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1.
Praxis (Bern 1994) ; 109(1): 42-46, 2020 Jan.
Artigo em Alemão | MEDLINE | ID: mdl-31910759

RESUMO

CME Rheumatology 20: Parvovirus B19-Induced Tenosynovitis? Abstract. In this case presentation we discuss diagnostic algorithms and differential diagnoses in undifferentiated tenosynovitis. We present a case of a patient with chronic tenosynovitis in the 4th extensor tendon compartment. With unremarkable anamnesis and due to normal laboratory results a «seronegative (RF), ACPA (CCP)-negative tenosynovitis without arthritis¼ had been reported. Diagnostic and therapeutic tenosynovectomy was performed. Histologic processing revealed a positive PCR for parvovirus B19. We discuss articular and extraarticular manifestations. A parvovirus B19-associated manifestation in the musculoskeletal system is usually self-limiting. The therapy should be carried out symptomatically. In our patient there was a marked local finding, so that the complete tenosynovectomy followed by a single steroid injection led to a persistent restitutio ad integrum.


Assuntos
Artrite , Infecções por Parvoviridae , Parvovirus B19 Humano , Tenossinovite , Diagnóstico Diferencial , Humanos , Infecções por Parvoviridae/complicações , Reação em Cadeia da Polimerase , Tenossinovite/virologia
2.
Food Chem ; 305: 125431, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31610425

RESUMO

Recent European regulations require safety assessments of food enzymes (FE) before their commercialization. FE are mainly produced by micro-organisms, whose viable strains nor associated DNA can be present in the final products. Currently, no strategy targeting such impurities exists in enforcement laboratories. Therefore, a generic strategy of first line screening was developed to detect and identify, through PCR amplification and sequencing of the 16S-rRNA gene, the potential presence of FE producing bacteria in FE preparations. First, the specificity was verified using all microbial species reported to produce FE. Second, an in-house database, with 16S reference sequences from bacteria producing FE, was constructed for their fast identification through blast analysis. Third, the sensitivity was assessed on a spiked FE preparation. Finally, the applicability was verified using commercial FE preparations. Using straightforward PCR amplifications, Sanger sequencing and blast analysis, the proposed strategy was demonstrated to be convenient for implementation in enforcement laboratories.


Assuntos
Bactérias/isolamento & purificação , Código de Barras de DNA Taxonômico , RNA Ribossômico 16S/análise , Bactérias/genética , Bactérias/metabolismo , Manipulação de Alimentos , Reação em Cadeia da Polimerase
3.
Sci Total Environ ; 700: 134447, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31677419

RESUMO

Identification of Cryptosporidium oocyst is essential in ensuring water quality fit for human use, consumption, and recreation. This communication proposes the supplemental analysis of substrate-associated biofilms, in particular, freshwater sponges in improving case finding of waterborne-protozoan pathogens (WBPP) in environmental aquatic samples. In this study, a small portion of a mature freshwater sponge under the Genus Spongilla was subjected to microscopic and molecular analysis to identify the presence of Cryptosporidium. Microscopic screening with modified Kinyoun's staining (MK) and microscopic confirmation using direct antibody fluorescent testing (IFT) returned with Cryptosporidium spp. positive findings. Molecular investigation resulted in the confirmation of Cryptosporidium hominis upon sequencing of PCR products and phylogenetic analysis. This is the first report of a pathogenic protozoan, C. hominis isolated from a freshwater sponge. The results of this study provide evidence of the value of expanding water quality assessment strategies to the analysis of substrate-associated biofilms and sponges in improving case finding of WBPP in natural aquatic environments.


Assuntos
Água Doce/microbiologia , Poríferos/parasitologia , Animais , Cryptosporidium/isolamento & purificação , Oocistos , Filogenia , Reação em Cadeia da Polimerase
4.
Biosci Biotechnol Biochem ; 84(1): 43-52, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31495297

RESUMO

To date, studies on the application of loop-mediated isothermal amplification (LAMP) in the detection of genetically modified organisms (GMOs) are stably increasing and demonstrates LAMP is a potential and promising method for on spot identification of GMOs. However, little information is known for detection of GM potato events by LAMP. In this report, we developed an optimized and visual LAMP assay with high specificity and sensitivity to rapidly amplify genomic DNA of potato EH92-527-1 within 45 min. The limit of detection of LAMP in our study is 10-fold higher than the conventional PCR. Furthermore, LAMP products can be directly observed via naked eyes by addition of SYBR Green I without gel electrophoresis analysis and PCR-based equipment. Therefore, the LAMP assay developed in this paper provides an efficient, convenient and cost-effective tool for the detection of GM potato EH92-527-1.


Assuntos
DNA de Plantas/genética , Plantas Geneticamente Modificadas/genética , Reação em Cadeia da Polimerase/instrumentação , Reação em Cadeia da Polimerase/métodos , Solanum tuberosum/genética , Sequência de Bases/genética , Percepção de Cores , Primers do DNA/genética , Enzimas de Restrição do DNA/genética , Eletroforese em Gel de Ágar , Corantes Fluorescentes/química , Contaminação de Alimentos/análise , Amplificação de Genes , Limite de Detecção , Compostos Orgânicos/química , Reação em Cadeia da Polimerase/economia , Sensibilidade e Especificidade , Temperatura Ambiente , Tempo
5.
Quintessence Int ; 51(1): 18-26, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31781689

RESUMO

OBJECTIVE: This study aimed to investigate the Epstein-Barr virus (EBV) prevalence and viral load in subgingival sites of human immunodeficiency virus type 1 (HIV-1) positive (HIV+) individuals, correlating subgingival EBV load to the clinical periodontal condition, HIV systemic load, EBV systemic load, and use of antiretroviral therapy (ART). METHOD AND MATERIALS: Ninety individuals were recruited and divided into three categories: those without periodontal disease (G1), with gingivitis (G2), and with periodontitis (G3). Subgingival biofilm and blood samples were analyzed by quantitative polymerase chain reactions (qPCR). A questionnaire was administered to collect general information about patients, and data regarding HIV and use of ART were accessed from their medical records. RESULTS: EBV was detected in 85.6% of the samples. Comparing subgingival and systemic load of EBV in G1, G2, and G3, there was a statistical difference only in G3 (3.93 log10 copies/mL and 5.47 log10 copies/mL, respectively; P = .014), where EBV load was higher in periodontal pockets than in the blood. All groups had high EBV loads in subgingival sites (> 2,000 copies/mL). A positive linear correlation between systemic HIV load and EBV subgingival load was found in G1 and G2 (r = 0.647; P < .001), but not in G3. Only G1 individuals using ART had lower subgingival EBV loads than those not using it (5.03 log10 copies/mL, and 7.14 log10 copies/mL, respectively; P = .0348). CONCLUSIONS: Subgingival sites, especially the periodontal pockets, are suggested to act as a reservoir of EBV in HIV+ individuals. Therefore, the identification of latent EBV infections in this easily accessible site might help to improve quality of life in patients with HIV by maintaining oral/periodontal health. In addition it might encourage new approaches in investigating EBV-associated disorders in HIV+ patients.


Assuntos
Infecções por HIV , Herpesvirus Humano 4 , Brasil , Estudos Transversais , DNA Viral , HIV , Humanos , Reação em Cadeia da Polimerase , Qualidade de Vida
6.
APMIS ; 128(1): 35-40, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31628868

RESUMO

Kingella kingae colonizes the upper airways in children and has been recognized as the most common causative agent of osteoarticular infections (OAI) in children below 4 years of age. This is the first Scandinavian study to investigate oropharyngeal K. kingae carriage in healthy children. From June 2015 to August 2016, we recruited 198 healthy children aged 11-14 months from routine consultations at health promotion centers in Hordaland County, Norway for a cross-sectional study. After their parents had provided informed consent; demographic data were registered, and an oropharyngeal swab was collected. The oropharyngeal swab was analyzed with a real-time PCR assay specific to K. kingae targeting the RTX toxin locus. Results showed an asymptomatic carriage rate of 12.6%. A striking and highly significant difference was observed between the children that had started attending day care facilities as compared with children still being at home (33.33% vs 8.5%; p < 0.001). K. kingae is prevalent in young children in Norway. This study emphasize that K. kingae should be considered an important etiological agent in OAI. Transmission seems to be facilitated in day care facilities. The correlation between oropharyngeal carriage and OAI needs to be further explored.


Assuntos
Portador Sadio/epidemiologia , Portador Sadio/microbiologia , Creches , Kingella kingae/isolamento & purificação , Infecções por Neisseriaceae/epidemiologia , Orofaringe/microbiologia , Infecções Assintomáticas/epidemiologia , Proteínas de Bactérias/genética , Estudos Transversais , Feminino , Humanos , Lactente , Kingella kingae/genética , Masculino , Noruega/epidemiologia , Osteomielite/diagnóstico , Osteomielite/microbiologia , Reação em Cadeia da Polimerase , Prevalência , Estudos Prospectivos
7.
J Water Health ; 17(6): 971-977, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31850903

RESUMO

Torque teno virus (TTV) is a single-stranded DNA virus which is predominantly transmitted by the fecal-oral route and may be excreted in the absence of the clinical symptoms. TTV was previously considered a probable cause of hepatitis, but further studies could not strongly connect TTV to any serious health problem. TTV is highly resistant to water and wastewater treatment processes and can be a useful indicator for determining the fecal contamination of water. The purpose of the present study was to assess the prevalence and molecular characterization of TTV in treated wastewater in Tehran. Thirteen effluent samples were collected monthly from the biggest wastewater treatment plant in Tehran, Iran (from September 2017 to August 2018). The presence of the TTV was monitored in the samples by the nested polymerase chain reaction (PCR) method. The TTV genome was found in 76.9% of the samples, and TTV of groups 1 and 3 were determined using phylogenetic analysis. Therefore, treated wastewater can play a key role in the transmission of TTV and the usage of treated wastewater as a source of potable water needs to be carefully controlled.


Assuntos
DNA Viral/genética , Torque teno virus/isolamento & purificação , Águas Residuárias/virologia , DNA Viral/isolamento & purificação , Humanos , Irã (Geográfico) , Filogenia , Reação em Cadeia da Polimerase/métodos , Prevalência , Torque teno virus/genética
8.
Biomed Khim ; 65(6): 477-484, 2019 Oct.
Artigo em Russo | MEDLINE | ID: mdl-31876518

RESUMO

Using random (combinatorial) DNA-libraries with various degrees of diversity, it was shown that their amplification by polymerase chain reaction in real time resulted in appearance of a maximum on amplification curves. The relative decrease of fluorescence after passing the maximum was directly proportional to the logarithm of the number of oligonucleotide sequence variants in the random DNA-library provided that this number was within in the interval from 1 to 104 and remained practically unaltered when the number of variants was in the interval from 105 to 108. The obtained dependence was used in the course of SELEX to evaluate changes in the diversity of random DNA-libraries from round to round in selection of DNA-aptamers to the recombinant SMAD4 protein. As a result, oligonucleotides containing sequences able to form a site of SMAD4-DNA interactions known as SBE (SMAD-binding element) have been selected thus indicating that the SMAD4-SBE interaction dominates the aptamer selection.


Assuntos
Aptâmeros de Nucleotídeos/química , DNA/química , Biblioteca Gênica , Técnica de Seleção de Aptâmeros , Reação em Cadeia da Polimerase
9.
Mikrobiyol Bul ; 53(4): 401-407, 2019 Oct.
Artigo em Turco | MEDLINE | ID: mdl-31709937

RESUMO

Acquired Immunodeficiency syndrome (AIDS) is an important global public health issue. Increasing HIV/AIDS cases reported each year has become a serious health problem for our country. The fourth generation enzyme immunoassay (EIA) test is the first step in the laboratory diagnosis of human immunodeficiency virus (HIV) infection. When the EIA test is repeatedly reactive, antibody-based tests such as immuno blot (IB), line immunoassay (LIA), HIV 1-2 antibody differentiation immunoassay, and HIV RNA tests for the early period of infection are used as confirmatory tests. The aim of this study was to evaluate the results of three different methods for the diagnosis of HIV infection. HIV 1-2 IB and quantitative HIV-1 RNA PCR tests were performed in 199 patient samples. These samples were detected as the reactive or gray zone with HIV 1-2 Ab+Ag EIA test between 2010 and 2015 at Akdeniz University Hospital, Microbiology Laboratory. HIV 1-2 Ab+Ag determination in serum samples was performed with the EIA method (Elecsys HIV combi PT test, Roche Diagnostics, Germany). A commercial kit (INNO-LIA HIV I-II Score, Innogenetics, Belgium) was used for HIV 1-2 IB method. The presence of HIV-1 RNA was investigated by automated nucleic acid extraction and real-time PCR method (Ampliprep/COBAS Tagman HIV-1 Test, Roche Diagnostics, Germany) in plasma samples. For statistical analysis, SPSS, Mann Whitney U test was used, ROC analysis was performed and p<0.05 value was considered statistically significant. HIV 1-2 Ab+Ag EIA COI (cut-off index) median value was higher with positive HIV 1-2 IB and HIV-1 RNA results than negative HIV 1-2 IB and HIV-1 RNA results. These values were 394 (range: 11.5-2272) and 1.79 (range: 1.01-83.3) respectively and this difference was statistically significant (p< 0.001). HIV-1 RNA test results were positive in one patient with gray zone and two patients with negative HIV 1-2 IB result (viral loads were > 10.000.000, > 10.000.000 and 5.040.000 copies/ml, respectively). For the kit that we used for HIV 1-2 Ab+Ag EIA COI ratio of >16.45 had a sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of 97.6%, 98.1%, 97.6% and 98.1%, respectively for the detection of HIV infection (r= 0.994, p< 0.001). HIV 1-2 Ab+Ag EIA S/CO ratio of < 9.26 had a sensitivity, specificity, PPV and NPV of 100%, 92.5%, 91.1% and 100% (p< 0.001). HIV infection is diagnosed if HIV 1-2 Ab+Ag EIA test result is repeatedly reactive and HIV 1-2 IB test and HIV-1 RNA tests are positive. In our study, HIV 1-2 Ab+Ag EIA COI median value was 394 (range: 11.5-2272) in this group of patients (p< 0.001). HIV-1 RNA PCR test was positive in three patients with > 10.000.000, 5.040.000 and > 10.000.000 copies/ml whose EIA tests were repeatedly reactive. HIV IB test was detected as the gray zone in one of them and as negative in the remaining two (HIV EIA S/CO values were 265, 9.5 and 131.8, respectively). These patients were diagnosed as acute HIV infection with clinical and laboratory findings. In conclusion, HIV RNA should also be performed and included in the diagnostic algorithm for acute HIV infection.


Assuntos
Infecções por HIV , Imunoensaio , Immunoblotting , Reação em Cadeia da Polimerase , Alemanha , Infecções por HIV/diagnóstico , HIV-1 , HIV-2 , Humanos , Imunoensaio/normas , Immunoblotting/normas , Reação em Cadeia da Polimerase/normas , RNA Viral/genética , Sensibilidade e Especificidade
10.
Mikrobiyol Bul ; 53(4): 408-418, 2019 Oct.
Artigo em Turco | MEDLINE | ID: mdl-31709938

RESUMO

Leishmaniasis is a parasitic disease that is transmitted to humans by the bites of infected female phlebotomine sandflies. In the diagnosis of cutaneous leishmaniasis (CL), in the smear samples, the demonstration of the parasite by microscope remains a gold standard method. However, it becomes difficult to diagnose the parasite since the number of amastigotes in chronic cases with a lesion of one year or longer is very low. Due to many factor such as patients primarily do not to take any notice these lesions in their bodies, do not apply to health institutions or late applied, receive wrong treatment; the diagnosis and treatment are delayed. In addition, it is been worse prognosis by add secondary infection to lesions and wounds become chronic. For this reason, molecular methods are used in addition to microscopic examination in chronic suspected CL cases. It was aimed to reveal of the molecular diagnostic value in chronic suspected CL cases by polymerase chain reaction (PCR) in the smear belonging to Turkish patients that reported to be evaluated clinically because it can not be seen Leishmania amastigotes in microscopic examination. Smear of 50 Turkish patients who were clinically reported of the evaluation of chronic CL were selected. These samples were smears belonging to suspected CL patients that applied Hatay Mustafa Kemal University, Faculty of Medicine, Parasitology laboratory from different polyclinics and were decided to be evaluated clinically as a result of microscopic examination because they came from endemic regions (such as Hassa, Altinözü, Yayladagi). DNA was isolated from selected samples and PCR was performed using 13A, 13B primers targeting the kinetoplastid DNA (kDNA) region. The samples found positive by PCR were typed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis using LITSR and L5.8S primers targeting internal transcribed spacer (ITS-1) region. Of the 50 smear samples, 17 (34%) were determined positive with 13A, 13B primers targeting the kinetoplastid DNA (kDNA) region. Positive samples were also found to be positive with LITSR and L5.8S primers targeting ITS-1 region. The PCR products obtained from PCR with ITS-1 gene region were digested with the restriction endonucleases BsuRI (HaeIII). As a result of PCR-RFLP analysis, it was determined that 11 of Leishmania tropica, one of Leishmania major and five of Leishmania infantum/donovani out of 17 samples. Chronic CL can be confused with skin diseases such as sarcoidosis, tuberculosis, malignant tumors. In particular, chronic CL cases can be escaped the attention for many reasons such as failure to diagnose correctly, insufficient microscope experience, fail to see due to low number of parasites. For this reason, it was concluded that PCR, which is a molecular method, should be used in chronic suspected CL samples which are negative for the parasite by microscopic examination.


Assuntos
DNA de Protozoário , Leishmania , Leishmaniose Cutânea , Reação em Cadeia da Polimerase , DNA de Protozoário/genética , Feminino , Humanos , Leishmania/genética , Leishmaniose Cutânea/diagnóstico , Microscopia , Polimorfismo de Fragmento de Restrição , Reprodutibilidade dos Testes , Turquia
11.
Mikrobiyol Bul ; 53(4): 434-441, 2019 Oct.
Artigo em Turco | MEDLINE | ID: mdl-31709940

RESUMO

Identification of viral agents causing central nervous system (CNS) infections increased by the application of nucleic acid tests. In this study, the results of polymerase chain reaction (PCR) for viral agents were evaluated in cerebrospinal fluid (CSF) samples taken from patients with CNS infection. CSF samples taken from 1185 patients between 2010 and 2017 were tested for the presence of Herpes simplex virus (HSV) type 1 and 2, Varicella Zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), adenovirus ve enterovirus by PCR in Dokuz Eylul University Hospital. Tests were performed according to the clinicians' orders and results were evaluated retrospectively. The number of tests performed were 1038 for HSV, 882 for adenovirus, 865 for enterovirus, 496 for VZV, 100 for EBV and 92 for CMV. Commercial tests were used for EBV, CMV (Artus QS-RGQ Kits, Qiagen, Germany) and enterovirus (GeneXpert, Cepheid, USA) while the other viruses (HSV, VZV, adenovirus) were tested by in-house real-time PCR assays. Ninety-one CSF (7.7%) samples were positive. The mean age was 13 (<1 to 76 years) while median was seven. The most frequently detected pathogens were enterovirus (63/91, 69%) and HSV-1 (14/91,15%). The number of patients positive for adenovirus, VZV, EBV and CMV were five, four, three and two, respectively. In one patient, both enterovirus (Ct: 29.5) and EBV (Ct: 38.53) were positive. The number of positive samples were increased in summer months. Enterovirus RNA positive patients (n= 60/63, 95.2%) were ≤ 18 years old while 29% were younger than one year of age. Enterovirus positive samples peaked in 2012 and 2014 and detected mainly in summer (60.3%) and autumn (20.6%) months. VZV was mostly detected in patients greater than 65 years of age. Mean Ct of the positive reactions was 31.87 ± 3.5 (22.88-40.32). The lowest and the highest Ct values were detected in HSV-1 assay. The mean Ct value of enterovirus assay (30.4; 25.7-35.9) was lower than the other pathogens' values. In the seven-year period, 7.7% of the1185 patients' CSF samples were positive for viral nucleic acids. As expected, enteroviruses were the most common pathogens in children and detected mainly in summer-autumn period. Syndromic approach in CNS infections could increase the viral pathogen detection.


Assuntos
Viroses do Sistema Nervoso Central , Adolescente , Adulto , Idoso , Viroses do Sistema Nervoso Central/líquido cefalorraquidiano , Viroses do Sistema Nervoso Central/epidemiologia , Viroses do Sistema Nervoso Central/virologia , Criança , Pré-Escolar , DNA Viral/genética , Alemanha/epidemiologia , Humanos , Lactente , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Estudos Retrospectivos , Vírus/genética , Adulto Jovem
12.
J Agric Food Chem ; 67(46): 12936-12944, 2019 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-31670953

RESUMO

An ultrasensitive bio-barcode competitive immunoassay method based on droplet digital polymerase chain reaction (ddPCR) was developed for the determination of triazophos. Gold nanoparticles (AuNPs) were coated with monoclonal antibodies (mAbs) and complementary double-stranded DNA (dsDNA), which included bio-barcode DNA and thiol-capped DNA. Magnetic nanoparticle (MNP) probes were constructed by modifying the MNPs with ovalbumin-hapten conjugates (OVA-hapten). The target pesticide and OVA-hapten on the surface of the MNP probes competed with the AuNP probes simultaneously, and then the bio-barcode DNA was released for quantification by ddPCR. The concentration of released DNA was inversely proportional to the concentration of pesticide to be tested. Under the optimum conditions, the competitive immunoassay exhibited a wide linear range of 0.01-20 ng/mL and a low detection limit of 0.002 ng/mL. Spike recovery tests were carried out using apple, rice, cabbage, and cucumber samples to verify the feasibility of the method. The recovery and relative standard deviations (RSDs) of the technique ranged from 76.9 to 94.4% and from 10.8 to 19.9%, respectively. To further validate the results, a linear correlation analysis was performed between the proposed method and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Consequently, the bio-barcode immunoassay based on nanoparticles and ddPCR, an ultrasensitive method, showed great potential for the determination of target pesticides in real samples.


Assuntos
Imunoensaio/métodos , Inseticidas/análise , Organotiofosfatos/análise , Reação em Cadeia da Polimerase/métodos , Triazóis/análise , DNA/genética , Ouro/química , Imunoensaio/instrumentação , Limite de Detecção , Magnetismo/métodos , Nanopartículas Metálicas/química
13.
Zhongguo Xue Xi Chong Bing Fang Zhi Za Zhi ; 31(5): 474-478, 2019 Oct 08.
Artigo em Chinês | MEDLINE | ID: mdl-31713374

RESUMO

OBJECTIVE: To investigate the prevalence and molecular features of Cryptosporidium in sheep and goats from Anhui Province and neighboring provinces. METHODS: A total 832 and 781 fresh fecal samples were collected from seven large-scale sheep farms and ten large-scale goat farms in Anhui Province and neighboring provinces of Henan, Jiangsu and Shandong. The prevalence and species of Cryptosporidium were investigated in the fecal samples from the sheep and goats in the study areas using nested PCR assay based on the Cryptosporidium-specific SSU rDNA gene, and the subgenotypes of C. parvum and C. ubiquitum were characterized by amplification and sequencing of the 60 kDa glycoprotein (gp60) gene. RESULTS: The overall prevalence of Cryptosporidium was 5.8% (48/832) in sheep and 8.7% (68/781) in goats in Anhui Province and neighboring provinces, respectively. The SSU rDNA gene-based PCR assay identified C. xiaoi and C. ubiquitum in sheep and C. parvum in goats, and subtyping revealed that all C. ubiquitum subgenotypes belonged to XIIa subtype 2 and C. parvum subgenotypes belonged to IIdA19G1. CONCLUSIONS: The identification of zoonotic C. ubiquitum XIIa subtype 2 and C. parvum subtype IIdA19G1 suggests that sheep and goats may serve as a potential source for human Cryptosporidium infections.


Assuntos
Criptosporidiose , Cryptosporidium , Doenças das Cabras , Doenças dos Ovinos , Animais , Criptosporidiose/epidemiologia , Criptosporidiose/parasitologia , Cryptosporidium/classificação , Cryptosporidium/genética , Fezes/parasitologia , Genótipo , Doenças das Cabras/epidemiologia , Doenças das Cabras/parasitologia , Cabras , Humanos , Reação em Cadeia da Polimerase , Prevalência , RNA Ribossômico 18S/genética , Ovinos , Doenças dos Ovinos/epidemiologia , Doenças dos Ovinos/parasitologia
14.
Dis Aquat Organ ; 137(1): 47-51, 2019 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-31777399

RESUMO

Panulirus argus virus 1 (PaV1) affects wild populations of Caribbean spiny lobsters. PaV1 can be lethal but shows predilection for juvenile lobsters. Because P. argus is one of the most valuable fisheries around the wider Caribbean region, monitoring disease prevalence in local populations is desirable. Diseased lobsters are easily recognized by their milky hemolymph, but this sign only becomes evident in advanced stages of infection. Other methods have been developed to detect PaV1, but are less practical for long-term monitoring of patterns of infection in populations. A previous study estimated the validity measures (sensitivity and specificity) of detection of PaV1 infection by observed clinical signs against endpoint PCR assays, using a representative sample of lobsters comprising mainly subadults and adults from a commercial fishing area. In the present study, these validity measures were estimated in a similar manner for a different population comprising mainly juveniles from a protected nursery area. We obtained virtually the same sensitivity and specificity values (0.48 and 1, respectively) for observed clinical signs as in the previous study (0.51 and 1, respectively), confirming the validity of applying a simple 2× correction factor to monitor the patterns of PaV1 infection over time based on more easily conducted visual assessments of a representative sample of the population.


Assuntos
Palinuridae , Animais , Pesqueiros , Hemolinfa , Reação em Cadeia da Polimerase
15.
Klin Lab Diagn ; 64(10): 635-640, 2019.
Artigo em Russo | MEDLINE | ID: mdl-31742959

RESUMO

To analyze the method for detecting HBV DNA in peripheral blood at low viral load and evaluate its significance in identifying HBsAg-negative viral hepatitis B. In this work, samples of blood and liver tissue biopsy material were used from 128 patients living in the Russian Federation and the Republic of Uzbekistan without CHB and with CHB confirmed detection of circle covalently closed HBV DNA in hepatocytes. Plasma viral load was measured using the «AmpliSens® HBV-Monitor-FL¼ kit. HBV at low viral load was detected by nested PCR. Analytical sensitivity was checked by step dilution. According to our method, at the first stage, an asymmetric PCR is carried out using extended oligonucleotide primers with different melting points, complementary to the hepatitis B different genotypes genomes greatest similarity region. To increase the sensitivity, a second PCR is performed using the first reaction amplification product and internal primers. The sensitivity of the method for DNA extraction from 100 µl of plasma was 5 IU / ml, specificity 100%. Since, in spite of the HBV genotypes characteristic geographical distribution, the detection of "alien" genovariants for certain territories is becoming more frequent, we tested the method in geographically remote but active international relations with the Russian Federation regions with a high frequency of hepatotropic viruses. The developed method for detecting HBV DNA in blood plasma at low viral load based on PCR technology allows the various HBV gene variants identification and genotyping, both characteristic and rare in the Russian Federation, circulating in other world regions. The method can be used to detect HBV in risk groups, in a population, as well as when screening blood donors in order to ensure the blood transfusions safety.


Assuntos
DNA Viral/sangue , Vírus da Hepatite B/isolamento & purificação , Hepatite B Crônica/sangue , Antígenos de Superfície da Hepatite B , Humanos , Reação em Cadeia da Polimerase , Federação Russa , Carga Viral
16.
Zhonghua Yu Fang Yi Xue Za Zhi ; 53(11): 1130-1135, 2019 Nov 06.
Artigo em Chinês | MEDLINE | ID: mdl-31683400

RESUMO

Objective: To understand the situation and genotype distribution of spotted fever group rickettsia (SFGR) in the border area of Tumen River Basin in free ticks in Yanbian Korean Autonomous Prefecture (Yanbian Prefecture), Jilin Province. Methods: From April to September, 2017, ticks were collected using flagging method from Hunchun, Tumen, Helong and Longjing cities in the Tumen River basin of Yanbian Prefecture. Outer membrane protein A (ompA) was detected by Polymerase Chain Reaction (PCR), then, the species were identified by gene sequencing and analyzed systematically. The positive rate of pools and MIR(minimum infection rate per 100 ticks,MIR) of SFGR were calculated, and the difference of positive rate of pools among ticks with different characteristics was compared by Chi-square test. Results: A total of 3 079 ticks were collected and divided into 536 pools. The positive rate of pools of SFGR nucleic acid was 39.7% (213 pools). The MIR of SFGR was 6.9%.The positive rate of pools of SFGR in Dermacentor silvarum, Haemaphysalis concinna, Haemaphysalis japonica, Haemaphysalis longicornis and Ixodes persulcatus were 80.4% (41/51), 14.0% (25/179), 20.2% (18/89), 78.9% (101/128) and 25.9% (21/81), and the difference was statistically significant (P<0.001). There was statistical difference in the positive rate of pools of SFGR in developmental stages of ticks (P<0.001); the positive rate of pools of female adults, male adults, nymph and larvae were 36.4% (95/261), 34.2% (67/196), 56.3% (40/71) and 7/8, and the MIR was 7.9%, 7.7%, 4.9% and 3.5%. The five genotype was detected which was Candidatus Rickettsia longicornii, Rickettsia raoultii, Rickettsia heilongjiangensis, Candidatus Rickettsia tarasevichiae,Rickettsia monacensis and have 98%-100% homology with known gene sequences. Candidatus Rickettsia longicornii, Rickettsia raoultii, Rickettsia heilongjiangensis and Candidatus Rickettsia tarasevichiae showed close evolutionary relationship with known specie (have 98%-100% homology with known gene sequences); Rickettsia monacensis showed Far from evolutionary relationship with known species (have 98% homology with known gene sequences). Conclusion: SFGR infection of ticks is common in the border areas of the Tumen River Basin. There was high diversity in SFGR species and tick species in the areas surveyed.


Assuntos
Proteínas da Membrana Bacteriana Externa/genética , Ixodidae/microbiologia , Rickettsia/classificação , Rickettsia/isolamento & purificação , Rickettsiose do Grupo da Febre Maculosa/diagnóstico , Carrapatos , Animais , China , Feminino , Ixodidae/classificação , Ixodidae/crescimento & desenvolvimento , Masculino , Reação em Cadeia da Polimerase , Rickettsia/genética , Rios , Análise de Sequência
17.
Georgian Med News ; (294): 37-41, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31687946

RESUMO

Premenstrual syndrome (PMS) is a common problem of women in reproductive age. Genetic aspects of this pathology are not completely clear. The aim of the article is devoted to the study of the frequency of ID polymorphism of angiotensin-converting enzyme gene ACE in patients with premenstrual syndrome. The object of the study were 50 women in reproductive age with the diagnosis of PMS, 25 of them had mild form of the disease, 25 - severe one. 25 persons without PMS were controls. Polymerase chain reaction was used to study ACE gene polymorphism. We determined an equal distribution of ACE gene genotypes between women with PMS and without this pathology (DD genotype was established in 24% of controls and 30% women with PMS, ID genotype - 60% and 46% respectively, II genotype - 16% and 24%). However, DD genotype was found in 2.17 times more often in patients with severe form of the disease (52%) compared to healthy persons. Thus, women with DD genotype of ACE gene have the tendency to the development of severe PMS (χ2=3.06, p=0.08; OR=3.43, 95% CI 1.02-11.47, p=0.045).


Assuntos
Predisposição Genética para Doença , Peptidil Dipeptidase A/genética , Polimorfismo Genético , Síndrome Pré-Menstrual/genética , Adulto , Estudos de Casos e Controles , Feminino , Frequência do Gene , Genótipo , Humanos , Masculino , Peptidil Dipeptidase A/metabolismo , Reação em Cadeia da Polimerase , Síndrome Pré-Menstrual/fisiopatologia
18.
Am J Forensic Med Pathol ; 40(4): 304-311, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31687979

RESUMO

Semen is crucial evidence for some sex crimes, with its sole confirmation being sperm detection. The success of sperm detection is dependent on all levels of preanalytic and analytic procedures. Specimen collection must be performed by well-trained and competent forensic physicians as well as forensic nurses, with preservation done properly before laboratory transfer. Laboratory procedures should consider archival sperm identification, by visualization, with adequate amounts separated from other cells to obtain male DNA profiles. Differential extraction is robust and accepted as the forensic standard but is time consuming and may result in male DNA loss. Thus, alternative methods and microdevices have been developed. Challenges in sperm isolation from vaginal or buccal epithelium mixes and discrimination in multiperpetrator cases have been overcome by single-cell profiling; however, problems inherent in identical twin discrimination and azoospermia have yet to be solved. Epigenetics and future molecular biomarkers may hold the key; therefore, all laboratory processes must consider DNA and RNA protection. Long-term specimen preservation should be done when possible in light of future confirmatory tests.


Assuntos
Ciências Forenses/métodos , Manejo de Espécimes , Espermatozoides/citologia , Separação Celular , Impressões Digitais de DNA , Metilação de DNA , Feminino , Humanos , Microdissecção e Captura a Laser , Masculino , Repetições de Microssatélites , Reação em Cadeia da Polimerase/métodos , Antígeno Prostático Específico/isolamento & purificação , RNA Mensageiro/isolamento & purificação , Proteínas Secretadas pela Vesícula Seminal/isolamento & purificação , Delitos Sexuais , Coloração e Rotulagem , Fatores de Tempo
19.
Medicine (Baltimore) ; 98(44): e17838, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31689873

RESUMO

RATIONALE: Recurrence of Klinefelter syndrome (KS) in non-twin brothers is very rare. This study examined the inheritance pattern of supernumerary X chromosomes in non-twin brothers. PATIENT CONCERNS: A 16-year-old man presented with small-sized testicles. During his diagnostic work-up, his brother, in his late 20's, also complained of small testes and erectile dysfunction. DIAGNOSIS: Chromosome analysis in peripheral blood revealed non-mosaic 47,XXY karyotype in both brothers. Their mother showed a normal 46,XX karyotype. INTERVENTIONS: To examine the inheritance pattern of supernumerary X chromosomes, quantitative-fluorescence PCR was performed with small tandem repeat markers. It revealed that their supernumerary X chromosomes were inherited from different parents. OUTCOMES: After the diagnosis of KS, 2 brothers started to receive testosterone treatment. CONCLUSION: This case report is the first to report differences in the origins of supernumerary X chromosomes in brothers with KS and furthers the current understanding of the cytogenetic mechanisms in KS.


Assuntos
Cromossomos Humanos X , Síndrome de Klinefelter/diagnóstico , Síndrome de Klinefelter/genética , Adolescente , Adulto , Disfunção Erétil/etiologia , Humanos , Síndrome de Klinefelter/tratamento farmacológico , Masculino , Pais , Reação em Cadeia da Polimerase/métodos , Irmãos , Sequências de Repetição em Tandem , Testosterona/uso terapêutico
20.
An Acad Bras Cienc ; 91(3): e20180487, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31618408

RESUMO

Authentication of cell lines is of paramount importance to validate the results from their use in biomedical research. Although isoenzyme polymorphism is the standard method, molecular methods based on mitochondrial DNA (mtDNA) have been developed to replace it. The aim of this study was the improvement of our isoenzyme electrophoretic analysis and the validation of one molecular technique targeted at mtDNA for the authentication of our animal cell lines. The combined method of cellular lysing through osmotic shock, followed by freezing-thawing in N2 to obtain isoenzyme extracts, and with 42 × 106 cells maintained the best efficiency. The superior electrophoretic conditions were PAGE run at 200 V. All cell lines had isoenzymatic mobility corresponding to their species to lactate dehydrogenase, malate-dehydrogenase, and glucose-6-phosphate dehydrogenase isoenzymes, and could be distinguished from each other. Two molecular techniques based on mtDNA were tested, one on the cytochrome b gene and other on cytochrome c oxidase I subunit gene. Due to difficulties in distinguishing all cell lines using only one these techniques, we merged the primers of two methods in such a way that there was a sufficient differentiation of all DNA fragments. The sequencing of these PCR products was also performed to validate these data.


Assuntos
Técnicas de Cultura de Células/métodos , DNA Mitocondrial/genética , Isoenzimas/análise , Animais , Linhagem Celular , Eletroforese , Glucosefosfato Desidrogenase/análise , L-Lactato Desidrogenase/análise , Malato Desidrogenase/análise , Reação em Cadeia da Polimerase
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