Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 178
Filter
1.
Braz. j. biol ; 81(4): 928-933, Oct.-Dec. 2021. tab, graf
Article in English | LILACS | ID: biblio-1153425

ABSTRACT

Abstract Species of Trichogramma Westwood, 1833 (Hymenoptera: Trichogrammtidae) are frequently used as biological control agents against Lepidoptera, but practical application of these egg endoparasitoids are complicated because of their complex taxonomy. This study aimed to compare sequences of internal transcribed spacer regions of ribosomal DNA (ITS2-rDNA) of Trichogramma accessions with those deposited in GenBank in order to access the reliability of the ITS2 as a barcode for discriminating species and evaluating the genetic diversity. ITS2-rDNA sequences obtained from seventeen specimens of Trichogramma confirmed previous identifications based on morphological characteristics. Multiple sequence alignment revealed the existence of highly conserved regions in ITS2 sequences while the neighbour-joining dendrogram indicated that the specimens formed three clusters comprising T. manicobai and T. marandobai (group I), T. galloi (group II) and T. pretiosum (group III). The ITS2 marker was shown to be a powerful DNA barcode for discriminating Trichogramma species and could be used to complement the morphological approach.


Resumo Espécies de Trichogramma Westwood, 1833 (Hymenoptera: Trichogrammatidae) são freqüentemente usadas como agentes de controle biológico contra Lepidoptera, esses endoparasitóides de ovos apresentam taxonomia complexa, o que dificulta sua aplicação prática. Este estudo teve como objetivo comparar seqüências de regiões espaçadoras internas transcritas de DNA ribossômico (ITS2-rDNA) de acessos de Trichogramma com aquelas depositadas no GenBank, a fim de avaliar a confiabilidade do ITS2 barcode para discriminar espécies e avaliar a diversidade genética. As seqüências de ITS2-rDNA obtidas de dezessete espécimes de Trichogramma confirmaram identidades anteriores com base em características morfológicas. O alinhamento de múltiplas sequências revelou a existência de regiões altamente conservadas nas sequências ITS2, enquanto o dendrograma indicou que os espécimes formavam três grupos compreendendo T. manicobai e T. marandobai (grupo I), T. galloi (grupo II) e T. pretiosum (grupo III). O marcador ITS2 mostrou ser um poderoso DNA barcode para discriminar espécies de Trichogramma podendo ser usado como complemento da abordagem morfológica.


Subject(s)
Animals , Hymenoptera/genetics , Phylogeny , Genetic Variation/genetics , DNA, Ribosomal/genetics , Reproducibility of Results , Sequence Analysis, DNA , DNA, Ribosomal Spacer/genetics
2.
Electron. j. biotechnol ; 44: 41-46, Mar. 2020. tab, ilus
Article in English | LILACS | ID: biblio-1087698

ABSTRACT

Background: The main objective of this study was to isolate fungi associated with Anthopleura xanthogrammica and measure their antimicrobial and enzymatic activities. A total of 93 fungal strains associated with A. xanthogrammica were isolated in this study, of which 32 isolates were identified using both morphological characteristics and internal transcribed spacer (ITS) sequence analysis. The antibacterial activities of 32 fungal isolates were tested against Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Edwardsiella tarda, Vibrio harveyi, Fusarium oxysporum, and Pyricularia oryzae by agar diffusion assay. Extracellular hydrolytic enzyme activities of the fungal isolates were determined by agar diffusion assays. Enzyme activities were detected from clear halo size. Results: The isolated fungi belonged to 18 genera within 7 taxonomic orders of 1 phylum. The genera Aspergillaceae were the most diverse and common. The antimicrobial activities of 32 isolates were evaluated, and 19 (59.4%) of fungi isolate displayed unique antimicrobial activities. All fungal strains displayed at least one enzyme activity. The most common enzyme activities in the fungi isolates were amylase and protease, while the least common were pectinase and xylanase. Conclusions: This is first report on the sea anemone-derived fungi with antimicrobial and enzyme activities. Results indicated that sea anemone is a hot spot of fungal diversity and a rich resource of bioactive natural products.


Subject(s)
Aspergillus/isolation & purification , Sea Anemones/microbiology , Anti-Bacterial Agents/isolation & purification , Peptide Hydrolases/metabolism , Phylogeny , Polygalacturonase/metabolism , Aspergillus/enzymology , Aspergillus/genetics , Bacteria/drug effects , DNA, Ribosomal Spacer , Biodiversity , Fungi/isolation & purification , Fungi/genetics , Amylases/metabolism , Anti-Bacterial Agents/pharmacology
3.
Rev. Soc. Bras. Med. Trop ; 53: e20190364, 2020. tab, graf
Article in English | LILACS | ID: biblio-1057277

ABSTRACT

Abstract The present report describes the first case of postpartum disseminated histoplasmosis in a 24-year-old HIV-negative woman. On the tenth day after vaginal delivery, the patient presented with dyspnea, fever, hypotension, tachycardia, and painful hepatomegaly. Yeast-like Histoplasma capsulatum features were isolated in the buffy coat. The phylogenetic analysis demonstrated that the fungal isolate was similar to other H. capsulatum isolates identified in HIV patients from Ceará and Latin America. Thus, histoplasmosis development in individuals with transitory immunosuppression or during the period of immunological recovery should be carefully examined.


Subject(s)
Humans , Female , Adult , DNA, Fungal/analysis , DNA, Ribosomal Spacer/genetics , Postpartum Period , Histoplasma/genetics , Histoplasmosis/diagnosis , Phylogeny , Polymerase Chain Reaction , Histoplasma/isolation & purification , Histoplasmosis/microbiology
4.
Rev. bras. parasitol. vet ; 28(2): 303-305, Apr.-June 2019. graf
Article in English | LILACS | ID: biblio-1042504

ABSTRACT

Abstract Leishmania spp. are important agents of human and animal leishmaniases that have an important impact on public health. In this study, we aimed to detect the circulation of Leishmania spp. in cattle from a visceral leishmaniasis non-endemic area of the state of São Paulo, Brazil. DNA was extracted from blood samples from 100 heifers in the municipality of Pirassununga and was amplified using primers specific for the first internal transcriber spacer (ITS1), to assess the presence of trypanosomatids. The assays revealed that one sample presented bands of between 300 and 350 base pairs. In GenBank, this sample matched 100% with Leishmania infantum (314 base pairs). The results suggest that cattle can be infected by Leishmania infantum in Brazil.


Resumo Leishmania spp. são agentes causadores das leishmanioses em humanos e em animais, gerando grande impacto à saúde pública. Este estudo objetivou detectar a circulação de Leishmania spp. em área não endêmica para leishmaniose visceral de São Paulo, Brasil. Foram extraídas amostras de DNA de 100 novilhas da cidade de Pirassununga. Estas amostras foram amplificadas com os iniciadores específicos para tripanosomatídeos Internal Transcriber Spacer 1 (ITS1). Os ensaios revelaram uma amostra com bandas entre 300 e 350 pares de base (pb). A amostra demonstrou 100% de identidade com Leishmania infantum (314 pb). Os resultados sugerem que o gado pode ser infectado por L. infantum no Brasil.


Subject(s)
Animals , Cattle , Cattle Diseases/diagnosis , Leishmania infantum/genetics , DNA, Ribosomal Spacer/genetics , Leishmaniasis, Visceral/veterinary , Polymerase Chain Reaction/veterinary , Leishmaniasis, Visceral/diagnosis
5.
Article in Chinese | WPRIM | ID: wpr-777511

ABSTRACT

DNA barcode technology was used to establish a rapid identification method of Chrysanthemum indicum based on ITS2 sequences. The total DNA was extracted from 22 collected samples,and the ITS2 sequence was amplified by PCR and sequenced,and the information of ITS2 sequence was obtained. Another 14 items of the same family or the same genus were downloaded from Gen Bank.We aligned all 36 sequences,calculated the intraspecific and interspecific distances,and constructed Neighbor Joining( NJ) phylogenetic tree,using MEGA 7. 0. The difference of the secondary structure between the ITS2 sequences was compared. The results showed that the genetic distance of Ch. indicum and Ch. morifolium was overlapped,but the maximum intraspecific distance was far less than the minimum interspecific distance between and among Ch. indicum and other species,with an obvious barcoding gap. The NJ tree showed that Ch. indicum and Ch. morifolium shared a clade,and most of Ch. morifolium with some Ch. indicum were shared a subclade,while Inula lineariifolia,Sinosenecio oldhamianus and Senecio scandens belonged to one clade separately. ITS2 secondary structures for I. lineariifolia,S. oldhamianus and S. scandens were significantly different enough to identify completely but Ch. indicum and Ch. morifolium shared two secondary structures of A and B. It was proved that Ch. indicum was one of the evolutionary sources of Ch.morifolium. Therefore ITS2 sequence as DNA barcode can identify Ch. indicum and its adulterants accurately and quickly. The study provides an important basis for Ch. indicum for the identification of germplasm resources and the safety of clinical medication.


Subject(s)
Chrysanthemum , DNA Barcoding, Taxonomic , DNA, Plant , DNA, Ribosomal Spacer , Drugs, Chinese Herbal , Phylogeny , Quality Control
6.
Article in Chinese | WPRIM | ID: wpr-773130

ABSTRACT

The endophytic fungi from root,main stem,branch and leaf of Scrophularia ningpoensis were isolated from Zhejiang,whether these strains could yield harpagide or harpagoside were tested by HPLC and LC-MS. According to the morphological characteristic and the similarity of the nucleotide sequence of internal transcribed spacer( ITS) between r DNAs,the strains producing harpagide or harpagoside were identified. The results showed that 210 strains were isolated from the samples,which were classified into 9 orders,13 families and 17 genera by morphological study. Harpagide was detected in endogenous fungi ZJ17 and harpagoside was detected in endogenous fungi ZJ25 by HPLC coupled with LC-MS. ZJ17 was identified as Alternaria alternate and ZJ25 was identified as A.gaisen by its morphology and authenticated by ITS( ITS4 and ITS5 regions and the intervening 5. 8 S rDNA region).


Subject(s)
China , DNA, Fungal , Genetics , DNA, Ribosomal Spacer , Genetics , Endophytes , Classification , Metabolism , Fungi , Classification , Metabolism , Glycosides , Iridoid Glycosides , Metabolism , Pyrans , Metabolism , Scrophularia , Microbiology
7.
Mem. Inst. Oswaldo Cruz ; 114: e180595, 2019. tab, graf
Article in English | LILACS | ID: biblio-1040622

ABSTRACT

The genetic information of ancient Paragonimus westermani, the oriental lung fluke infecting over 20 million people worldwide, has not been thoroughly investigated thus far. We analysed genetic markers (COI and ITS2) of P. westermani from coprolite specimens (n = 6) obtained from 15th to 18th century Korean mummies. Our results indicated that all P. westermani sequences were generally distinct from the other species of the genus Paragonimus. The sequences were clustered into three groups: Group I for East Asia; Group II for South and Southeast Asia; and Group III for India and Sri Lanka. In this study, we found that ancient P. westermani sequences in Korea belong to Group I, adding invaluable information to the existing knowledge of Paragonimus paleogenetics.


Subject(s)
Humans , Animals , Mummies/parasitology , Electron Transport Complex IV/genetics , DNA, Helminth/genetics , DNA, Ribosomal Spacer/genetics , Paragonimus westermani/isolation & purification , Feces/parasitology , Paleodontology , Parasite Egg Count , Phylogeny , Asia , Paragonimus westermani/genetics
8.
Braz. j. med. biol. res ; 52(9): e8224, 2019. tab, graf
Article in English | LILACS | ID: biblio-1019569

ABSTRACT

Leishmaniasis is a neglected disease that affects a large part of the world population. Knowing the sand fly fauna of a region is of fundamental importance for guiding health surveillance actions related to the prevention and control of leishmaniasis. A total of 86 specimens of sand flies (60 females and 26 males) were collected. Using the classification proposed by Galati (2003), the following species were identified: Lutzomyia longipalpis (Lutz & Neiva, 1912), Migonemyia migonei (França, 1920), Evandromyia cortelezzi (Brethes, 1923), Ev. sallesi (Galvão & Coutinho, 1939), Nyssomyia whitmani (Atunes & Coutinho, 1939), Psathyromyia lutziana (Costa Lima, 1932), Ev. lenti (Mangabeira, 1938), Brumptomyia sp. (França and Parrot, 1921), and Pressatia sp. (Mangabeira, 1942). Using PCR with internal transcribed spacer target to identify infected sand flies, five Lu. longipalpis females were infected with Leishmania spp. Despite the small number of specimens collected, considerable species diversity was found in the study area.


Subject(s)
Animals , Male , Female , Psychodidae/classification , Psychodidae/parasitology , RNA, Protozoan/genetics , Insect Vectors/classification , Insect Vectors/parasitology , Leishmania/isolation & purification , Brazil , Leishmaniasis/transmission , Polymerase Chain Reaction , DNA, Ribosomal Spacer/genetics , Leishmania/genetics
9.
Article in Chinese | WPRIM | ID: wpr-775405

ABSTRACT

Fructus Arctii is a traditional Chinese medicine. The main counterfeit species are the seeds of Arctium tomentosum, Onopordum acanthium, Silybum marianum, Saussurea costus, Amorpha fruticosa. Traditional identification methods or molecular barcoding techniques can identify Fructus Arctii and its counterfeit species. However, the identification of the mixture of it and its spurious species is rarely reported. In this paper, we sequenced the ITS2 sequences of Fructus Arctii and 5 kinds of spurious species mix powder by high-throughput sequencing to identify the mixed powder species and providing new ideas for the identification of Fructus Arctii mix powder. The total DNA in mixed powder was extracted, and the ITS2 sequences in total DNA was amplified. Paired-end sequencing was performed on the DNA fragment of the community using the Illumina MiSeq platform. The sequence was analyzed by the software FLASH, QIIME and GraPhlAn etc. The results showed that the high quality ITS2 sequences of 39910 mix samples were obtained from the mixed samples, of which the total ITS2 sequence of the samples genus was 34 935. Phylogenetic analysis showed that the samples contained Fructus Arctii, A. tomentosum, O. acanthium, S. marianum, S. costus and A. fruticosa. Using ITS2 sequences as DNA barcodes, high-throughput sequencing technology can be used to detect the Fructus Arctii and its spurious specie in mixed powder, which can provide reference for the quality control, safe use of medicinal materials of Fructus Arctii and the identification of mixed powder of traditional Chinese medicine.


Subject(s)
Arctium , Chemistry , Classification , DNA Barcoding, Taxonomic , DNA, Plant , Genetics , DNA, Ribosomal Spacer , Genetics , Drug Contamination , Drugs, Chinese Herbal , Reference Standards , Fabaceae , Fruit , High-Throughput Nucleotide Sequencing , Milk Thistle , Onopordum , Phylogeny , Saussurea
10.
Mem. Inst. Oswaldo Cruz ; 112(3): 214-219, Mar. 2017. tab, graf
Article in English | LILACS | ID: biblio-1040568

ABSTRACT

Since the description of Candida orthopsilosis and C. metapsilosis in 2005, several methods have been proposed to identify and differentiate these species from C. parapsilosis sensu stricto. Species-specific uniplex polymerase chain reaction (PCR) was performed and compared with sequencing of the D1/D2 region of the LSU 28S rDNA gene, microsatellite typing of C. parapsilosis sensu stricto, and PCR-restriction fragment length polymorphism patterns in the ITS1-5.8S-ITS2 region of the rDNA gene. There was agreement between results of testing of 98 clinical isolates with the four PCR-based methods, with 59 isolates identified as C. parapsilosis sensu stricto, 37 as C. orthopsilosis, and two as C. metapsilosis.


Subject(s)
Humans , Candida/isolation & purification , Mycological Typing Techniques/methods , Polymorphism, Restriction Fragment Length , Candida/classification , Candida/genetics , DNA, Fungal/analysis , Polymerase Chain Reaction , DNA Fingerprinting , Sequence Analysis, DNA , DNA, Ribosomal Spacer/genetics , Genotype
11.
Mem. Inst. Oswaldo Cruz ; 112(2): 140-145, Feb. 2017. graf
Article in English | LILACS | ID: biblio-841762

ABSTRACT

BACKGROUND Fluorescence in situ hybridisation (FISH) associated with Tyramide Signal Amplification (TSA) using oligonucleotides labeled with non-radioactive fluorophores is a promising technique for detection and differentiation of fungal species in environmental or clinical samples, being suitable for microorganisms which are difficult or even impossible to culture. OBJECTIVE In this study, we aimed to standardise an in situ hybridisation technique for the differentiation between the pathogenic species Paracoccidioides brasiliensis and Paracoccidioides lutzii, by using species-specific DNA probes targeting the internal transcribed spacer-1 (ITS-1) of the rRNA gene. METHODS Yeast and mycelial phase of each Paracoccidioides species, were tested by two different detection/differentiation techniques: TSA-FISH for P. brasiliensis with HRP (Horseradish Peroxidase) linked to the probe 5’ end; and FISH for P. lutzii with the fluorophore TEXAS RED-X® also linked to the probe 5’ end. After testing different protocols, the optimised procedure for both techniques was accomplished without cross-positivity with other pathogenic fungi. FINDINGS The in silico and in vitro tests show no reaction with controls, like Candida and Cryptococcus (in silico) and Histoplasma capsulatum and Aspergillus spp. (in vitro). For both phases (mycelial and yeast) the in situ hybridisation showed dots of hybridisation, with no cross-reaction between them, with a lower signal for Texas Red probe than HRP-TSA probe. The dots of hybridisation was confirmed with genetic material marked with 4’,6-diamidino-2-phenylindole (DAPI), visualised in a different filter (WU) on fluorescent microscopic. MAIN CONCLUSION Our results indicated that TSA-FISH and/or FISH are suitable for in situ detection and differentiation of Paracoccidioides species. This approach has the potential for future application in clinical samples for the improvement of paracoccidioidomycosis patients prognosis.


Subject(s)
Paracoccidioides/classification , Paracoccidioides/genetics , DNA, Fungal , DNA, Ribosomal Spacer , Species Specificity , Oligonucleotide Probes , In Situ Hybridization, Fluorescence , Fluorescence , Fluorescent Dyes
12.
Braz. j. microbiol ; 47(1): 172-176, Jan.-Mar. 2016. tab
Article in English | LILACS | ID: lil-775126

ABSTRACT

Abstract Candida species, especially C. albicans, are commensals on human mucosal surfaces, but are increasingly becoming one of the important invasive pathogens as seen by a rise in its prevalence in immunocompromised patients and in antibiotic consumption. Thus, an accurate identification of Candida species in patients with pulmonary symptoms can provide important information for effective treatment. A total of 75 clinical isolates of Candida species were obtained from the bronchoalveolar lavage fluid of both immunocompromised and immunocompetent patients with pulmonary symptoms. Candida cultures were identified based on nuclear ribosomal Internal Transcribed Spacer (ITS1-ITS2 rDNA) sequence analysis by polymerase chain reaction–restriction fragment length polymorphisms (PCR-RFLP). Molecular identification indicated that the isolates belonged predominantly to C. albicans (52%), followed by C. tropicalis (24%), C. glabrata (14.7%), C. krusei (5.3%), C. parapsilosis (1.3%), C. kefyr (1.3%) and C. guilliermondii (1.3%). Given the increasing complexity of disease profiles and their management regimens in diverse patients, rapid and accurate identification of Candida species can lead to timely and appropriate antifungal therapy.


Subject(s)
Humans , Bronchoalveolar Lavage Fluid/microbiology , Candida/isolation & purification , Candidiasis/diagnosis , Lung Diseases, Fungal/diagnosis , Candida/classification , Candida/genetics , DNA, Fungal/chemistry , DNA, Fungal/genetics , DNA, Ribosomal Spacer/chemistry , DNA, Ribosomal Spacer/genetics , Polymerase Chain Reaction , Polymorphism, Restriction Fragment Length , Sequence Analysis, DNA , Time Factors
13.
Article in English | WPRIM | ID: wpr-36483

ABSTRACT

The aim of the present study is to determine the characteristics of genotype and phenotype of Echinococcus granulosus derived from wild sheep and to compare them with the strains of E. granulosus sensu stricto (sheep-dog) and E. granulosus camel strain (camel-dog) in Iran. In Khojir National Park, near Tehran, Iran, a fertile hydatid cyst was recently found in the liver of a dead wild sheep (Ovis orientalis). The number of protoscolices (n=6,000) proved enough for an experimental infection in a dog. The characteristics of large and small hooks of metacestode were statistically determined as the sensu stricto strain but not the camel strain (P=0.5). To determine E. granulosus genotype, 20 adult worms of this type were collected from the infected dog. The second internal transcribed spacer (ITS2) of the nuclear ribosomal DNA (rDNA) and cytochrome c oxidase 1 subunit (COX1) of the mitochondrial DNA were amplified from individual adult worm by PCR. Subsequently, the PCR product was sequenced by Sanger method. The lengths of ITS2 and COX1 sequences were 378 and 857 bp, respectively, for all the sequenced samples. The amplified DNA sequences from both ribosomal and mitochondrial genes were highly similar (99% and 98%, respectively) to that of the ovine strain in the GenBank database. The results of the present study indicate that the morpho-molecular features and characteristics of E. granulosus in the Iranian wild sheep are the same as those of the sheep-dog E. granulosus sensu stricto strain.


Subject(s)
Animals , DNA, Helminth/genetics , DNA, Ribosomal Spacer/genetics , Dogs , Echinococcosis/parasitology , Echinococcus granulosus/anatomy & histology , Electron Transport Complex IV/genetics , Genotype , Iran , Phenotype , Phylogeny , Polymorphism, Restriction Fragment Length , Sheep , Sheep Diseases/parasitology , Species Specificity
14.
Article in English | WPRIM | ID: wpr-36478

ABSTRACT

A study of 426 rabbits from 3 cities in Jilin province (Changchun City and Jilin City) and Liaoning province (Shenyang City) was conducted between May and June 2015. The overall prevalence of E. bieneusi in rabbits was 0.94% (4/426), with 0% (0/116), 1.72% (3/174), and 0.74% (1/136) in Jilin, Changchun, and Shenyang City, respectively. Only 3 farms (farm 1 and farm 3 in Changchun City, farm 8 in Shenyang City) were PCR-positive for E. bieneusi. Moreover, rabbits of more than 6 months (1.72%) had the highest E. bieneusi prevalence, followed by rabbits of 4-6 months (1.26%), 2-3 months (0.58%), and less than 1 month (0%). Analysis of ITS gene of E. bieneusi suggested that all 4 E. bieneusi isolates were genotype D, and were classified as group 1a. The present results first demonstrated the existence of zoonotic E. bieneusi in domestic rabbits in China. Effective control measures should be implemented to prevent E. bieneusi infection in domestic rabbits, other animals, and humans.


Subject(s)
Animals , China/epidemiology , DNA, Ribosomal Spacer/genetics , Enterocytozoon/genetics , Genotype , Microsporidiosis/epidemiology , Rabbits/microbiology , Zoonoses/microbiology
15.
Article in English | WPRIM | ID: wpr-36473

ABSTRACT

We performed a molecular genetic study on the sequences of 18S ribosomal RNA (ITS1 region) gene in 4-day-old adult worms of Macroorchis spinulosus recovered in mice experimentally infected with metacercariae from crayfish in Jeollanam-do Province, Korea. The metacercariae were round, 180 µm in average diameter, encysted with 2 layers of thick walls, but the stylet on the oral sucker was not clearly seen. The adult flukes were oval shape, and 760-820 µm long and 320-450 µm wide, with anterolateral location of 2 large testes. The phylogenetic tree based on ITS1 sequences of 6 M. spinulosus samples showed their distinguished position from other trematode species in GenBank. The most closely resembled group was Paragonimus spp. which also take crayfish or crabs as the second intermediate host. The present study is the first molecular characterization of M. spinulosus and provided a basis for further phylogenetic studies to compare with other trematode fauna in Korea.


Subject(s)
Animals , DNA, Ribosomal Spacer/genetics , Metacercariae/classification , Mice , Phylogeny , RNA, Ribosomal, 18S/genetics , Trematoda/classification
16.
Braz. j. infect. dis ; 19(6): 563-570, Nov.-Dec. 2015. tab, graf
Article in English | LILACS | ID: lil-769620

ABSTRACT

ABSTRACT The diagnosis of cryptococcosis is usually performed based on cultures of tissue or body fluids and isolation of the fungus, but this method may require several days. Direct microscopic examination, although rapid, is relatively insensitive. Biochemical and immunodiagnostic rapid tests are also used. However, all of these methods have limitations that may hinder final diagnosis. The increasing incidence of fungal infections has focused attention on tools for rapid and accurate diagnosis using molecular biological techniques. Currently, PCR-based methods, particularly nested, multiplex and real-time PCR, provide both high sensitivity and specificity. In the present study, we evaluated a nested PCR targeting the gene encoding the ITS-1 and ITS-2 regions of rDNA in samples from a cohort of patients diagnosed with cryptococcosis. The results showed that in our hands, this Cryptococcus nested PCR assay has 100% specificity and 100% sensitivity and was able to detect until 2 femtograms of Cryptococcus DNA.


Subject(s)
Humans , Cryptococcosis/diagnosis , Cryptococcus gattii/genetics , Cryptococcus neoformans/genetics , DNA, Fungal/analysis , Cryptococcosis/microbiology , Cryptococcus gattii/isolation & purification , Cryptococcus neoformans/isolation & purification , DNA, Ribosomal Spacer , Real-Time Polymerase Chain Reaction , Sensitivity and Specificity , Sequence Analysis, DNA
17.
Braz. j. biol ; 75(4): 974-982, Nov. 2015. tab
Article in English | LILACS | ID: lil-768195

ABSTRACT

Abstract ITS2 (Internal transcribed spacer 2) sequences have been used in systematic studies and proved to be useful in providing a reliable identification of Trichogramma species. DNAr sequences ranged in size from 379 to 632 bp. In eleven T. pretiosum lines Wolbachia-induced parthenogenesis was found for the first time. These thelytokous lines were collected in Peru (9), Colombia (1) and USA (1). A dichotomous key for species identification was built based on the size of the ITS2 PCR product and restriction analysis using three endonucleases (EcoRI, MseI and MaeI). This molecular technique was successfully used to distinguish among seventeen native/introduced Trichogramma species collected in South America.


Resumo Sequências do Espaço Transcrito Interno 2 (ITS2) têm sido utilizadas em estudos taxonômicos e sua utilidade constatada pela confiabilidade que o método confere à identificação das espécies de Trichogramma. Esta técnica molecular foi bem sucedida em distinguir dezessete espécies nativas e introduzidas de Trichogramma, coletadas na América do Sul. As sequências do DNAr variaram de 379 a 632 pb. Em 11 linhagens de T. pretiosum estudadas, o endosinbionte Wolbachia foi detectado pela primeira vez. Estas linhagens telítocas foram encontradas no Peru (9), Colômbia (1) e Estados Unidos (1). Uma chave dicotômica para identificação de espécies foi construída baseada no tamanho do produto da PCR do ITS2 e em análises de restrição utilizando-se três endonucleases (EcoRI, MseI and MaeI).


Subject(s)
Animals , Wasps/classification , Wasps/physiology , DNA, Ribosomal Spacer/genetics , Molecular Sequence Data , Parthenogenesis , Sequence Analysis, DNA , South America , Wasps/genetics , Wasps/microbiology , Wolbachia/physiology
18.
Braz. j. microbiol ; 46(2): 359-366, Apr-Jun/2015. tab, graf
Article in English | LILACS | ID: lil-749714

ABSTRACT

Endophytes are being considered for use in biological control, and the enzymes they secrete might facilitate their initial colonization of internal plant tissues and direct interactions with microbial pathogens. Microbial proteases are also biotechnologically important products employed in bioremediation processes, cosmetics, and the pharmaceutical, photographic and food industries. In the present study, we evaluated antagonism and competitive interactions between 98 fungal endophytes and Alternaria alternata, Colletotrichum sp., Phyllosticta citricarpa and Moniliophthora perniciosa. We also examined the proteolytic activities of endophytes grown in liquid medium and conducted cup plate assays. The results showed that certain strains in the assemblage of P. hispidum endophytes are important sources of antifungal properties, primarily Lasiodiplodia theobromae JF766989, which reduced phytopathogen growth by approximately 54 to 65%. We detected 28 endophytes producing enzymatic halos of up to 16.40 mm in diameter. The results obtained in the present study highlight the proteolytic activity of the endophytes Phoma herbarum JF766995 and Schizophyllum commune JF766994, which presented the highest enzymatic halo diameters under at least one culture condition tested. The increased activities of certain isolates in the presence of rice or soy flour as a substrate (with halos up to 17.67 mm in diameter) suggests that these endophytes have the potential to produce enzymes using agricultural wastes.


Subject(s)
Antifungal Agents/metabolism , Endophytes/isolation & purification , Endophytes/metabolism , Fungi/isolation & purification , Fungi/metabolism , Peptide Hydrolases/metabolism , Piper/microbiology , DNA, Fungal/chemistry , DNA, Fungal/genetics , DNA, Ribosomal Spacer/chemistry , DNA, Ribosomal Spacer/genetics , Fungi/classification , Fungi/drug effects , Microbiological Techniques , Molecular Sequence Data , Sequence Analysis, DNA
19.
Braz. j. microbiol ; 46(1): 59-65, 05/2015. tab, graf
Article in English | LILACS | ID: lil-748250

ABSTRACT

Thermophilic and thermotolerant micro-organisms strains have served as the natural source of industrially relevant and thermostable enzymes. Although some strains of the Trametes genus are thermotolerant, few Trametes strains were studied at the temperature above 30 °C until now. In this paper, the laccase activity and the mycelial growth rate for Trametes trogii LK13 are superior at 37 °C. Thermostability and organic cosolvent tolerance assays of the laccase produced at 37 °C indicated that the enzyme possessed fair thermostability with 50% of its initial activity at 80 °C for 5 min, and could remain 50% enzyme activity treated with organic cosolvent at the concentration range of 25%–50% (v/v). Furthermore, the test on production of laccase and lignocellulolytic enzymes showed the crude enzymes possessed high laccase level (1000 U g−1) along with low cellulose (2 U g−1) and xylanase (140 U g−1) activity. Thus, T. trogii LK13 is a potential strain to be applied in many biotechnological processes.


Subject(s)
Laccase/metabolism , Trametes/enzymology , Trametes/growth & development , Cluster Analysis , DNA, Fungal/chemistry , DNA, Fungal/genetics , DNA, Ribosomal Spacer/chemistry , DNA, Ribosomal Spacer/genetics , Enzyme Stability , Laccase/chemistry , Microscopy , Molecular Sequence Data , Phylogeny , Sequence Analysis, DNA , Solvents , Temperature , Trametes/cytology , Trametes/radiation effects
20.
Braz. j. biol ; 75(2): 391-395, 05/2015. tab, graf
Article in English | LILACS | ID: lil-749700

ABSTRACT

The objective of this work was the identification and differentiation of Trichogramma exiguum Pinto and Platner species, T. pretiosum Riley, and T. galloi Zucchi using sequences of the ITS2 region of ribosomal DNA. After extracting DNA from the studied species, a PCR reaction was performed, where the amplified samples were subjected to sequencing. The sequences obtained were submitted to a similarity search in GenBank (NCBI - National Center for Biotechnology Information) using the BLAST program, aiming to determine the similarity of these sequences with the species already deposited in the referenced database, and then multiple sequences were aligned using version 2.0 of the ClustalX program. According to the results of the multiple alignments of all sequences obtained, it was possible to observe the differences between the T. pretiosum, T. galloi and T. exiguum species. It was concluded that using the sequences of the ITS2 region of the ribosomal DNA was efficient in the differentiation of the studied Trichogramma species, which suggests a strong inter-specific variation among species.


O objetivo deste trabalho foi realizar a identificação e diferenciação das espécies Trichogramma exiguum Pinto e Platner, T. pretiosum Riley e T. galloi Zucchi utilizando o sequenciamento da região ITS2 do DNA ribossomal. Após a extração do DNA das espécies estudadas, foi realizada a reação de PCR, onde as amostras amplificadas foram submetidas ao sequenciamento. As sequências obtidas foram submetidas à busca por similaridade no GenBank (NCBI – National Center for Biotechnology Information) por meio do programa BLAST visando-se determinar a similaridade destas com sequências das espécies já depositadas no referido banco de dados e em seguida foi feito o alinhamento múltiplo das sequências com o auxílio do programa CLUSTALX, versão 2.0. De acordo com os resultados do alinhamento múltiplo de todas as sequências obtidas, foi possível verificar as diferenças entre as espécies de T.pretiosum, T. galloi e T. exiguum. Isto permitiu concluir que a utilização do sequenciamento da região ITS2 do DNA ribossomal foi eficiente na diferenciação das espécies de Trichogramma estudadas, o que sugere uma forte variação inter-específica entre as espécies.


Subject(s)
Animals , DNA, Ribosomal Spacer/genetics , Hymenoptera/genetics , Hymenoptera/classification , Polymerase Chain Reaction , Sequence Analysis, DNA
SELECTION OF CITATIONS
SEARCH DETAIL