Papers by Keyword: RT-PCR

Abstract: Rift Valley Fever (RVF) is a notifiable multiple species diseases in the OIE list, and causes human and agricultural losses in endemic regions. To develop the rapid method for detecting of RVF, 2 specific primers for reverse transcriptase polymerase chain reaction (RT-PCR) and 7 overlapping oligo primers were designed according to the nucleotide sequence information of RVFV published in GenBank, and a DNA fragment about 318 bp of the segment S was synthesized in vitro by overlap extension PCR to construct the recombinant plasmid pMD19-T-RVFVS. Then, the 2 specific primers were evaluated via a serial of tests, including reaction temperature optimization test, sensitivity and specificity tests. The results showed that the 2 designed primers are suitable for RVFV RT-PCR detection which is a rapid method with good specificity and sensitivity, the detection limit was approximately 85 copies of the cloned viral genomic fragments (pMD19-T-RVFVS) as well as resulted in no cross-reaction for peste des petits ruminants virus (PPRV), Epidemic encephalitis B virus, E.coli , Salmonella and Pasteurella multocida etc common pathogens isolated from ruminants detection.

Abstract: Present paper describes early findings from the study of Ti-6Al-4V scaffolds additively manufactured using electron beam melting (EBM^®) technology and the influence of surface topography on the initial stages of cell acceptance. The surface topography of the components made by additive manufacturing (AM) processes including EBM^® are often hard to control within the desired feature size range without post-processing. Two groups of experiments studying the behavior of human osteoblast-like cells (MG63) on samples with different surface roughness were carried out in vitro: Ti-6Al-4V samples only powder-blasted, and Ti-6Al-4V samples additionally electrochemically polished. The cell migration into powder-blasted Ti-6Al-4V 3D scaffolds with different shapes and dimensions of the lattice structures were studied.

Abstract: Protein tyrosine phosphatase receptor-type O (PTPRO) has been described in several forms of cancer as a new member of the PTP family. The tumor suppressor function of PTPRO was evaluated by design and synthesis the 10-23 deoxyribozyme (DRz), thio-modified DRz (DRz-s) and antisense oligonucleotide (asON) of the PTPRO genomic mRNA to detect the catalytic cleavage activity. Firstly, the cDNA fragment of PTPRO gene was amplified from total cellular RNA of the HepG2.2.15 cells by reverse transcription PCR (RT-PCR). Subsequently, the fragments were cloned to pcDNA3.1(+) plasmids and generated a recombinant plasmids, then sifted the positive recombinant plasmids out to amplify. The expression vector of PTPRO mRNA was obtained in vitro transcription by using T7 RNA polymerase. The results of transfection indicated that when PTPRO mRNA gamyed with deoxyribozyme which activity enhanced, so DRz-s were detected with more intensive specific catalytic cleavage activity than DRz by cells transfecting. And the asON wasn't detected with the property.

Abstract: Objective: To investigate the correlation among CD4⁺CD25⁺ Treg cell frequency, CTLA-4 expression level in the peripheral blood mononuclear cells (PBMC), and disease progression of HIV/AIDS patents. Methods: Fifty HIV/AIDS-positive samples were confirmed by both Zhejiang Provincial and Hangzhou Center for Disease Control and Prevention. Ten samples of healthy volunteers were randomly selected as controls. Immune cells were counted using a flow cytometer (FACS), and the expression level of CTLA-4 mRNA analyzed using RT-PCR. Results: CD4⁺CD25⁺ Treg cells in PBMC of HIV/AIDS patients were markedly higher than those of healthy volunteers£ ̈P = 0.04j, and CTLA-4 mRNA was highly expressed in the CD4⁺CD25⁺ Treg subpopulation (P = 0.002), compared to healthy adults as well as AIDS patients treated with HAART. Conclusion: CTLA-4 expressed on CD4⁺CD25⁺ Treg cells can be a specific marker of the progression of HIV/AIDS, and the inhibitory receptor may play an important role in the regulation and pathogenesis of HIV/AIDS.

Abstract: Heart disease is the second most common cause of mortality in Japan. Most cases of late stage heart failure can only be effectively treated by a heart transplant. Cardiac tissue engineering is emerging both as a new approach for improving the treatment of heart failure and for developing new cardiac drugs. Apatite-fiber scaffold (AFS) was originally designed as a substitute material for bone. AFS contains two sizes of pores and is appropriate for the three dimensional proliferation and differentiation of osteoblasts. To establish engineered heart tissue, a pluripotent embryonal carcinoma cell line, P19.CL6, was cultured in AFS. P19.CL6 cells seeded into AFS proliferated well. Generally, cardiac differentiation of P19.CL6 cells is induced by treating suspension-cultured cells with dimethyl sulfoxide (DMSO), after which the cells form spheroids. However, our results showed that P19.CL6 cells cultured in AFS differentiated into myocytes without forming spheroidal aggregates, and could be cultured for at least one month. Thus, we conclude that AFS is a good candidate as a scaffold for cardiac tissue engineering.

Abstract: α-TCP/HAp functionally graded porous beads were prepared for use as a scaffold for bone regeneration. In this research, porous beads which have compositionally graded layer, from TCP of inner area to HAp of surface area, were fabricated. It is known that the dissolution rate of TCP is fast in compare with that of HAp in living body, thus the inner area would dissolve prior to the surface area. These beads to allow the penetration of cells and blood vessel into the inner area of beads following new bone regeneration. At present, conditions to fabricate the functionally graded porous beads have been established already, and experiments using cells [MC3T3-E1] are in progress.

Abstract: Swine and canine influenza, which is acute viral respiratory infectious disease, can do great threat to the health of people and animals. What’s more, swine as an intermediate host for the transmission of avian influenza viruses to humans, it’s very important to supervise the epidemiological situation of swine influenza virus (SIV) infections all over the world. The major aim of this paper was to investigate the epidemiological situation of influenza A viruses in laboratory swine and dogs in shanghai and surrounding area from January 2010 to March 2011. There were 109 throat swabs collected from swine and 35 from dogs. Based on RT-PCR, we have got only one swine influenza virus-positive sample and none canine influenza virus-positive. In addition, this survey not only gives a data about the epidemiological situation of swine and canine influenza, but also provides a basis and guarantee for taking a timely and effective security measures.

Abstract: In this pepar we investigated the developmental patterns of expression of growth hormone (GH) gene in pituitary tissue in pigs of different breeds and their effects on the carcass fat contents. 3 Jinhua gilts and 3 Landrace gilts were sampled at 35, 80 and 125 days of age, respectively. Carcass fat contents were determined. Pituitary tissue was sampled and total RAN was extracted to determine GH mRNA expression levels by semi-quantitative RT-PCR. The results showed that the contents of carcass fat increased with growth and showed significant differences (P﹤0.05) between different age groups in the two breeds. Furthermore, carcass fat contents in Jinhua gilts were higher than that in Landrace gilts during growth (P﹤0.05). GH mRNA expression levels decreased with age and displayed breed differences. Jinhua gilts showed lower abundance of GH mRNA compared with Landrace gilts at 35, 80 and 125 days of age (P﹤0.05). In addition, GH mRNA expression level was negatively related to carcass fat content in Jinhua and Landrace gilts (r = -0.790 (P = 0.01), r = -0.755 (P = 0.02), respectively).

Abstract: Metallothionein (MT) is one kind of proteins induced in organism under the stress of heavy metal. It plays a great pole in detoxification and tolerance of heavy metal. In this study, according to the rich conservative sequence in MT, we designed corresponding primer, then cloned MT mRNA gene induced by As in earthworm E. andrei and got its nucleotide sequence. The results proved the presence of MT in Eisenia andrei, and showed that roxarsone could induce the expression of arsenic MT gene.

Abstract: To develop the reverse transcriptase polymerase chain reaction (RT-PCR) for the detection of European brown hare syndrome virus (EBHSV), 3 specific primers and 9 overlapping oligo primers were designed to amplify a fragment of 335 nucleotides from the genome of EBHSV according to the genome sequences published in GenBank, and a 359 bp DNA fragment of the capsid protein(VP60)gene of EBHSV was synthesized in vitro by using overlap extension PCR to construct the recombinant plasmids pGM-T-EBHSV, then the RT-PCR method was described through the optimization of reaction conditions, sensitivity and specificity tests. Results showed the RT-PCR method was supplied for the detection of EBHSV with good specificity and sensitivity. The sensitivity of the RT-PCR could reach about 50 copies of cloned viral genomic fragments (pGM-T-EBHSV), and no amplifications for RHDV, Pasteurella multocida, E.coli and Salmonella from rabbits detection by this approach.