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摘要:A loop-mediated isothermal amplification （LAMP） assay was developed for rapid visual detection of Brucella. A set of six specific primers specific to eight regions of OMP25 gene were designed. The reaction was performed in a single tube at 63℃ with the addition ofhydroxynaphthol blue （HNB） dye prior to amplification and the result was determined by the color change of HNB. The sensi- tivity and specificty of LAMP method were evaluated with the optimized reaction system and conditions. Meanwhile, sixty bovine bru- cellosis positive serum samples diagnosed by rose bengal precipitation test （RBT） were detected by LAMP and B4/B5-PCR. The results showed the detection limit of the LAMP assay was about 17 fg genomic DNA, about 100 times higher than conventional PCR method. Also, the specificity of the LAMP assay showed there was no cross reactivity with other related bacteria including Escherichia coli K99, Pasteurella multocida C48-1, Streptococcus ST171, Pseudomonas aeruginosa. Furthermore, the LAMP assay was evaluated by 60 seropositive samples from brucellosis epidemic areas, and the results showed 53 positive samples including all 43 B4/B5-PCR positive samples. The coincidence rate of two methods was 85.0%. These results suggested that the LAMP assay with HNB dye provided a useful tool for the rapid detection of Brucella.

摘要:With the variable middle region of the omlA gene and the specificlty of the apx gene and the cps gene, a typing system of three multiplex PCRs was established. By the PCR system, 11 out of the 12 reference strains of Actinobacillus pleuropneumoniae were differentiated except distinguishing serotype 9 from 11. Three wild strains serotyped from the serological tests were consistent with the typing system serotyping results, and the serotyping results of artificially infected specimens were also accurate, including 12 tonsils, 12 lungs, 12 nasal swabs. The method can also be used directly to detect A. pleuropneumoniae from the clinical suspicion samples, and 2 positive results detected from 35 clinical suspicion samples were both ascribed to serotype 7.And 5 positive results detected from 350 tonsils of healthy swine were serotype 4, serotypel2, serotype3 and two serotype7.

摘要:In this study, the Hly gene ofListeria monocytogenes train C53005 was amplified by PCR using designed primers. Then, the gene was cloned into pMD18-T vector, and indentified by digestion with restriction endonuclease, PCR and sequencing. After the sequences were confirmed correct, a prokaryotic expression vector was constructed by inserting the gene into pET-28a vector. The recombinant plasmid pET-28a- silly was transformed into E.coli BL21 （DE3） competent cells, and was induced with 1PTG. The expressed product was analysed by SDS-PAGE and Western-blot. In result, the Hly gene was highly expressed in E.coli and the expressed protein was 65 Ku in size as expected. Western-blot test demonstrated that the expressed protein could specifically react with rabbit anti-LM serum. The results showed the expressed protein was in soluble form with satisfactory antigenicity.

摘要:The F3L gene sequence ofmonkeypox virus was synthesized and cloned into the plasmid to be used as a target. A pair of PCR primers were designed and synthesized based on the nucleotide sequences of MPV in GenBank. After optimization of the detection methods, they were used to amplify MPV. The results showed that the primers could get the specific bands. The high specificity of the methods was demonstrated by detecting 14 viruses, such as chickenpox, goalpox and so on. The maximal detection limits by the primers were approximately 0.3 pg of plasmid DNA. Therefore, PCR method developed here for MPV detection had the characteristic of high efficiency, speediness, specificity and sensitivity. They can be used to entry-exit quarantine detection ofmonkeypox virus.

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