RESUMO
For field-identification of taeniid cestodes in canine animals in Tibetan area, loop-mediated isothermal amplification (LAMP) assays for Echinococcus multilocularis, E. shiquicus, Taenia hydatigena, T. multiceps, T. pisiformis and T. crassiceps were developed and evaluated along with the reported assay for E. granulosus. The LAMP assays showed specific reaction with their corresponding target species DNA with the detection limit of 1 to 10 pg. Moreover, the assays for E. granulosus, E. multilocularis, T. hydatigena and T. multiceps could detect DNA extracted from 3 or more eggs of their corresponding target species. Then, the LAMP assays were applied on samples containing 3 to 35 taeniid eggs obtained from 61 field-collected canine feces in Qinghai, and the result was compared with a reported multiplex PCR and sequence analysis. The LAMP assays and the PCR detected single species DNA of E. granulosus, E. shiquicus, T. hydatigena and T. multiceps in 5, 2, 44 and 2 samples, respectively. In the rest 8 samples, DNA of both E. granulosus and T. hydatigena were detected by the PCR but the LAMP assays detected those DNAs in 2 samples and only T. hydatigena DNA in 6 samples. It was assumed that less than 3 E. granulosus eggs were mixed in the samples although the samples contained 21 to 27 eggs in total. In conclusion, the LAMP assays were less sensitive than the multiplex PCR, but would have adequate sensitivity for field use in Tibetan area.
Assuntos
Doenças do Cão/parasitologia , Técnicas de Amplificação de Ácido Nucleico/veterinária , Teníase/veterinária , Animais , DNA de Helmintos/genética , Doenças do Cão/diagnóstico , Doenças do Cão/epidemiologia , Cães , Equinococose/diagnóstico , Equinococose/epidemiologia , Equinococose/veterinária , Echinococcus/genética , Echinococcus multilocularis/genética , Reação em Cadeia da Polimerase Multiplex/veterinária , Técnicas de Amplificação de Ácido Nucleico/métodos , Sensibilidade e Especificidade , Taenia/genética , Teníase/diagnóstico , Teníase/epidemiologia , Tibet/epidemiologiaRESUMO
Objective: To clone and express the Tibetan Sheep-origin Echinococcus granulosus Antigen B8/2 Gene, and immunologically identify the encoded protein. Methods: The cDNA of EgAgB8/2 gene was amplified by RT-PCR. The prokaryotic expression vector pET-EgAgB8/2 was constructed and transformed into E. coli BL21ï¼DE3ï¼ for expression. Proteins were extracted, separated in SDS-PAGE and identified by Western blotting. Results: The cloned EgAgB8/2 gene was 335 bp in length, and had a 98%-100% sequence homology with the reported cDNA sequence of EgAgB8/2, indicating the successful construction of the pET-EgAgB8/2 vector. SDS-PAGE revealed large amount of proteins in supernatant. Western blotting further confirmed the expression of the target protein. Conclusion: The EgAgB8/2 gene of Tibetan Sheep-origin in Qinghai is successfully cloned, and the constructed pET-EgAgB8/2 vector can be used to express the target protein.
