Determination of neutral polymorphisms (frameworks) of the human beta globin gene in beta thalassaemias by PCR/DGGE

Purpose: Considering the importance of type beta thalassaemias as hereditary syndromes of high significance in different populations of Mediterranean origin and, by extension, in the Brazilian population, the objective of the present study was to determine by PCR/DGGE the gene structures responsible for neutral polymorphisms (frameworks) observed in the human beta globin gene associated with the mutations responsible for type beta thalassaemias in a sample of the Brazilian population and, more specifically, of the population of the State of São Paulo. Patients and methods: Thirty individuals with beta thalassaemic mutations were analyzed: 22 mutations were in codon 39 (C->T), 5 in IVS1-110 (G->A), 2 in IVS1-6 (T->C) and 1 in IVS1-1 (G->A). DNA was extracted and selective amplification was performed by PCR extending from position IVS1 nt 46 to IVS2 nt 126 (474 pb). The product was then analyzed by polyacrylamide gel electrophoresis on a denaturing 10-60% urea/formamide gradient. Results: The results demonstrated that, as expected, the mutations responsible for type beta thalassaemia observed in this population are of Mediterranean origin, with 73% distribution represented by codon 39,17% by IVS1-110, 7% by IVS1-6 and 3% by IVS1-1. In turn, framework distribution seems to indicate a higher frequency of Fr 1-1 in codon 39 and IVS1-110, of Fr 1-3 in IVS1-6 and of Fr 1-2 in IVS1-1. Conclusions: These results permit us to conclude that gene amplification by PCR followed by DGGE is an appropriate method for the separation of DNA molecules that differ even by a single base change and therefore can be utilized to detect the alterations observed in the human beta globin gene. This methodology shows that, using only a pair of primers, it is possible to define the frameworks that are observed in the beta globin gene.

The detection of Toxoplasma gondii by comparing cytology, histopathology, bioassay in mice, and the polymerase chain reaction (PCR)

The objective of this study was to compare the different methods of detecting Toxoplasma gondii in sheep tissue, tested serologically positive by the indirect immunofluorescent antibody test (IFAT). Brain, diaphragm, and blood samples were collected from 522 sheep slaughtered at the São Manuel abattoir, São Paulo State, Brazil. Brain and diaphragm samples from IFAT seropositive animals were digested by both trypsin and pepsin and then injected into mice. Part of the digested samples was used to prepare slides for Giemsa staining and in the polymerase chain reaction (PCR). Tissue fragments were fixed in formalin and examined using hematoxilin-eosin (HE). Forty of the sheep (7.7%) were IFAT positive. T. gondii was isolated in 23 (59.0%) of the 39 mice with pepsin-digested brain samples and in 27 (69.0%) of the 39 with trypsin-digested brain samples. Injection of diaphragm samples led to T. gondii isolation in 26 (66.7%) of the 39 pepsin-digested samples and 21 (53.8%) of the 39 trypsin-digested samples. Cytological and hystopathological examination of both brains and diaphragms was negative in all examined sheep. PCR was positive in 7 (17.9%) of the trypsin and 2 (5.1%) of the pepsin-digested samples, while 9 (23.1%) of the trypsin and 3 (7.7%) of the pepsin-digested samples showed T. gondii DNA. T. gondii isolation rate in mice (n = 34; 85.0%) was significantly higher than detection by PCR (n = 15; 37.5%). © 2001 Elsevier Science B.V.

Comparison of a multiplex-PCR assay with mycolic acids analysis and conventional methods for the identification of Mycobacteria

A fast, sensitive and cost-effective multiplex-PCR assay for Mycobacterium tuberculosis complex (MTC) and Mycobacterium avium (M. avium) identification for routine diagnosis was evaluated. A total of 158 isolates of mycobacteria from 448 clinical specimens from patients with symptoms of mycobacterial disease were analyzed. By conventional biochemical methods 151 isolates were identified as M. tuberculosis, five as M. avium and two as Mycobacterium chelonae (M. chelonae). Mycolic acid patterns confirmed these results. Multiplex-PCR detected only IS6110 in isolates identified as MTC, and IS1245 was found only in the M. avium isolates. The method applied to isolates from two patients, identified by conventional methods and mycolic acid analysis, one as M. avium and other as M. chelonae, resulted positive for IS6110, suggesting co-infection with M. tuberculosis. These patients were successfully submitted to tuberculosis treatment. The multiplex-PCR method may offer expeditious identification of MTC and M. avium, which may minimize risks for active transmission of these organisms and provide useful treatment information.

Clonagem e expressão da glicoproteína transmembrana do retrovírus HTLV-1 em células de mamíferos

The retrovirus HTLV-1 is the etiological agent of the adult T-cell leukemia and HTLV-1 associated myelopathy/tropical spastic paraparesis. The proviral genome has 9,032 base pairs, showing regulatory and structural genes. The env gene encodes for the transmembrane glycoprotein gp 21. The development of methodologies for heterologous protein expression, as well as the acquisition of a cellular line that constituently expresses the recombinant, were the main goals of this work. The DNA fragment that encodes for gp 21 was amplified by nested-PCR and cloned into a pCR2.1-TOPO vector. After which, a sub-cloning was realized using the expressing vector pcDNA3.1+. The transfection of mammalian cells HEK 293 was performed transitorily and permanently. Production of the recombinant gp 21 was confirmed by flux cytometry experiments and the cell line producing protein will be used in immunogenicity assays.

Rapid molecular diagnosis of tuberculosis and other mycobacteriosis in smear-negative clinical specimens

Delay in diagnosis of pulmonary and other forms of tuberculosis (TB) can be fatal, particularly in HIV-infected patients. Hence, techniques based on nucleic acid amplification, which are both rapid and of high specificity and sensitivity, are now widely used and recommended for laboratories that diagnose TB. In the present study, diagnostic methods based on mycobacterial DNA amplification were evaluated in comparative trials alongside tradicional bacterial methods, using negative smear samples from patients with clinically-suspected TB (sputum samples from 25 patients with suspected pulmonary TB, urine samples from two patients with suspected renal TB and cerebrospinal fluid samples from one patient with suspected meningeal TB). A specificity of 100% was achieved with DNA amplification methods and tradicional culture/identification methods, in relation to clinical findings and treatment results. For the smear-negative sputa, conventional PCR for M. tuberculosis was positive in 62% of suspected lung TB case, showing the same sensitivity as bacterial identification. Both techniques failed in the detection of extra-pulmonary samples. Nested PCR showed, after species-specific amplification, a sensitivity of 100% for M. avium and 85% for M. tuberculosis. For extra-pulmonary smear-negative samples, only Nested PCR detected M. tuberculosis and all cases were confirmed clinically. Nested PCR, in which two-step amplification reactions are performed, can identify the two most important mycobacteria in human pathology quickly and directly from clinical spicimens.

Incidence and transplacental transmission of Neospora caninum in primiparous females from Bos indicus slaughtered in Presidente Prudente, São Paulo, Brazil

To produce an epidemiological map of neosporosis in Brazil and identify the types of transmission of this disease, the present study evaluated the occurrence of Neospora caninum in Nelore cattle (Bos indicus) in Presidente Prudent, west region of Sao Paulo state; its vertical transmission; and the early stage in which fetuses are infected. To achieve this, serum samples from 518 slaughtered pregnant heifers and their fetuses were tested by ELISA technique and fetal brain tissues subjected to PCR. One hundred and three heifers (19.88%) had antibodies to N. caninum, as well as 38 (36.8%) of fetuses from 4 months of gestation. The conventional PCR failed to detect N. caninum DNA. These findings show that neosporosis occurs in the area studied and that it may be transmitted the transplacental route, althought N. caninum had not detected in brain tissue from non-aborted fetuses. The use of nested PCR it would be applied to increase the sensitivy of test.

Heminested reverse-transcriptase polymerase chain reaction (hnRT-PCR) as a tool for rabies virus detection in stored and decomposed samples

Background. The use of methods, both sensitive and specific, for rabies diagnosis are important tools for the control and prophylaxis of the disease. Reverse-Transcriptase Polymerase Chain Reaction (RT-PCR) has been used in rabies diagnosis with good results, even in decomposed materials. Additionally, molecular techniques have been used for epidemiological studies and to gain a better knowledge of viral epidemiology. Findings. The aim of this work was to evaluate the RT-PCR and hnRT-PCR for rabies virus detection in original tissues stored at -20°C for different periods considering their use for rabies virus detection in stored and decomposed samples. RT-PCR and hnRT-PCR were evaluated in 151 brain samples from different animal species, thawed and left at room temperature for 72 hours for decomposition. The RT-PCR and hnRT-PCR results were compared with previous results from Direct Fluorescent Antibody Test and Mouse Inoculation Test. From the 50 positive fresh samples, 26 (52%) were positive for RT-PCR and 45 (90%) for hnRT-PCR. From the 48 positive decomposed samples, 17 (34, 3%) were positive for RT-PCR and 36 (75%) for hnRT-PCR. No false-positives results were found in the negatives samples evaluated to the molecular techniques. Conclusion. These results show that the hnRT-PCR was more sensitive than RT-PCR, and both techniques presented lower sensibility in decomposed samples. The hnRT-PCR demonstrated efficacy in rabies virus detection in stored and decomposed materials suggesting it's application for rabies virus retrospective epidemiological studies. © 2008 Arajo et al; licensee BioMed Central Ltd.

Assessing genetic variability in bat species of Emballonuridae, Phyllostomidae, Vespertilionidae and Molossidae families (Chiroptera) by RFLP-PCR

A PCR-RFLP analysis of the restriction pattern in nuclear (RAG2) and mitochondrial (12S/16S) gene sequences of bat species from the Molossidae, Phyllostomidae, Vespertilionidae, and Emballonuridae families produced a large number of fragments: 107 for RAG2 and 155 for 12S/16S combined in 139 and 402 haplotypes, respectively. The values detected for gene variation were low for both sequences (0.13 for RAG2 and 0.15 for 12S/16S) and reflected their conservative feature, reinforced by high values of inter- and intraspecies genetic identity (70-100%). The species with a high gene divergence were variable in the analyses of RAG2 (Eumops perotis, Artibeus lituratus, and Carollia perspicillata) and of 12S/16S (Nyctinomops laticaudatus, C. perspicillata, and Cynomops abrasus), and furthermore, one of them, C. perspicillata, also showed the highest intraspecific variation. The species that exhibited the lowest variation for both genes was Molossus rufus. In the families, the highest variation was observed in the Molossidae and this can be attributed to variation exhibited by Eumops and Nyctinomops species. The variations observed were interpreted as a natural variability within the species and genus that exhibited a conserved pattern in the two gene sequences in different species and family analyzed. Our data reinforce the idea that the analyses of mitochondrial and nuclear genes contribute to our knowledge of the diversity of New World bats. The genetic variability found in different taxa suggests that an additional diversity, unnoticed by other methods, can be revealed with the use of different molecular strategies. ©FUNPEC-RP.

Multiplex PCR use for Staphylococcus aureus identification and oxacillin and mupirocin resistance evaluation

Oxacillin-resistant Staphylococcus aureus represents a serious problem in hospitals worldwide, increasing infected patients' mortality and morbidity and raising treatment costs and internment time. In this study, the results of using the Multiplex PCR technique to amplify fragments of the genes femA (specific-species), mecA (oxacillin resistance) and ileS-2 (mupirocin resistance) were compared with those of tests conventionally used to identify S. aureus isolates and ascertain their resistance to drugs. Fifty S. aureus strains were isolated from patients receiving treatment at UNOESTE University Hospital in Presidente Prudente, SP, Brazil. The 686 bp fragment corresponding to the gene femA was amplified and detected in all the isolates. On the other hand, the 310 bp fragment corresponding to the mecA gene was amplified in 29 (58%) of the isolates. All of the isolates showed sensitivity to mupirocin in the agar diffusion test, which was corroborated by the lack of any amplicon of the 456 bp fragment corresponding to the ileS-2 gene, in the PCR bands. The conventional tests to identify S. aureus and detect resistance to oxacillin and mupirocin showed 100% agreement with the PCR Multiplex results. The use of techniques for rapid and accurate identification of bacteria and assessment of their resistance may be valuable in the control of infection by resistant strains, allowing the rapid isolation and treatment of an infected patient. However, the results demonstrate that traditional phenotypic tests are also reliable, though they take more time.

Genetic polymorphism, molecular characterization and relatedness of Macrobrachium species (Palaemonidae) based on RAPD-PCR.

The prawn genus Macrobrachium belongs to the family Palaemonidae. Its species are widely distributed in lakes, reservoirs, floodplains, and rivers in tropical and subtropical regions of South America. Globally, the genus Macrobrachium includes nearly 210 known species, many of which have economic and ecological importance. We analyzed three species of this genus (M. jelskii, M. amazonicum and M. brasiliense) using RAPD-PCR to assess their genetic variability, genetic structure and the phylogenetic relationship between them and to look for molecular markers that enable separation of M. jelskii and M. amazonicum, which are closely related syntopic species. Ten different random decamer primers were used for DNA amplification, yielding 182 fragments. Three of these fragments were monomorphic and exclusive to M. amazonicum or M. jelskii and can be used as specific molecular markers to identify and separate these two species. Similarity indices and a phylogenetic tree showed that M. amazonicum and M. jelskii are closest to each other, while M. brasiliense was the most differentiated species among them; this may be attributed to the different habitat conditions to which these species have been submitted. This information will be useful for further studies on these important crustacean species.

Use of reverse transcriptase polymerase chain reaction (RT-PCR) in molecular screening of Newcastle disease virus in poultry and free-living bird populations

The aim of this study was to evaluate a simple molecular method of reverse transcriptase polymerase chain reaction (RT-PCR) to differentiate Newcastle disease virus strains according to their pathogenicity, in order to use it in molecular screening of Newcastle disease virus in poultry and free-living bird populations. Specific primers were developed to differentiate LaSota-LS-(vaccine strain) and Sao Joao do Meriti-SJM-strain (highly pathogenic strain). Chickens and pigeons were experimentally vaccinated/infected for an in vivo study to determine virus shedding in feces. Validation of sensitivity and specificity of the primers (SJM and LS) by experimental models used in the present study and results obtained in the molecular analysis of the primers by BLAST made it possible to generalize results. The development of primers that differentiate the level of pathogenicity of NDV stains is very important, mainly in countries where real-time RT-PCR is still not used as a routine test. These primers were able to determine the presence of the agent and to differentiate it according to its pathogenicity. © 2012 Springer Science+Business Media B.V.

Exequibility of differential gene expression analysis by DDRT-PCR in murine bone marrow cells

Model of study: Experimental study. Introduction: Recently, stem cell research has generated great interest due to its applicability in regenerative medicine. Bone marrow is considered the most important source of adult stem cells and the establishment of new methods towards gene expression analysis regarding stem cells has become necessary. Thus Differential Display Reverse Transcription Polymerase Chain Reaction (DDRT-PCR) may be an accessible tool to investigate small differences in the gene expression of different stem cells in distinct situations. Aim: In the present study, we investigated the exequibility of DDRT-PCR to identify differences in global gene expression of mice bone marrow cells under two conditions. Methods: First, bone marrow cells were isolated fresh and a part was cultivated during one week without medium replacement. Afterwards, both bone marrow cells (fresh and cultivated) were submitted to gene expression analyses by DDRT-PCR. Results: Initially, it was possible to observe in one week-cultured bone marrow cells, changes in morphology (oval cells to fibroblastic-like cells) and protein profile, which was seen through differences in band distribution in SDS-Page gels. Finally through gene expression analysis, we detected three bands (1300, 1000 and 225 bp) exclusively expressed in the fresh bone marrow group and two bands (400 and 300 bp) expressed specifically in the cultivated bone marrow cell group. Conclusions: In summary, the DDRT-PCR method was proved efficient towards the identification of small differences in gene expression of bone marrow cells in two defined conditions. Thus, we expect that DDRT-PCR can be fast and efficiently designed to analyze differential gene expression in several stem cell types under distinct conditions.

Detection of Paracoccidioides spp. in environmental aerosol samples

Taking into account that paracoccidioidomycosis infection occurs by inhalation of the asexual conidia produced by Paracoccidioides spp. in its saprobic phase, this work presents the collection of aerosol samples as an option for environmental detection of this pathogen, by positioning a cyclonic air sampler at the entrance of armadillo burrows. Methods included direct culture, extinction technique culture and Nested PCR of the rRNA coding sequence, comprising the ITS1-5.8S-ITS2 region. In addition, we evaluated one armadillo (Dasypus novemcinctus) as a positive control for the studied area. Although the pathogen could not be isolated by the culturing strategies, the aerosol sampling associated with molecular detection through Nested PCR proved the best method for discovering Paracoccidioides spp. in the environment. Most of the ITS sequences obtained in this investigation proved to be highly similar with the homologous sequences of Paracoccidioides lutzii from the GenBank database, suggesting that this Paracoccidioides species may not be exclusive to mid-western Brazil as proposed so far. © 2013 ISHAM.