ABSTRACT
Objective To analyse the feasibility of detecting F1 antibody to Yersinia pestis in flushing fluid of heart blood of Rhombomys opimus with enzyme linked immunosorbent assay(ELISA) method and its application value in surveillance of the disease. Methods Serum, flushing fluid of heart blood and infusion fluid of liver and spleen of Rhombomys opimus, which were caught by capture in the plague focus of Zunger basin in 2007, were taken to carry out detection for F1 antibodies to Yersinia pestis with ELISA method. The data were processed with SPSS 17.0. Results Positive rate and average titer of serum were 12.35%(11/162) and 25.35, of flushing fluid of heart blood were 10.49%(17/162) and 23.75 and of the infusion fluid of liver and spleen 6.79%(17/162) and 2240,respectively. No statistical difference was found in positive detection rate when it was compared between serum and flushing fluid of heart blood(χ2 = 1.333, P ＞ 0.05), but it was obviously different between serum and infusion fluid of liver and spleen(χ2 = 7.111, P ＜ 0.01 ) and between flushing fluid of heart blood and infusion fluid of liver and spleen(x2 = 6.250, P ＜ 0.05). There was a significant difference in average titer between serum, flushing fluid of heart blood and infusion fluid of liver and spleen(t = 2.290, 3.612, P ＜ 0.05 or ＜ 0.01 ). The plague F1 antibody positive coincidence rate of serum and flushing fluid of heart blood was 85.0%(17/20), of serum and infusion fluid of liver and spleen was 55.0% (11/20), and of flushing fluid of heart blood and infusion fluid of liver and spleen was 64.7%(11/17). Conclusions The ELISA method can detect Fl antibody in flushing fluid of heart blood,and the method is feasible in plague surveillance.
ABSTRACT
Objective To compare the effect of three methods in diagnosis of plague by detecting of Yersina pestis F1 antigen. Methods In natural foci of plague, wild animal samples, such as blood, liver, spleen,and lymphoid tissue were collected, and the three methods of enzyme linked immunosorbent assay (ELISA),reverse indirect hemagglutination assay(RIHA) and gold-immunochromatography assay(GICA) were employed to detect F1 antigen of Yersina pestis. Results Total of 414 infused organ samples of natural death and captured wild animals in natural foci of plague were determined. Positive samples detected by GICA and ELISA were the same,the positive rates were 5.31%(22/414), both positive and negative coincidence rates were consistently 100%. Only 18 samples were positive by retrial in 186 samples with more than 2 holes aggregation by preliminary examination of RIHA, with nonspecific agglutination rate of 40.6% (168/414) and positive rate of 4.35% (18/414). The positive coincidence rate was 81.82% (18/22) between RIHA with GICA and ELISA, and negative coincidence rate was statistically significant(t = 4.379, P ＜ 0.01). Conclusions ELISA, RIHA and GICA can be used for early diagnosis of plague by detecting F1 antigen. The results of RIHA have quantitative significance, with higher non-specific agglutination rate, and heavy workload of re-examination; GICA and ELISA has the same specificity and sensitivity, but the results of GICA is only qualitative. ELISA excluded the defect of RIHA and GICA, and combines the advantages of both methods.
ABSTRACT
CCK correlates with the generation and progression of pancreatic cancer. The research aims to construct eukaryotic expression plasmid pIRES2-EGFP/CCK (CCK pDNA) and transiently express it in COS-7 cells. Total RNA was extracted from porcine intestinal mucosa. RT-PCR was used to amplify the aimed segments CCKcDNA which was then digested with EcoR1 and BamH1 and inserted into a eukaryotic expression plasmid pIRES2-EGFP to construct CCK pDNA. The constructed plasmid was transfected into COS-7 cells by lepofectamine TM2000-mediated transfer method.The expression of CCK in transfected COS-7 cells was detected 24, 48 and 72 h post-transfection with fluorescence microscopy and the expression level of CCK mRNA in transfected COS-7 cells was assayed by using RT-PCR. The results showed CCK pDNA was successfully constructed and expressed transiently in COS-7 cells. Green fluorescent protein could be detected in the COS-7 cells transfected with porcine CCK pDNA 24 h post-transfection. At 48th h post-transfection, the number of positive cells was increased significantly and much brighter green fluorescence could be detected.And 72 h post-transfection, the green fluorescence of positive cells became even stronger, while no green fluorescence was detected in the control group. The expression of CCK mRNA in the cells was detectable by using RT-PCR. In COS-7 cells transfected with CCK pDNA a high level of porcine CCK mRNA was detected while no expression of porcine CCKmRNA was found in the cells transfected with null plasmid. It was concluded CCK pDNA was expressed successfully in COS-7 cells,which lays a foundation for further research on the relationship between CCK and tumor.