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Objective:To investigate the species types and phylogenetic relationship of canine Leishmania in Diebu County, Gansu Province, so as to provide a basis for exploring new methods of prevention and control of canine visceral leishmaniasis. Methods:DNA was extracted from blood samples of eight asymptomatic Leishmania-infected dogs in Luoda administrative village in Diebu County, Gansu Province. Ribosomal internal transcribed spacer 1 (ITS-1) gene fragments were amplified and isolated by PCR, and then the amplified target fragments were sequenced. The MEGA 7.0 software was used for multiple sequence alignment, and a phylogenetic tree was constructed by neighbor-joining method to analyze the phylogenetic relationship of canine Leishmania in Diebu County, Gansu Province. Results:Fragments of about 320 bp corresponding in size to the target sequence ITS-1 were isolated from all of the eight asymptomatic Leishmania-infected dogs blood samples. ITS-1 sequence alignment showed that the sequence homology between 8 samples and Leishmania infantum MG969403, MN648755 strains was 99.1% - 100.0%; phylogenetic tree showed that all 8 samples were clustered into one branch with Leishmania infantum. Conclusion:Leishmania infantum is identified from all of the eight asymptomatic Leishmania-infected dogs blood samples in Diebu County, Gansu Province.
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Objective To develop a fluorescent recombinase-aided isothermal amplification (RAA)-based nucleic acid assay for detection of Leshimania. Methods Specific primers and probes were designed targeting Leishmania internal transcribed spacer 1 (ITS1) gene for RAA assay, and a fluorescent RAA assay was developed for detection of Leishmania following screening of primer pairs and optimization of primer and probe concentrations. The sensitivity of RAA assay for detection of Leishmania was evaluated using recombinant plasmid containing Leishmania ITS1 gene sequences at different copies and Leshimania genomic DNA at different concentrations as templates, and the specificity of RAA assay for detection of Leishmania was evaluated using the genomic DNA of transfusion-transmitted parasites, including Babesia microti, Toxoplasma gondii, Plamodium vivax, P. ovale, P. falciparum, P. malariae, L. donovani and L. infantum. Results After the optimal primer pair was screened from 9 pairs of primer combinations, the final primer and probe concentrations were optimized as 0.3 μmol/L and 0.08 μmol/L, respectively. Nucleic acid detection of Leishmania was completed by the fluorescent RAA assay at an isothermal temperature of 39 °C within 20 min. Remarkable florescent signals were seen within 5 min following RAA detection of genomic DNA of L. donovani and L. infantum, and no cross-reactions were observed with B. microti, T. gondii, P. vivax, P. ovale, P. falciparum or P. malariae. The lowest limitation of detection of the fluorescent RAA assay was 10 copies/μL recombinant plasmid containing Leishmania ITS1 gene sequences and 1 fg/μL Leishmania genomic DNA. Conclusions A rapid, simple, sensitive and specific fluorescent RAA assay is successfully developed for detection of L. donovani and L. infantum, which is effective for field screening of leishmaniasis.
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La leishmaniasis es una enfermedad producida por protozoarios parásitos del género Leishmania. Estos parásitos infectan a hospedadores mamíferos, entre los cuales los perros han sido implicados como reservorios del parásito. Este trabajo planteó estandarizar la técnica de la PCR-RFLP luego de la amplificación de la región ITS1 de Leishmania spp, como herramienta útil para la detección y caracterización molecular. Se utilizaron promastigotes de cultivo y muestras de biopsias procedentes de perros con leishmaniasis visceral previamente diagnosticados en el Centro Antirrábico Nacional. La región ITS1 del ADN genómico nuclear de Leishmania spp. fue amplificada utilizando los cebadores LITSR y L5,8S. La técnica ITS1 PCR-RFLP aplicada, permitió la detección de Leishmania (L) infantum en 10/10 aislados de parásitos mantenidos en medio NNN, en 10/18 muestras de bazo y 10/18 muestras de ganglio linfático poplíteo. Las condiciones óptimas de reacción fueron 0,2 mM de dNTPs, 0,1 pmol de cada cebador y 1U de Taq polimerasa. La sensibilidad de la PCR fue de 3 ng/µL de ADN en aislados de cultivo NNN y 60 ng/µL de ADN en muestras de biopsias, mientras que la especificidad fue de 100% para la detección de Leishmania sp. La enzima de restricción Hae III, determinó fragmentos de 184, 72 y 55 pb., que resultaron específicos para la especie Leishmania (L.) infantum. El marcador utilizado resultó confiable para la detección y caracterización de Leishmania sp. en perros procedentes de zonas endémicas, lo cual podría ser útil para verificar las especies de parásitos circulantes entre los perros.
Leishmaniasis is a disease caused by protozoan parasites of the genus Leishmania. The separasites infect to mammalian hosts, including canines that have been implicated as reservoirs of the parasite. The aim of this research was to standardize the technique of PCR RFLP after amplification of the ITS1 region of Leishmania (Leishmania) infantum, as a useful tool for detection and molecular characterization. Promastigotes from culture and biopsies from dogs with visceral Leishmaniasis previously diagnosed by the Centro Antirrábico Nacional. The ITS1 region of the genomic DNA of Leishmania sp. was amplified using LITSR and L5,8S primers. The technique ITS1 PCR-RFLP applied, allowed the detection of Leishmania (L.) infantum in 10/10 of the isolates from parasites maintained in NNN culture medium, in 10/18 samples from spleen and 10/18 samples from popliteal lymph node. Optimal reaction conditions were 0.2 mM dNTPs, 0.1 pmol of each primer and 1U of Taq polymerase. The sensitivity of PCR was 3 ng/µL DNA in isolates of parasites from NNNculture medium and 60 ng/µL DNA in biopsy samples while the specificity was 100% for the detection of DNA of Leishmania sp. The restriction enzyme Hae III determined fragments of184, 72 and 55 bp., which were specific to Leishmania (L.) infantum. The marker used isreliable for the detection and characterization of Leishmania sp. in dogs from endemic areas, which could be useful to verify the species of parasites circulating among animals.
Subject(s)
Animals , Eukaryota , Polymerase Chain Reaction , DogsABSTRACT
BACKGROUND: Trichophyton mentagrophytes is a complex species and 3 perfect states, Arthroderma benhamiae, A. vanbreuseghemii, and A. simii have been described. In Korea and Japan, all of the isolates of T. mentagrophytes of which the perfect states determined were known as A. vanbreuseghemii till recently. However, in Japan, one isolate from a monkey was reported as A. simii and several strains from rabbits or patients were A. benhamiae. Therefore, it is necessary to confirm the existence of A. simii or A. benhamiae in Korea. OBJECTIVES: We performed this study to determine the teleomorphs of 19 strains of T. mentagrophytes isolated from Korean patients infected from pets and 4 from the skin lesions of rabbits. METHODS: DNA sequences of internal transcribed spacer 1 (ITS1) area of the 23 isolates and 3 reference strains were determined and matched with the data registered in GenBank nucleotide sequence database. RESULTS: The sequencing data of 22 isolates were very similar to those of A. vanbreuseghemii and 1 to T. mentagrophytes var. interdigitale respectively. CONCLUSION: Although all of the clinical isolates were related with A. vanbreuseghemii in this study, the possibility of the existence of A. simii and A. benhamiae in Korea is still high and further study is needed.
Subject(s)
Animals , Humans , Rabbits , Arthrodermataceae , Base Sequence , Databases, Nucleic Acid , Haplorhini , Japan , Korea , Skin , TrichophytonABSTRACT
Thirty strains of seven Candida species from CICC(China Center of Industrial Culture Collection)were studied. The strains were differentiated by ITS1 region PCR-SSCP fingerprinting analysis and ITS2 region PCR-SSCP fingerprinting analysis. Results showed that both ITS1 region and ITS2 region were able to differentiate the seven species of Candida clearly. Contrasting the maps and effects on the identification of Candida species of ITS1 region with that of ITS2 region, result indicated that on the identification of Candida species the application of ITS2 region was better than ITS1 region.