A sensitive and rapid single step real time （rt） RT-PCR was standardized using one-step Brilliant SYBR Green kit for detection and semi-quantitation ofpeste des petitis ruminants virus （PPRV） using the virus RNA a...A sensitive and rapid single step real time （rt） RT-PCR was standardized using one-step Brilliant SYBR Green kit for detection and semi-quantitation ofpeste des petitis ruminants virus （PPRV） using the virus RNA and matrix （M） protein gene-specific primers and compared with established conventional RT-PCR and TaqMan RT-PCR. The assay amplifies a 124 bp fragment of the PPRV M gene with Tm of 78.28 to 78.50. The assay was linear within a range of 50 ng to 0.5 fg total virus RNA with a detection limit （sensitivity） of 0.5 fg. Based on the serial dilution of the live-attenuated PPR vaccine virus, the detection limit was -0.0001 cell culture infectious dose 50% units （TCID50）. Additionally, swab materials spiked with known titre of vaccine virus were equally well detected in the assay. The standardized rt RT-PCR was easily employed for the detection of PPRV nucleic acid directly in the field and experimental clinical samples. The assay detected the PPRV nucleic acid as early as 3 day post infection （dpi） and up to 20 dpi in swab materials from the experimental samples. The assay was rapid and more sensitive than TaqMan and conventional RT-PCR in the detection of PPRV nucleic acid from the PPR suspected clinical samples of sheep and goats. Therefore, the established, simplified SYBR green rt RT-PCR is an alternative test to the already existing various diagnostic assays and could be useful for rapid clinical diagnosis with advantage in reducing risk of contamination.展开更多
Objective:The aim of this study was to investigate the expression of ABCG2 in human gastric carcinoma and its clinical significance.Methods:Expression of ABCG2 was examined with immunohistochemical technique in the sp...Objective:The aim of this study was to investigate the expression of ABCG2 in human gastric carcinoma and its clinical significance.Methods:Expression of ABCG2 was examined with immunohistochemical technique in the specimens from 45 gastric carcinoma tissues and 30 surrounding normal tissues.The mRNA expression of ABCG2 was measured by RT-PCR and real-time quantitative PCR in 30 cases of gastric carcinoma and normal gastric mucosa, respectively.Results:ABCG2 expression was observed in 28 of 45(62.2%) cases by immunohistochemical analysis.In ABCG2-positive tumors, adjacent non-neoplastic tissue was similarly analyzed, revealed that ABCG2 was up-regulated in gastric carcinoma.ABCG2 expression in poorly differentiated/undifferentiated carcinoma was significantly higher than that in well/moderately-differentiated carcinoma(P < 0.05).The mRNA expression of ABCG2 was significantly higher than that in normal gastric mucosa(P < 0.05).Conclusion:ABCG2 plays an important role in the multi-drug resistance of gastric carcinoma.ABCG2 might be an important factor in the research of gastric cancer stem cell.展开更多
基金Indian Council of Agricultural Research,New Delhi,India under Niche Area of Excellence:Production and Quality control of Veterinary Immunodiganostics and immunoprophylactics(F.No.10(11)2005-EP&D.dated 15.12.2005)
文摘A sensitive and rapid single step real time （rt） RT-PCR was standardized using one-step Brilliant SYBR Green kit for detection and semi-quantitation ofpeste des petitis ruminants virus （PPRV） using the virus RNA and matrix （M） protein gene-specific primers and compared with established conventional RT-PCR and TaqMan RT-PCR. The assay amplifies a 124 bp fragment of the PPRV M gene with Tm of 78.28 to 78.50. The assay was linear within a range of 50 ng to 0.5 fg total virus RNA with a detection limit （sensitivity） of 0.5 fg. Based on the serial dilution of the live-attenuated PPR vaccine virus, the detection limit was -0.0001 cell culture infectious dose 50% units （TCID50）. Additionally, swab materials spiked with known titre of vaccine virus were equally well detected in the assay. The standardized rt RT-PCR was easily employed for the detection of PPRV nucleic acid directly in the field and experimental clinical samples. The assay detected the PPRV nucleic acid as early as 3 day post infection （dpi） and up to 20 dpi in swab materials from the experimental samples. The assay was rapid and more sensitive than TaqMan and conventional RT-PCR in the detection of PPRV nucleic acid from the PPR suspected clinical samples of sheep and goats. Therefore, the established, simplified SYBR green rt RT-PCR is an alternative test to the already existing various diagnostic assays and could be useful for rapid clinical diagnosis with advantage in reducing risk of contamination.
基金Supported by a grant of the National Natural Science Foundation of China (No. 30570522)
文摘Objective:The aim of this study was to investigate the expression of ABCG2 in human gastric carcinoma and its clinical significance.Methods:Expression of ABCG2 was examined with immunohistochemical technique in the specimens from 45 gastric carcinoma tissues and 30 surrounding normal tissues.The mRNA expression of ABCG2 was measured by RT-PCR and real-time quantitative PCR in 30 cases of gastric carcinoma and normal gastric mucosa, respectively.Results:ABCG2 expression was observed in 28 of 45(62.2%) cases by immunohistochemical analysis.In ABCG2-positive tumors, adjacent non-neoplastic tissue was similarly analyzed, revealed that ABCG2 was up-regulated in gastric carcinoma.ABCG2 expression in poorly differentiated/undifferentiated carcinoma was significantly higher than that in well/moderately-differentiated carcinoma(P < 0.05).The mRNA expression of ABCG2 was significantly higher than that in normal gastric mucosa(P < 0.05).Conclusion:ABCG2 plays an important role in the multi-drug resistance of gastric carcinoma.ABCG2 might be an important factor in the research of gastric cancer stem cell.
