method was proved to be as low as 10 copies of target molecules in optimized systems, and to be 10-100 times more sensitive than the conventional PCR. Furthermore, fluorescent dye SYBR Green I was used to visualize the color changes of LAMP products by naked eyes in daylight, which resulted in distinct colors between positive and negative reactions. For the detection of transgenes, all the transgenic samples collected from hLTF and hLALBA-induced cattle were amplified by LAMP in 1 h, followed by direct visual SYBR Green I dying or gel electrophoresis. Results showed that transgenic and non-transgenic samples exhibited distinct properties in colors or electrophoresis profiles. Thus, all the results indicated that the LAMP assay was a simple and convenient method for the test of transgenic animals.

Molecular characterization of transcriptome-wide interactions between highly pathogenic porcine reproductive and respiratory syndrome virus and porcine alveolar macrophages in vivo.

Porcine reproductive and respiratory syndrome virus (PRRSV) infects mainly the porcine alveolar macrophages (PAMs) and causes porcine reproductive and respiratory syndrome (PRRS). Previous studies have analyzed the global gene expression profiles of lung tissue in vivo and PAMs in vitro following infection with PRRSV, however, transcriptome-wide understanding of the interaction between highly pathogenic PRRSV (HP-PRRSV) and PAMs in vivo has not yet been established. In this study, we employed Affymetrix microarrays to investigate the gene expression patterns of PAMs isolated from Tongcheng piglets (a Chinese indigenous breed) after infection with HP-PRRSV. During the infection, Tongcheng piglets exhibited typical clinical signs, e.g. fever, asthma, coughing, anorexia, lethargy and convulsion, but displayed mild regional lung damage at 5 and 7 dpi. Microarray analysis revealed that HP-PRRSV infection has affected PAMs in expression of the important genes involved in cytoskeleton and exocytosis organization, protein degradation and folding, intracellular calcium and zinc homeostasis. Several potential antiviral strategies might be employed in PAMs, including upregulating IFN-induced genes and increasing intracellular zinc ion concentration. And inhibition of the complement system likely attenuated the lung damage during HP-PRRSV infection. Transcriptomic analysis of PAMs in vivo could lead to a better understanding of the HP-PRRSV-host interaction, and to the identification of novel antiviral therapies and genetic components of swine tolerance/susceptibility to HP-PRRS.

Molecular characterization, chromosomal localization, expression profile and association analysis with carcass traits of the porcine dickkopf homolog1 gene.

DKK1 (dickkopf homolog 1) is a potent inhibitor of the canonical Wnt/ß-cantenin signalling pathway, which plays a pivotal role in myogenesis, adipogenesis, and many other crucial biological processes. In this study, DKK1 was assigned to porcine 14q25-26 by using the radiation hybrid (IMpRH) panel. A G1757A single nucleotide polymorphism site by Csp6I PCR-RFLP was identified. Association analysis showed that different genotypes were associated with loin muscle area (P = 0.0281). Semi-quantitative-RT-PCR analysis revealed that DKK1 was highly expressed in spleen and lymph node at two developmental stages, while in skeletal muscle, further real-time PCR quantified that DKK1 was down-regulated in Large White pigs compared to Tongcheng pigs, accompanied by the down-regulation of CTTNB1 and TCF4, the up-regulation of LRP6, suggesting that the phenotypic difference between lean and obese pigs might be correlated with the activity of Wnt/ß-cantenin signalling pathway.