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1.
Neotrop. ichthyol ; 19(2): e200045, 2021. graf
Article in English | LILACS, VETINDEX | ID: biblio-1279481

ABSTRACT

Characidium sp. aff. C. vidali is a species found in coastal streams in southeastern Brazil, which has karyotypic explanatory elements as the occurrence of microstructural variations, keeping the chromosomal macrostructure of the genus. The objective of this study was to apply cytomolecular tools in the chromosomes of Characidium sp. aff. C. vidali to identify characteristics in their karyotype contributing to cytogenetic definition of this species, adding information about the evolution of the chromosomal structure of the group. The species showed 2n = 50 chromosomes and from 1 to 4 additional B microchromosomes. FISH technique showed histone H3 and H4 genes in the short arm of pair 10, and microsatellites (CA)15, (CG)15, (GA)15 and (TTA)10 clustered in the subtelomeric portions of all A chromosomes, with total accumulation by supernumerary. The telomeric probe marked terminal regions of all chromosomes, in addition to the interstitial portion of four pairs, called ITS sites, with these markings being duplicated in two pairs, hence the double-ITS classification. C-banding revealed that supernumerary chromosomes are completely heterochromatic, that ITS sites are C-banding positive, but double-ITS sites are C-banding negative. So, throughout the evolution to Characidium, genomic events are occurring and restructuring chromosomes in populations.(AU)


Characidium sp. aff. C. vidali é uma espécie encontrada em riachos costeiros do sudeste do Brasil, que apresenta elementos cariotípicos elucidativos quanto à ocorrência de variações microestruturais, conservando a macroestrutura cromossômica do gênero. O objetivo deste estudo foi aplicar ferramentas citomoleculares para identificar características no cariótipo de Characidium sp. aff. C. vidali, que contribuam para a definição citogenética desta espécie, agregando informações quanto à evolução da estruturação cromossômica do grupo. A espécie apresentou 2n = 50 cromossomos, além de 1 a 4 microcromossomos B por célula. A FISH mostrou os genes de histona H3 e H4 sintênicos no braço curto do par 10, e os microssatélites (CA)15, (CG)15, (GA)15 e (TTA)10 clusterizados nas porções subteloméricas de todos os cromossomos do complemento A, com grande acúmulo nos supranumerários. A sonda telomérica identificou marcações terminais em todos os cromossomos, além de quatro pares marcados intersticialmente, chamados de sítios ITS, e dois pares com duas marcações intersticiais, chamados de double-ITS. O bandamento C revelou que os cromossomos supranumerários são completamente heterocromáticos, que os sítios ITS são banda C positivos, mas os sítios double-ITS são banda C negativos. Então, ao longo da evolução de Characidium, eventos genômicos estão ocorrendo e reestruturando cromossomos nas populações.(AU)


Subject(s)
Animals , Biomarkers/analysis , Cytogenetics , Characiformes/genetics , DNA Probes
2.
Chinese Journal of Biotechnology ; (12): 3323-3333, 2021.
Article in Chinese | WPRIM | ID: wpr-921428

ABSTRACT

Vibrio splendidus is an opportunistic pathogen in aquaculture. It can infect a variety of aquaculture animals and has caused huge losses to the aquaculture industry. In this study, a novel and efficient method for detecting V. splendidus was developed by combining the exonuclease Ⅲ amplification strategy with a nucleic acid test strip developed based on gold nanoparticles-labeled DNA probe. The results could be directly visualized by naked eyes, and this system overcame the difficulty in preparation of the monoclonal antibody used in conventional immunostrip. Upon optimization of experimental conditions, the detection limit of the strip was 5 ng/mL for the synthetic oligonucleotide DNA fragment and 10 ng/mL for the actual genomic DNA sample of V. splendidus. This test strip was more sensitive compared with the PCR method and was specific for the detection of V. splendidus. The rapid preparation of nucleic acid strip and the efficient detection of V. splendidus open a new way for the prevention and control of aquatic diseases.


Subject(s)
Animals , DNA Probes , Gold , Metal Nanoparticles , Vibrio/genetics
3.
J. appl. oral sci ; 28: e20190266, 2020. graf
Article in English | LILACS, BBO | ID: biblio-1056586

ABSTRACT

Abstract Objective: The microbial composition of pericoronitis (Pc) is still controversial; it is not yet clear if the microbial profile of these lesions is similar to the profile observed in periodontitis (Pd). Therefore, the aim of the present study was to describe the microbial profile of Pc lesions and compare it directly with that of subjects with Pd. Methodology: Subjects with Pc and Pd were selected, and subgingival biofilm samples were collected from (i) third molars with symptomatic Pc (Pc-T), (ii) contralateral third molars without Pc (Pc-C) and (iii) teeth with a probing depth >3 mm from subjects with Pd. Counts and proportions of 40 bacterial species were evaluated using a checkerboard DNA-DNA hybridization technique. Results: Twenty-six patients with Pc and 18 with Pd were included in the study. In general, higher levels of microorganisms were observed in Pd. Only Actinomyces oris and Eubacterium nodatum were present in higher mean counts in the Pc-T group in comparison with the Pc-C and Pd-C groups (p<0.05). The microbiota associated with Pc-T was similar to that found in Pc-C. Sites with Pc lesions had lower proportions of red complex in comparison with the Pd sites. Conclusion: The microbiota of Pc is very diverse, but these lesions harbour lower levels of periodontal pathogens than Pd.


Subject(s)
Humans , Male , Female , Adult , Middle Aged , Aged , Pericoronitis/microbiology , Periodontitis/microbiology , Bacteria/isolation & purification , Reference Values , Activation Analysis , DNA Probes , Cross-Sectional Studies , Biofilms , Bacterial Load , Gingiva/microbiology
4.
J. appl. oral sci ; 28: e20190100, 2020. tab, graf
Article in English | LILACS, BBO | ID: biblio-1056594

ABSTRACT

Abstract Objective: This clinical study sought to evaluate the effectiveness of passive ultrasonic activation (PUA) in eliminating microorganisms in primary endodontic infection (PEI) after instrumentation of root canals using microbiological culture and checkerboard DNA-DNA hybridization. Methodology: Twenty root canals with PEI and apical periodontitis were selected. The root canals were instrumented and then randomly divided into 2 groups, according to the irrigation method: PUA and conventional needle irrigation (CNI). Microbiological samples were collected before instrumentation (S1), after instrumentation (S2) and after irrigation with 17% EDTA (S3). The samples were subjected to anaerobic culture technique and checkerboard DNA-DNA hybridization analysis. Results: A statistically significant difference was found between CNI (23.56%) and PUA (98.37%) regarding the median percentage values for culturable bacteria reduction (p<0.05). In the initial samples, the most frequently detected species was S. constellatus (50%), and after root canal treatment was E. faecalis (50%). Conclusion: Both treatments significantly decreased the number of bacterial species compared with the initial sample. However, no statistical difference in the total microbial load between PUA and CNI groups was detected. The number of cultivable anaerobic bacteria reduced significantly using PUA, and the bacterial composition and number of bacterial species after using either CNI or PUA was similar.


Subject(s)
Humans , Male , Female , Adolescent , Adult , Middle Aged , Periapical Periodontitis/therapy , Root Canal Therapy/instrumentation , Ultrasonic Therapy/instrumentation , Dental Pulp Cavity/microbiology , Root Canal Irrigants/therapeutic use , Root Canal Therapy/methods , Sodium Hypochlorite/therapeutic use , Bacteria/isolation & purification , Ultrasonic Therapy/methods , Colony Count, Microbial , DNA Probes , Linear Models , Analysis of Variance , Treatment Outcome , Cone-Beam Computed Tomography , Bacterial Load , Therapeutic Irrigation/instrumentation , Therapeutic Irrigation/methods
5.
J. appl. oral sci ; 27: e20180426, 2019. tab, graf
Article in English | LILACS, BBO | ID: biblio-1002406

ABSTRACT

Abstract Objectives Enamel demineralization is among the main topics of interest in the orthodontic field. Self-ligating brackets have been regarded as advantageous in this aspect. The aim of this study was to evaluate the break homeostasis in the oral environment and the levels of microorganisms associated with dental caries among the different types of brackets. Material and Methods Twenty patients received two self-ligating brackets: In-Ovation®R, SmartClipTM, and one conventional GeminiTM. Saliva was collected before bonding (S0), 30 (S1) and 60 (S2) days after bonding. One sample of each bracket was removed at 30 and 60 days for the in situ analysis. Checkerboard DNA-DNA Hybridization was employed to evaluate the levels of microbial species as-sociated with dental caries. Data were evaluated by nonparametric Friedman and Wilcoxon tests at 5% significance level. Results The salivary levels of L. casei (p=0.033), S. sobrinus (p=0.011), and S. sanguinis (p=0.004) increased in S1. The in situ analyses showed alteration in S. mutans (p=0.047), whose highest levels were observed to the In-Ovation®R. Conclusions The orthodontic appliances break the salivary homeostasis of microorganisms involved in dental caries. The contamination pattern was different between self-ligating and conventional brackets. The In-Ovation®R presented worse performance considering the levels of cariogenic bacterial species.


Subject(s)
Humans , Male , Female , Child , Adolescent , Saliva/microbiology , Orthodontic Brackets/microbiology , Dental Caries/microbiology , Time Factors , DNA Probes , Dental Bonding , Orthodontic Brackets/standards , Orthodontic Appliance Design , Statistics, Nonparametric , Homeostasis
6.
Braz. oral res. (Online) ; 33(supl.1): e065, 2019. tab, graf
Article in English | LILACS | ID: biblio-1039317

ABSTRACT

Abstract Additive manufacturing (AM) is an emerging process for biomaterials and medical devices. Direct Laser Metal Sintering (DLMS) is an AM technique used to fabricate Ti-6Al-4V implant materials with enhanced surface-related properties compared with wrought samples; thus, this technique could influence microbial adsorption and colonization. Therefore, this in vitro study was conducted to evaluate the impact of different implant production processes on microbial adhesion of periodontal pathogens. Titanium discs produced using two different processes—conventional and AM—were divided into three groups: conventional titanium discs with machined surface (G1), AM titanium discs with chemical treatment (G2) and AM titanium discs without chemical treatment (G3). Subgingival biofilm composed of 32 species was formed on the titanium discs, and positioned vertically in 96-well plates, for 7 days. The proportions of microbial complexes and the microbial profiles were analyzed using a DNA-DNA hybridization technique, and data were evaluated using Kruskal-Wallis and Dunnett tests (p < 0.05). Lower proportions of the red complex species were observed in the biofilm formed in G2 compared with that in G1 (p < 0.05). Moreover, the proportions of the microbial complexes were similar between G2 and G3 (p > 0.05). Compared with G1, G2 showed reduced levels of Porphyromonas gingvalis , Actinomyces gerencseriae, and Streptococcus intermedius , and increased levels of Parvimonas micra , Actinomyces odontolyticus, and Eikenella corrodens (p < 0.05). The microbial profile of G3 did not differ from G1 and G2 (p > 0.05). The results of this in vitro study showed that titanium discs produced via AM could alter the microbial profile of the biofilm formed around them. Further clinical studies should be conducted to confirm these findings.


Subject(s)
Titanium/pharmacology , Titanium/chemistry , Biofilms/growth & development , Reference Values , Surface Properties , Time Factors , Bacteria/drug effects , Microscopy, Electron, Scanning , DNA Probes , Reproducibility of Results , Analysis of Variance , Statistics, Nonparametric , Microscopy, Atomic Force , Biofilms/drug effects , Photoelectron Spectroscopy
7.
An. acad. bras. ciênc ; 90(1): 41-47, Mar. 2018. graf
Article in English | LILACS | ID: biblio-886917

ABSTRACT

ABSTRACT Chromosome-specific probes have been widely used in molecular cytogenetics, being obtained with different methods. In this study, a reproducible protocol for construction of chromosome-specific probes is proposed which associates in situ amplification (PRINS), micromanipulation and degenerate oligonucleotide-primed PCR (DOP-PCR). Human lymphocyte cultures were used to obtain metaphases from male and female individuals. The chromosomes were amplified via PRINS, and subcentromeric fragments of the X chromosome were microdissected using microneedles coupled to a phase contrast microscope. The fragments were amplified by DOP-PCR and labeled with tetramethyl-rhodamine-5-dUTP. The probes were used in fluorescent in situ hybridization (FISH) procedure to highlight these specific regions in the metaphases. The results show one fluorescent red spot in male and two in female X chromosomes and interphase nuclei.


Subject(s)
Humans , Polymerase Chain Reaction/methods , DNA Primers/genetics , Primed In Situ Labeling/methods , Cytogenetic Analysis/methods , DNA Probes/genetics , Reproducibility of Results , In Situ Hybridization, Fluorescence/methods , Chromosomes, Human, X/genetics , Microdissection/methods
8.
Article in Chinese | WPRIM | ID: wpr-775380

ABSTRACT

In this study, the specific primers and probes of Panax quinquefolius were designed for a quantitative real-time PCR, and the rapid identification method of P. quinquefolius was established by optimizing conditions. The method was used to validate 43 samples of the traditional Chinese medicine,and the results showed that 22 samples of P. quinquefolius were identified accurately. The limit of detection of the method can be reach to 1×10⁻⁴ ng. The method is accurate, fast, sensitive and specifically.


Subject(s)
DNA Primers , DNA Probes , Drugs, Chinese Herbal , Reference Standards , Panax , Genetics , Real-Time Polymerase Chain Reaction
9.
Arq. bras. oftalmol ; 80(2): 84-87, Mar.-Apr. 2017. tab
Article in English | LILACS | ID: biblio-838786

ABSTRACT

ABSTRACT Objective: Bacterial keratitis occurs worldwide, and despite recent developments, it remains a potentially blinding condition. This study assesses the presence of herpes simplex virus (HSV-1 and -2) and varicella zoster virus (VZV) by quantitative real-time polymerase chain reaction (qPCR) in corneal scrapings from patients with bacterial keratitis. Methods: A total of 65 patients with clinical diagnoses of infectious corneal ulcers prospectively underwent clinical eye examinations. Corneal scrapings were investigated by Gram staining, Giemsa staining, culture, and qPCR (the study group). Risk factors and epidemiological data were recorded. The control group comprising 25 eyes with typical herpes dendritic keratitis was also analyzed by qPCR. Results: From the study group (n=65), nine patients (13.8%) had negative smears, cultures, and qPCR findings. Fifty-six (86.2%) patients had positive cultures: 51 for bacteria, 4 for fungi, and 1 for amoebae. Of the patients who had positive bacterial cultures, qPCR identified 10 patients who were also positive for virus: one for VZV and nine for HSV-1. Of the 25 patients in the control group, 21 tested positive for HSV-1 by qPCR analysis. Conclusions: Herpes may be present in patients with bacterial corneal ulcers, and qPCR may be useful in its detection.


RESUMO Objetivo: Ceratites bacterianas ocorrem mundialmente e apesar dos novos desenvolvimentos permanece como uma condição que pode levar à cegueira. Avaliar a presença de herpes simples (-1 e -2) e vírus varicella zoster (VZV) por reação em cadeia quantitativa de polimerase em tempo real (qPCR) em raspados corneanos de pacientes com ceratite bacteriana. Métodos: Sessenta e cinco pacientes com ceratite infecciosa foram submetidos a raspados corneanos estudados para gram, Giemsa, cultura e qPCR (grupo de estudo). Foram avaliados fatores de risco e epidemiológicos. O grupo controle foi composto por 25 casos de úlcera dendrítica típica por herpes analisados por qPCR. Resultados: Do grupo de estudo (n=65), nove pacientes (13,8%) apresentaram cultura, qPCR e raspado negativos. Cinquenta e seis (86,2%) pacientes apresentaram cultura positiva, 51 para bacteria, 4 para fungo e 1 para ameba. A qPCR identificou 10 pacientes do grupo de cultura positiva para bactéria que também foram positivos para vírus, um VZV e 9 para HSV-1. Dos 25 pacientes que compunham o grupo controle, 21 apresentaram qPCR positivo para HSV-1. Conclusão: Herpes pode estar presente em pacientes com úlceras de córnea bacterianas e a qPCR pode ser útil na sua detecção.


Subject(s)
Humans , Male , Female , Child , Adolescent , Adult , Middle Aged , Aged , Aged, 80 and over , Young Adult , Keratitis, Dendritic/microbiology , Herpesvirus 2, Human/isolation & purification , Herpesvirus 1, Human/isolation & purification , Herpesvirus 3, Human/isolation & purification , Cornea/virology , Real-Time Polymerase Chain Reaction/methods , Keratitis/microbiology , DNA Probes , Eye Infections, Bacterial/microbiology , Keratitis, Dendritic/diagnosis , Keratitis, Dendritic/virology , Prospective Studies , Keratitis/diagnosis , Keratitis/virology
10.
Braz. oral res. (Online) ; 31: e21, 2017. tab, graf
Article in English | LILACS | ID: biblio-839522

ABSTRACT

Abstract The aim of this randomized, single blinded clinical trial was to evaluate the effect of a pre-procedural mouthwash containing cetylpyridinium chloride (CPC), zinc lactate (Zn) and sodium fluoride (F) in the reduction of viable bacteria in oral aerosol after a dental prophylaxis with ultrasonic scaler. Sixty systemically healthy volunteers receiving dental prophylaxis were randomly assigned to one of the following experimental groups (15 per group): (i) rinsing with 0.075% CPC, 0.28% Zn and 0.05% F (CPC+Zn+F), (ii) water or (iii) 0.12% chlorhexidine digluconate (CHX), and (iv) no rinsing. Viable bacteria were collected from different locations in the dental office on enriched TSA plates and anaerobically incubated for 72 hours. The colonies were counted and species were then identified by Checkerboard DNA–DNA Hybridization. The total number of colony-forming units (CFUs) detected in the aerosols from volunteers who rinsed with CPC+Zn+F or CHX was statistically significantly (p<0.05) lower than of those subjects who did not rinse or who rinsed with water. When all locations were considered together, the aerosols from the CPC+Zn+F and CHX groups showed, respectively, 70% and 77% fewer CFUs than those from the No Rinsing group and 61% and 70% than those from the Water group. The mean proportions of bacterial species from the orange complex were statistically significantly (p<0.05) lower in aerosols from the CPC+Zn+F and CHX groups compared with the others two groups. In conclusion, the mouthwash containing CPC+Zn+F, is effective in reducing viable bacteria in oral aerosol after a dental prophylaxis with ultrasonic scaler.


Subject(s)
Humans , Male , Female , Adult , Middle Aged , Young Adult , Aerosols , Anti-Infective Agents, Local/therapeutic use , Bacteria/drug effects , Mouthwashes/therapeutic use , Mouth/microbiology , Cetylpyridinium/therapeutic use , Colony Count, Microbial , DNA Probes , DNA, Bacterial , Lactates/therapeutic use , Mouthwashes/chemistry , Reproducibility of Results , Single-Blind Method , Sodium Fluoride/therapeutic use , Statistics, Nonparametric , Time Factors , Treatment Outcome , Zinc/therapeutic use
11.
J. appl. oral sci ; 24(2): 181-185, Mar.-Apr. 2016. tab, graf
Article in English | LILACS | ID: lil-779905

ABSTRACT

ABSTRACT Objective The aim of this study was to evaluate the association of Porphyromonas endodontalis, Filifactor alocis and Dialister pneumosintes with the occurrence of periodontitis. Material and Methods Thirty subjects with chronic periodontitis (ChP) and 10 with periodontal health (PH) were included in the study. Nine subgingival biofilm samples were collected as follows: i) PH group - from the mesial/buccal aspect of each tooth in two randomly chosen contralateral quadrants; ii) ChP group - from three sites in each of the following probing depth (PD) categories: shallow (≤3 mm), moderate (4-6 mm) and deep (≥7 mm). Checkerboard DNA-DNA hybridization was used to analyze the samples. Results We found the three species evaluated in a higher percentage of sites and at higher levels in the group with ChP than in the PH group (p<0.05, Mann-Whitney test). We also observed these differences when the samples from sites with PD≤4 mm or ≥5 mm of subjects with ChP were compared with those from subjects with PH (p<0.05, Mann-Whitney test). In addition, the prevalence and levels of D. pneumosintes, and especially of F. alocis were very low in healthy subjects (0.12x105 and 0.01x105, respectively). Conclusion F. alocis and D. pneumosintes might be associated with the etiology of ChP, and their role in the onset and progression of this infection should be further investigated. The role of P. endodontalis was less evident, since this species was found in relatively high levels and prevalence in the PH group.


Subject(s)
Humans , Male , Female , Adult , Middle Aged , Peptostreptococcus/pathogenicity , Porphyromonas endodontalis/pathogenicity , Veillonellaceae/pathogenicity , Chronic Periodontitis/microbiology , Peptostreptococcus/isolation & purification , Brazil , DNA, Bacterial , Colony Count, Microbial , DNA Probes , Case-Control Studies , Statistics, Nonparametric , Biofilms , Porphyromonas endodontalis/isolation & purification , Dental Plaque/microbiology , Veillonellaceae/isolation & purification , Gingiva/microbiology
12.
Article in English | WPRIM | ID: wpr-59846

ABSTRACT

BACKGROUND: Intrachromosomal amplification of chromosome 21 (iAMP21) is known to be associated with poor prognosis in B-cell ALL (B-ALL). To determine the frequency and clinical characteristics of iAMP21 in Korean B-ALL patients, we performed FISH and multiplex ligation-dependent probe amplification (MLPA) analyses. METHODS: A total of 102 childhood B-ALL patients were screened with ETV6-RUNX1 FISH probes (Abbott Molecular, USA). The presence of an iAMP21 was confirmed by using MLPA P327 iAMP21-ERG probemix (MRC Holland, The Netherlands). RESULTS: iAMP21 was detected in one of the screened B-ALL patients (1/102 patients, 1.0%) who presented the ALL immunophenotype and complex karyotype at initial diagnosis. The patient relapsed twice after bone marrow transplantation. MLPA showed 12.5-Mb and 4.28-Mb regions of amplification and deletion, respectively. CONCLUSIONS: The frequency of iAMP21 is considerable in Korean pediatric patients. Our report suggests that iAMP21 in childhood B-ALL has very unfavorable impact on patient's prognosis. Additional methods such as MLPA analysis is essential to rule out patients with equivocal interphase FISH results.


Subject(s)
Adolescent , Asians/genetics , B-Lymphocytes/metabolism , Child , Child, Preschool , Chromosomes, Human, Pair 21 , Core Binding Factor Alpha 2 Subunit/genetics , DNA Probes/metabolism , Female , Humans , Immunophenotyping , In Situ Hybridization, Fluorescence , Infant , Infant, Newborn , Male , Multiplex Polymerase Chain Reaction , Precursor Cell Lymphoblastic Leukemia-Lymphoma/diagnosis , Proto-Oncogene Proteins c-ets/genetics , Repressor Proteins/genetics , Republic of Korea , Translocation, Genetic , Young Adult
13.
Article in English | IMSEAR | ID: sea-158446

ABSTRACT

Background & objectives: Wilson’s disease (WD) is an autosomal recessive disorder caused by mutations in ATP7B, a copper transporter gene, leading to hepatic and neuropsychiatric manifestations due to copper accumulation. If diagnosed early, WD patients can be managed by medicines reducing morbidity and mortality. Diagnosis of this disease requires a combination of tests and at times is inconclusive due to overlap of the symptoms with other disorders. Genetic testing is the preferred alternative in such cases particularly for individuals with a family history. Use of DNA microarray for detecting mutations in ATP7B gene is gaining popularity because of the advantages it offers in terms of throughput and sensitivity. This study attempts to establish the quality analysis procedures for microarray based diagnosis of Wilson’s disease. Methods: A home-made microarrayer was used to print oligonucleotide based low-density microarrays for addressing 62 mutations causing Wilson’s disease reported from Indian population. Inter- and intra- array comparisons were used to study quality of the arrays. The arrays were validated by using mutant samples generated by site directed mutagenesis. Results: The hybridization reaction were found to be consistent across the surface of a given microarray. Our results have shown that 52 °C post-hybridization wash yields better reproducibility across experiments compared to 42 °C. Our arrays have shown > 80 per cent sensitivity in detecting these 62 mutations. Interpretation & conclusions: The present results demonstrate the design and evaluation of a low-density microarray for the detection of 62 mutations in ATP7B gene, and show that a microarray based approach can be cost-effective for detecting a large number of mutations simultaneously. This study also provides information on some of the important parameters required for microarray based diagnosis of genetic disorders.


Subject(s)
DNA Probes , Hepatolenticular Degeneration/diagnosis , Hepatolenticular Degeneration/etiology , Hepatolenticular Degeneration/genetics , Humans , Mutation , Oligonucleotide Array Sequence Analysis
14.
Article in English | WPRIM | ID: wpr-188227

ABSTRACT

BACKGROUND: The conventional method for decalcification of bone specimens uses hydrochloric acid (HCl) and is notorious for damaging cellular RNA, DNA, and proteins, thus complicating molecular and immunohistochemical analyses. A method that can effectively decalcify while preserving genetic material is necessary. METHODS: Pairs of bilateral bone marrow biopsies sampled from 53 patients were decalcified according to protocols of two comparison groups: EDTA versus HCl and RDO GOLD (RDO) versus HCl. Pairs of right and left bone marrow biopsy samples harvested from 28 cases were allocated into the EDTA versus HCl comparison group, and 25 cases to the RDO versus HCl comparison group. The decalcification protocols were compared with regards to histomorphology, immunohistochemistry, and molecular analysis. For molecular analysis, we randomly selected 5 cases from the EDTA versus HCl and RDO versus HCl groups. RESULTS: The decalcification time for appropriate histomorphologic analysis was the longest in the EDTA method and the shortest in the RDO method. EDTA was superior to RDO or HCl in DNA yield and integrity, assessed via DNA extraction, polymerase chain reaction, and silver in situ hybridization using DNA probes. The EDTA method maintained intact nuclear protein staining on immunohistochemistry, while the HCl method produced poor quality images. Staining after the RDO method had equivocal results. RNA in situ hybridization using kappa and lambda RNA probes measured RNA integrity; the EDTA and RDO method had the best quality, followed by HCl. CONCLUSIONS: The EDTA protocol would be the best in preserving genetic material. RDO may be an acceptable alternative when rapid decalcification is necessary.


Subject(s)
Biopsy , Bone Marrow , Decalcification Technique , DNA , DNA Probes , Edetic Acid , Humans , Hydrochloric Acid , Immunohistochemistry , In Situ Hybridization , Nuclear Proteins , Polymerase Chain Reaction , RNA , RNA Probes , Silver
15.
Article in English | IMSEAR | ID: sea-154552

ABSTRACT

The diagnosis of any pathology is fundamentally based on the microscopic structure of cells and tissues and this remains as the standard by which all other diagnostic tests are measured. In this era, the pathologists are relying on the examination of tissue section stained by histochemical means and it is supported by the advanced immunological, biochemical and molecular techniques. This review will provide the information about one of the way that can be followed to unravel the molecular mechanism in spotting the disease process. Technologies used to study the cellular process are same for the normal and the abnormal cell. Experimental strategy briefed here is also applicable for both. The cellular process can be studied either from protein to gene or from gene to protein. Earlier days biochemical analysis (isolation of protein, protein sequencing) was separate and genetic analysis (genomic mapping) was separate. But now with advent of recombinant DNA technology it is possible to have a link between the biochemical and genetic analysis. Intermediary step of development of oligonucleotide synthesis, complementary DNA probe and cloning has revolutionized the research process. Identified gene can be compared with the normal gene by comparative genomics or expressed proteins by expression proteomics.


Subject(s)
DNA Probes/genetics , Gene Expression Profiling , Genes/genetics , Genetic Variation/analysis , Proteins/genetics , Review Literature as Topic
16.
São Paulo; s.n; 2014. 88 p. ilus, tab, graf.
Thesis in Portuguese | LILACS | ID: lil-750092

ABSTRACT

Introdução O diagnóstico de derrame pleural maligno (DPM) se baseia no achado de células tumorais no líquido ou no tecido pleural. Resultados falsos positivos ou falsos negativos influenciam na escolha da melhor conduta terapêutica a ser tomada, além de alterar substancialmente o prognóstico desses pacientes. A sensibilidade do exame citológico é geralmente inferior a 70%, motivo pelo qual, métodos complementares são frequentemente associados. Fatores como tipo histológico, sítio primário e grau de invasibilidade do tumor são os principais responsáveis por esta variação. Dentre os exames complementares propostos, destacam-se a dosagem de marcadores tumorais no líquido pleural (LP), as técnicas citoquímicas, imunocitoquímicas e de marcadores de proliferação celular em células do LP, a análise da ploidia de DNA por citometria de fluxo (CF) ou estática (CE) e, mais recentemente, as técnicas de citogenética e de biologia molecular, como a técnica de hibridação in situ por fluorescência (FISH) e a técnica de amplificação multiplex por sondas ligação - dependentes (MLPA) estas, capazes de detectar alterações em regiões gênicas consideradas "alvo" para o desfecho neoplásico. Objetivos 1) Padronizar as técnicas de DNA ploidia, FISH e MLPA em amostras frescas de líquido pleural; 2) Testar a eficiência diagnóstica dos métodos da DNA ploidia e da FISH no diagnóstico de derrame pleural maligno e 3) Avaliar alterações no número de cópias no gene EGFR em metástases pleurais utilizando a técnica de MLPA. Métodos Foram incluídos 200 pacientes adultos portadores de derrame pleural (DP) com indicação de toracocentese. O diagnóstico histológico foi o padrão ouro para malignidade. Características clínicas, radiológicas, histológicas e de seguimento clínico foram considerados para a exclusão de malignidade, de maneira que 130 casos foram classificados como malignos e 70 como benignos. As 200 amostras de LP foram submetidas ao exame citológico e à FISH utilizando sondas...


Introduction The diagnosis of malignant pleural effusion (MPE) is based on the finding of tumor cells in the pleural fluid or tissue. False positive or false negative results influence the choice of the best therapeutic approach to be used with these patients and substantially change their prognosis. The sensitivity of the cytology is generally lesser than 70%, for which complementary methods are often associated. Factors such as tumor histological type, staging, primary site and potential of invasiveness are responsible for this variation. Among the proposed ancillary tests, we highlight the dosage of tumor markers in pleural fluid (PF), the cytochemical and immunocytochemical techniques, including markers of cell proliferation, DNA ploidy analysis by flow cytometry (FC) or static cytometry (EC) and more recently, the cytogenetics and molecular techniques, as the fluorescence in situ hybridization (FISH) and the multiplex ligation - dependent probe amplification (MLPA), capable of detecting changes in gene regions considered "target" for the neoplastic outcome. Objectives 1) To standardize the techniques of DNA ploidy, FISH and MLPA in fresh samples of pleural fluid; 2) To test the diagnosis efficiency of DNA ploidy and FISH in the diagnosis of malignant pleural effusion and 3) To evaluate changes in the copy number of the EGFR gene by using the MLPA technique in cases of pleural metastases. Methods We included 200 adult patients with pleural effusion and clinical indication for thoracentesis. The histological diagnosis was considered the gold standard for malignancy. Clinical follow-up, radiological and histological characteristics were considered for exclusion of malignancy, which ranked de cases as 130 malignant effusions and 70 as benign ones. All cases were submitted to cytology and FISH using centromeric probes for the chromosomes 11 and 17. Analysis of DNA ploidy by FC was performed in 45 cases and the MLPA for epidermal...


Subject(s)
Humans , Male , Female , Cytogenetic Analysis/methods , Cytodiagnosis , DNA Copy Number Variations , DNA Probes , Flow Cytometry , Genes, erbB-1 , In Situ Hybridization, Fluorescence , Body Fluids/cytology , Pleural Effusion, Malignant
17.
Rio de Janeiro; s.n; s.n; 2014. 83 p. ilus.
Thesis in Portuguese | LILACS, BBO | ID: biblio-907144

ABSTRACT

O presente trabalho tem por objetivo investigar a microbiota de canais radiculares, buscando a identificação e a quantificação destes microrganismos. Foram selecionados 31 dentes com infecção primária devido a traumatismo dentário. As amostras microbiológicas foram coletadas dos canais com o auxílio de limas tipo Hedströen e cones de papel absorvente estéril. A técnica do Checkerboard DNA-DNA hybridization foi utilizada para detecção de até 38 espécies bacterianas em cada amostra, utilizando sondas de DNA específicas. Os dados microbiológicos foram expressos em percentagem média (prevalência), nível médio (contagem) e proporção de cada espécie em cada amostra. Os testes t independente e de correlação de Pearson foram usados para correlacionar as bactérias testadas com os tipos de trauma (p≤ 0,05). Foi encontrada uma média de 13,74 espécies por amostra. As espécies mais prevalentes foram P. melaninogenica (84%), E. faecalis (77%), C. gracilis (71%) e F. nucleatum sp. vicentii (71%). Algumas espécies demonstraram baixa prevalência, sendo elas A. odontolyticus (26%), P. acnes (26%), E. corrodens (23%), A. israelii (16%), A. gerencseriae (16%), P. endodontalis (16%) e A. naeslundii (13%). As espécies F. nucleatum sp. vicentii, P. nigrescens, T. denticola, C. gingivalis, C. rectus e P. gingivalis apresentaram níveis médios significativamente maiores entre os casos de trauma dentário e trauma de tecidos de suporte (P < 0,05). As bactérias T. denticola, C. gingivalis e P. gingivalis também apresentaram proporções significativamente mais elevados no casos de trauma de tecidos de suporte (p>0,05). Baseado nos resultados obtidos é possível concluir que o perfil da microbiota presente em dentes com periodontite apical primária causada por traumatismos dentários pode variar de acordo com a ocorrência de dano aos tecidos de suporte.


This study aims to investigate the microbiota of root canals, seeking to identify and quantify these microorganisms. Thirty one teeth with related primary infection due to dental trauma were selected. Microbiological samples were collected from the root canal using Hedströen files and sterile absorbent paper points. The checkerboard DNA-DNA hybridization molecular technique was used to detect up to 38 bacterial species in each sample using specific DNA probes. Microbiological data were expressed as mean percentage (prevalence), mean level (score) and the proportion of each species in each sample. The independent and Pearson correlation tests were used to correlate the bacterias tested with the types of trauma (p ≤ 0.05). An average of 13.74 species per sample was found. The most prevalent species were P. melaninogenica (84%), E. faecalis (77%), C. gracilis (71%) and F. nucleatum sp. vicentii (71%). The species that showed low prevalence were A. odontolyticus (26%), P. acnes (26%), E. corrodens (23%), A. israelii (16%), A. gerencseriae (16%), P. endodontalis (16%) and A. naeslundii (13%). The species F. nucleatum sp. vicentii, P. nigrescens, T. denticola, C. gingivalis, C. rectus and P. gingivalis showed significantly higher mean levels between cases of trauma to the supporting tissues and cases of dental trauma (P <0.05). T. denticola, P. gingivalis and C. gingivalis also showed significant higher ratios in the trauma to the supporting tissues cases (p> 0.05). Based on the obtained results, it can be concluded that the bacterial profile of pulpal necrosis caused by traumactic injuries may vary depending on the occurrence of damage to the supporting tissues.


Subject(s)
Humans , Male , Female , Child , Adolescent , Adult , Middle Aged , Aged , Aged, 80 and over , Endodontics , Infections/microbiology , Microbiota , Periapical Periodontitis/microbiology , Tooth Injuries/complications , Dental Pulp Cavity/microbiology , DNA Probes , Periapical Periodontitis/etiology , Root Canal Therapy
18.
Genomics & Informatics ; : 289-291, 2013.
Article in English | WPRIM | ID: wpr-84014

ABSTRACT

Human papillomavirus (HPV) infection is the leading cause of cancer mortality among women worldwide. The molecular understanding of HPV proteins has significant connotation for understanding their intrusion in the host and designing novel protein vaccines and anti-viral agents, etc. Genomic, proteomic, structural, and disease-related information on HPV is available on the web; yet, with trivial annotations and more so, it is not well customized for data analysis, host-pathogen interaction, strain-disease association, drug designing, and sequence analysis, etc. We attempted to design an online reserve with comprehensive information on HPV for the end users desiring the same. The Human Papillomavirus Proteome Database (hpvPDB) domiciles proteomic and genomic information on 150 HPV strains sequenced to date. Simultaneous easy expandability and retrieval of the strain-specific data, with a provision for sequence analysis and exploration potential of predicted structures, and easy access for curation and annotation through a range of search options at one platform are a few of its important features. Affluent information in this reserve could be of help for researchers involved in structural virology, cancer research, drug discovery, and vaccine design.


Subject(s)
DNA Probes , Drug Design , Drug Discovery , Female , Genome , Host-Pathogen Interactions , Humans , Mortality , Proteome , Residence Characteristics , Sequence Analysis , Statistics as Topic , Vaccines , Virology
19.
Article in Korean | WPRIM | ID: wpr-184777

ABSTRACT

The objective of this study was to develop PCR primers that are specific for Streptococcus sanguinis, Streptococcus parasanguinis, and Streptococcus gordonii. We designed the S. sanguinis-, S. parasanguinis-, and S. gordonii-specific primers, Ssa21-F3/Ssa21-R2, Spa17-F/Spa17-R, and Sgo41-F1/Sgo41-R1 respectively, based on the nucleotide sequences of the Ssa21, Spa17, and Sgo41 DNA probes that were screened using inverted dot blot hybridization (IDBH). The species-specificity of these primers was assessed against 43 strains of mitis group streptococci, including clinical strains of S. sanguinis, S. parasanguinis, and S. gordonii. The resulting PCR data revealed that species-specific amplicons had been obtained from all strains of the target species tested, and that none of these amplicons occurred in any other strains from other species. These results suggest that the Ssa21-F3/Ssa21-R2, Spa17-F/Spa17-R, and Sgo41-F1/Sgo41-R1 primers may be useful in detecting S. sanguinis, S. parasanguinis, and S. gordonii at the species level, respectively.


Subject(s)
Base Sequence , Chimera , DNA , DNA Probes , Polymerase Chain Reaction , Streptococcus , Streptococcus gordonii
20.
Journal of Kerman University of Medical Sciences. 2013; 20 (5): 450-459
in Persian | IMEMR | ID: emr-140977

ABSTRACT

Around the world, cervical cancer is the second most common cancer in women. Today, screening programs have reduced morbidity and mortality rates of this disease. Epidemiological and molecular studies have shown that certain types of the human papillomavirus are carcinogen types and the primary cause of cervical cancer. HPV type 16 and 18 are the most common high-risk types. In this study, frequency of different HPV genotypes in women who referred for a routine visit to an outpatient clinic of Kerman University of Medical Sciences, Iran, has been obtained by DNA probe technique. Our study is a cross-sectional, analytic study on 20000 Pap smear samples over four consecutive years among women in reproductive ages [15-50 years] referred to University centers and private institutions in Kerman, Iran. All samples were collected in the laboratory of Afzalipour, and Bahonar Hospitals, and private institutions. The typical samples of dysplasia and cancer were reviewed by two pathologists and a pathology assistant according to the World Health Organization standards. The samples were examined after DNA extraction and molecular DNA probe technique. 62 cases of 82 Pap smear samples were dysplastic and 20 samples were diagnosed as squamous cell carcinoma [SCC]. Moreover, 20 cases [32.2%] of dysplastic Pap smears and 12 cases [60%] of SCC samples were HPV positive. A total of 32 patients [39%] were positive for HPV. Of all samples only two were genotype 18 [25.6%], one was a mixture of 16 and 31 genotypes, and the remaining were all genotype 16 [93.75%]. In the comparison between dysplasia severity [mild, moderate, and severe] and the HPV status [+ or -], and also the relation between age and status of HPV and the severity of dysplasia no relations were found. However, there was a significant relation between detection [dysplasia, SCC] and the HPV status, and also the relation between age and type of lesion diagnosis. Based on the findings of our study and the Iranian culture, prevalence of HPV infection among women with cervical cancer is less common than in other countries. HPV type 16, which is a carcinogenic genotype, was the predominant genotype


Subject(s)
Humans , Female , Papanicolaou Test , Genotype , Genotyping Techniques , DNA Probes , Uterine Cervical Neoplasms , Cross-Sectional Studies , Carcinoma, Squamous Cell
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