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1.
Vet Parasitol ; 277: 109020, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31896019

RESUMO

Haemonchus contortus is one of the most important gastrointestinal nematodes (GINs) infecting sheep, goats, and cattle worldwide. We developed a SYBR Green real-time PCR (qPCR) assay for detection and quantification of H. contortus by using specific primers based on a conserved region of the mitochondrial cytochrome oxidase subunit I (mt-COI) gene, and evaluated this technique in the detection of H. contortus infections in cattle in Central Anatolia Region of Turkey. The newly developed qPCR assay successfully discriminated H. contortus from other GIN species infecting cattle in the specificity evaluations, with a specific melt peak of 77.5 °C. Our results revealed the efficient amplification of the proposed target COI region within the range of plasmid copies, from 2 × 106 to 2 × 101 per µl, with 96.9 % efficiency, R² value of 0.999, and a slope of -3.398. Among the 920 cattle fecal samples from the field, 58 samples (6.3 %) were positive with qPCR assay, whereas 45 samples (4.9 %) were positive, as determined by larval culture, suggesting the utility of SYBR Green qPCR. Phylogenetic characterization of the partial COI gene of H. contortus isolates was also evaluated for 100 eggs and third stage larvae recovered from positive cattle faecal samples, which were verified with the qPCR assay prior to analyses. COI sequences were classified into three haplotypes (THC1 to THC3) with intraspecific nucleotide differences of 0.50 to 0.76 %. Phylogenetic analyses revealed that the haplotypes grouped with H. contortus isolates from several countries in a monophyletic cluster, with evidence of at least two main haplogroups. Overall, the SYBR Green qPCR assay was highly specific and sensitive, suggesting that it can be used for screening of H. contortus infections in livestock populations in epidemiological studies and the control of this important parasite.


Assuntos
Criação de Animais Domésticos/métodos , Doenças dos Bovinos/diagnóstico , Doenças dos Bovinos/parasitologia , Complexo IV da Cadeia de Transporte de Elétrons/genética , Hemoncose/veterinária , Haemonchus/genética , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Animais , Bovinos , Hemoncose/diagnóstico , Hemoncose/parasitologia , Compostos Orgânicos/análise , Especificidade da Espécie , Turquia
2.
J Insect Sci ; 20(1)2020 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-31925425

RESUMO

Diaphania caesalis (Walker) is an important boring insect mainly distributed in subtropical and tropical areas and attacked tropical woody grain crops, such as starchy plants of Artocarpus. Quantitative real-time polymerase chain reaction (qRT-PCR) is a powerful approach for investigating target genes expression profiles at the transcriptional level. However, the identification and selection of internal reference genes, which is often overlooked, is the most vital step before the analysis of target gene expression by qRT-PCR. So far, the reliable internal reference genes under a certain condition of D. caesalis have not been investigated. Therefore, this study evaluated the expression stability of eight candidate reference genes including ACT, ß-TUB, GAPDH, G6PDH, RPS3a, RPL13a, EF1α, and EIF4A in different developmental stages, tissues and sexes using geNorm, NormFinder and BestKeeper algorithms. To verify the stability of the recommended internal reference genes, the expression levels of DcaeOBP5 were analyzed under different treatment conditions. The results indicated that ACT, RPL13a, ß-TUB, RPS3a, and EF1α were identified as the most stable reference genes for further studies on target gene expression involving different developmental stages of D. caesalis. And ACT and EIF4A were recommended as stable reference genes for different tissues. Furthermore, ACT, EF1α, and RPS3a were ranked as the best reference genes in different sexes based on three algorithms. Our research represents the critical first step to normalize qRT-PCR data and ensure the accuracy of expression of target genes involved in phylogenetic and physiological mechanism at the transcriptional level in D. caesalia.


Assuntos
Expressão Gênica , Genes de Insetos , Mariposas/genética , Animais , Perfilação da Expressão Gênica , Larva/genética , Larva/crescimento & desenvolvimento , Mariposas/crescimento & desenvolvimento , Óvulo/crescimento & desenvolvimento , Pupa/genética , Pupa/crescimento & desenvolvimento , Reação em Cadeia da Polimerase em Tempo Real
3.
Isr Med Assoc J ; 22(1): 48-52, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31927806

RESUMO

BACKGROUND: Nasal polyps are three-dimensional structures arising from the mucosa of the upper airway. Due to their complexity, the reliability of single-layer cell cultures and animal systems as research models is limited. OBJECTIVES: To evaluate the feasibility of an ex vivo organ culture of human polyps, preserving tissue structure and function. METHODS: Nasal polyps were excised during routine endoscopic sinus surgery for chronic rhinosinusitis and polyposis. Fresh tissue samples were used for pathological evaluation and for the preparation of 250-500 µm sections, which were incubated in culture media. Tissue viability was assessed by visualisation of cilia motility, measurement of glucose uptake, and an infectivity assay. Cytokine secretion was evaluated by enzyme-linked immunosorbent assay and real-time polymerase chain reaction before and after the introduction of steroids. RESULTS: Polyp tissue viability was retained for 2-3 days as demonstrated by cilia motility, glucose uptake and preserved cellular composition. Tissue samples maintained their capacity to respond to infection by herpes simplex virus 1 and adenovirus. Introduction of dexamethasone to cultured tissue samples led to suppression of interferon-g production. CONCLUSIONS: The ex vivo nasal polyp organ culture reproduces the physiological, metabolic, and cellular features of nasal polyps. Furthermore, it shows a preserved capacity for viral infection and response to drugs. This system is a useful tool for the investigation nasal-polyps and for the development of novel therapies.


Assuntos
Pólipos Nasais/diagnóstico , Técnicas de Cultura de Órgãos/métodos , Adulto , Quimiocinas/metabolismo , Citocinas/metabolismo , Glucose/metabolismo , Humanos , Pólipos Nasais/metabolismo , Pólipos Nasais/patologia , Pólipos Nasais/cirurgia , Reação em Cadeia da Polimerase em Tempo Real
4.
Braz Oral Res ; 33: e117, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31939498

RESUMO

The aim of this study was to evaluate the effect of mineral trioxide aggregate (MTA) and Brazilian propolis on the cell viability, mineralization, anti-inflammatory ability, and migration of human dental pulp cells (hDPCs). The cell viability was evaluated with CCK-8 kit after 1, 5, 7, and 9 days. The deposition of calcified matrix and the expression of osteogenesis-related genes were evaluated by Alizarin Red staining and real-time PCR after incubation in osteogenic medium for 21 days. The expression of inflammation-related genes in cells was determined after exposure to 1 µg/mL LPS for 3 h. Finally, the numbers of cells that migrated through the permeable membranes were compared during 15 h. Propolis and MTA significantly increased the viability of hDPCscompared to the control group on days 7 and 9. In the propolis group, significant enhancement of osteogenic potential and suppressed expression of IL-1ß and IL-6 was observed after LPS exposure compared to the MTA and control groups. The number of migration cells in the propolis group was similar to that of the control group, while MTA significantly promoted cell migration. Propolis showed comparable cell viability to that of MTA and exhibited significantly higher anti-inflammatory and mineralization promotion effects on hDPCs.


Assuntos
Compostos de Alumínio/farmacologia , Anti-Inflamatórios/farmacologia , Compostos de Cálcio/farmacologia , Polpa Dentária/citologia , Polpa Dentária/efeitos dos fármacos , Óxidos/farmacologia , Própole/farmacologia , Silicatos/farmacologia , Antraquinonas , Brasil , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Combinação de Medicamentos , Humanos , Interleucina-1beta/análise , Interleucina-6/análise , Odontoblastos/efeitos dos fármacos , Reação em Cadeia da Polimerase em Tempo Real , Reprodutibilidade dos Testes , Estatísticas não Paramétricas , Fator de Necrose Tumoral alfa/análise
5.
Food Microbiol ; 85: 103307, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31500711

RESUMO

Cold atmospheric-gaseous plasma (CAP) is an emerging non-thermal technology for decontamination of foodborne bacterial and viral pathogens. We obtained a >5 log10 reduction in the titer (TCID50) of feline calicivirus (FCV) on stainless steel discs and Romaine lettuce leaves after 3 min wet exposure to air plasma generated by a two-dimensional array of integrated coaxial-microhollow dielectric barrier discharge (2D-AICM-DBD). However, when human norovirus (HuNoV GII.4) was treated for 5 min under the same conditions, ~2.6 log10 (>99.5%) reduction in genome copy number was observed as measured by ethidium monoazide-coupled RT-qPCR (EMA-RT-qPCR). To assess this discrepancy, we studied CAP's effect on FCV by the cell culture method and by the EMA-coupled RT-qPCR method. It was found that the molecular titration method (EMA-RT-qPCR) underestimates the level of virus reduction by CAP. Additionally, the fecal matter present in HuNoV samples partially suppressed virucidal activity of CAP. Assuming that the lower virus reduction measured by EMA-RT-qPCR method compared to cell culture method for FCV is the same as for HuNoV, we can conclude that FCV may be used as a surrogate for HuNoV to assess the virucidal effect of CAP. CAP is able to inactivate 3.5 Log10 units of HuNoV at low titers after 2 min of exposure.


Assuntos
Fezes/virologia , Norovirus/efeitos dos fármacos , Gases em Plasma/farmacologia , Inativação de Vírus/efeitos dos fármacos , Azidas , Calicivirus Felino/efeitos dos fármacos , Calicivirus Felino/genética , Desinfecção/métodos , Humanos , Alface/virologia , Norovirus/genética , Reação em Cadeia da Polimerase em Tempo Real , Aço Inoxidável
6.
Food Chem ; 305: 125426, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31522124

RESUMO

Genetically modified (GM) Atlantic salmon, AquAdvantage (AquAd), was the first GM animal approved officially for human consumption. Many countries monitor the use of this product under their GM regulations, but a pragmatic system for AquAd-specific detection is needed. Here, we developed a real-time polymerase chain reaction method with high sensitivity for detection of AquAd in foods. This method showed high specificity for the AquAd transgene and the detection limit was 12.5-25 targeted DNA copies per test reaction. An inter-laboratory study using the method developed demonstrated reproducibility at >0.1% (w/w) AquAd content.


Assuntos
Alimentos Geneticamente Modificados , Reação em Cadeia da Polimerase em Tempo Real/métodos , Salmo salar/genética , Alimentos Marinhos/análise , Animais , Animais Geneticamente Modificados , Reprodutibilidade dos Testes
7.
Biosci Biotechnol Biochem ; 84(1): 53-62, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31483222

RESUMO

Large numbers of miRNAs are found in biofluid exosomes. We isolated ~50-200 nm diameter exosomes from four types of porcine biofluid (urine, plasma, semen, and bile) using serial centrifugation and ultracentrifugation procedures. A total of 42.15 M raw data were generated from four small RNA libraries. This produced 40.17 M map-able sequences, of which we identified 204 conserved miRNAs, and 190 novel candidate miRNAs. Furthermore, we identified 34 miRNAs specifically expressed in only one library, all with well-characterized immune-related functions. A set of five universally abundant miRNAs (miR-148a-3p, miR-21-5p, let-7f-5p, let-7i-5p, and miR-99a-5p) across all four biofluids was also found. Function enrichment analysis revealed that the target genes of the five ubiquitous miRNAs are primarily involved in immune and RNA metabolic processes. In summary, our findings suggest that porcine biofluid exosomes contain a large number of miRNAs, many of which may be crucial regulators of the immune system.


Assuntos
Secreções Corporais , Líquidos Corporais , Exossomos/genética , Imunidade Inata , MicroRNAs/genética , MicroRNAs/imunologia , Suínos/genética , Animais , Sequência de Bases/genética , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/imunologia , Sequenciamento de Nucleotídeos em Larga Escala , Microscopia de Força Atômica , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de RNA
8.
Medicine (Baltimore) ; 98(51): e17944, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31860949

RESUMO

To investigate the difference in messenger ribonucleic acid (mRNA) and protein expression of growth arrest DNA damage-inducible gene 45α (GADD45α), mouse double minute 2 homolog (MDM2), and P73 in cancer and cancer-adjacent tissues in patients with non-small-cell lung carcinoma (NSCLC).We compared the mRNA expression of GADD45α and MDM2 and the protein expression of GADD45α, MDM2, and P73 in lung cancer and cancer-adjacent tissues in NSCLC patients by quantitative real-time PCR, immunohistochemistry (IHC), and Western Blot (WB). We analyzed GADD45α, MDM2, and P73 expression in patients with different pathological types of NSCLC, and the correlation of these genes with gender, smoking history, and TNM/T stages.IHC results suggested that MDM2 protein expression significantly increased in cancer tissues in female patients (P = .01), but not in male patients. In addition, WB results indicated that P73 protein expression significantly decreased in cancer tissues in patients with adenocarcinoma (P = .03), but not squamous carcinoma.MDM2 and P73 protein levels were differentially regulated in cancer and cancer-adjacient tissues in patients with sub types of NSCLC. There was no significant difference in GADD45α expression between cancer and cancer-adjacent tissues in NSCLC patients.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Proteínas de Ciclo Celular/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/genética , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteína Tumoral p73/genética , Adenocarcinoma/genética , Adenocarcinoma/patologia , Idoso , Animais , Biópsia por Agulha , Western Blotting , Carcinoma Pulmonar de Células não Pequenas/patologia , Estudos de Coortes , Dano ao DNA/genética , Seguimentos , Humanos , Imuno-Histoquímica , Neoplasias Pulmonares/patologia , Camundongos , Pessoa de Meia-Idade , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real/métodos , Valores de Referência
9.
Medicine (Baltimore) ; 98(51): e18110, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31860959

RESUMO

OBJECTIVE: To study the potential diagnostic value of plasma miR-200c-3p, miR-100-5p, and miR-1826 levels in knee osteoarthritis (KOA). METHODS: Real-time quantitative PCR (RT-PCR) was used to measure the expression levels of serum miR-200c-3p, miR-100-5p, and miR-1826 in 150 KOA patients and 150 control controls. In addition, the levels of DNMT3A, ZEB1, MMP13, and CTNNB1 mRNAs in the synovial fluid were also measured by RT-PCR. RESULTS: The expression levels of miR-100-5p, miR-200c-3p, and miR-1826 in the synovial fluid of 150 KOA patients were significantly lower than those in 54 controls (P < .001). In the synovial fluid, the miR-100-5p and DNMT3A mRNA levels, miR-100-5p and ZEB1 mRNA levels, miR-200c-3p and MMP13 mRNA levels, and miR-1826 and CTNNB1 mRNA levels were all negatively correlated (r = -0.83, -0.81, -0.83, -0.58, respectively). The AUCs of the diagnosis for KOA using the plasma levels of miR-200c-3p, miR-100-5p, and miR-1826 were 0.755, 0.845, and 0.749, respectively. CONCLUSION: The plasma levels of miR-200c-3p, miR-100-5p, and miR-1826 are of potentially high value in the diagnosis of KOA.


Assuntos
Regulação da Expressão Gênica , MicroRNAs/genética , Osteoartrite do Joelho/diagnóstico , Osteoartrite do Joelho/genética , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Área Sob a Curva , Biomarcadores/sangue , Western Blotting , Estudos de Coortes , Progressão da Doença , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Osteoartrite do Joelho/sangue , Prognóstico , Curva ROC , Reação em Cadeia da Polimerase em Tempo Real/métodos , Estudos Retrospectivos , Medição de Risco , Índice de Gravidade de Doença , Fatores Sexuais
10.
Zhonghua Zhong Liu Za Zhi ; 41(12): 918-922, 2019 Dec 23.
Artigo em Chinês | MEDLINE | ID: mdl-31874549

RESUMO

Objective: To investigate the expression level of antisense transcript of pseudogene, general transcription factor Ⅱi psedugen23 (GTF2IP23), in breast cancer and its effect on the host gene general transcription factor Ⅱi (GTF2I). Methods: The expressions of GTF2IP23 and GTF2I were detected in 40 cases of invasive breast cancer tumors and their counterparts by using quantitative real-time polymerase chain reaction (qRT-PCR). The effects of GTF2IP23 on the expression of GTF2I gene and cell proliferation and migration were analyzed by overexpression of GTF2IP23 in breast cancer cells. Results: The expression of GTF2IP23 mRNA in breast cancer tissues was significantly higher than that in adjacent tissues (P<0.001), while the expression of GTF2I mRNA was significantly lower than that in adjacent tissues (P=0.007). The expression of GTF2IP23 was negatively correlated with GTF2I (r=-0.335, P=0.025). The expression of GTF2IP23 in breast cancer cells was significantly higher than in normal breast cells (P<0.01), while GTF2I expression in breast cancer cells was significantly lower than that in normal breast cells (P<0.01). Overexpression of GTF2IP23 in ZR-75-30 cells significantly reduced the expression of GTF2I (P=0.034) and enhanced cell proliferation (P=0.017) and migration (P=0.026) capacity. Conclusions: GTF2IP23 is distinctly upregulated in breast cancer, it inhibits the expression of real gene GTF2I and promotes the proliferation of breast cancer cells.


Assuntos
Neoplasias da Mama/sangue , Proteínas Musculares/metabolismo , Proteínas Nucleares/metabolismo , Transativadores/metabolismo , Fatores de Transcrição TFII/genética , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Proteínas Musculares/genética , Proteínas Nucleares/genética , Reação em Cadeia da Polimerase em Tempo Real , Transativadores/genética , Fatores de Transcrição TFII/metabolismo
11.
Zhonghua Xue Ye Xue Za Zhi ; 40(11): 889-894, 2019 Nov 14.
Artigo em Chinês | MEDLINE | ID: mdl-31856435

RESUMO

Objective: To investigate the current status and real performance of the detection of RUNX1-RUNX1T1 fusion transcript levels and WT1 transcript levels in China through interlaboratory comparison. Methods: Peking University People's Hospital (PKUPH) prepared the samples for comparison. That is, the fresh RUNX1-RUNX1T1 positive (+) bone morrow nucleated cells were serially diluted with RUNX1-RUNX1T1 negative (-) nucleated cells from different patients. Totally 23 sets with 14 different samples per set were prepared. TRIzol reagent was added in each tube and thoroughly mixed with cells for homogenization. Each laboratory simultaneously tested RUNX1-RUNX1T1 and WT1 transcript levels of one set of samples by real-time quantitative PCR method. All transcript levels were reported as the percentage of RUNX1-RUNX1T1 or WT1 transcript copies/ABL copies. Spearman correlation coefficient between the reported transcript levels of each participated laboratory and those of PKUPH was calculated. Results: ①RUNX1-RUNX1T1 comparison: 9 samples were (+) and 5 were (-) , the false negative and positive rates of the 20 participated laboratories were 0 (0/180) and 5% (5/100) , respectively. The reported transcript levels of all 9 positive samples were different among laboratories. The median reported transcript levels of 9 positive samples were from 0.060% to 176.7%, which covered 3.5-log. The ratios of each sample's highest to the lowest reported transcript levels were from 5.5 to 12.3 (one result which obviously deviated from other laboratories' results was not included) , 85% (17/20) of the laboratories had correlation coefficient ≥0.98. ②WT1 comparison: The median reported transcript levels of all 14 samples were from 0.17% to 67.6%, which covered 2.6-log. The ratios of each sample's highest to the lowest reported transcript levels were from 5.3-13.7, 62% (13/21) of the laboratories had correlation coefficient ≥0.98. ③ The relative relationship of the reported RUNX1-RUNX1T1 transcript levels between the participants and PKUPH was not always consistent with that of WT1 transcript levels. Both RUNX1-RUNX1T1 and WT1 transcript levels from 2 and 7 laboratories were individually lower than and higher than those of PKUPH, whereas for the rest 11 laboratories, one transcript level was higher than and the other was lower than that of PKUPH. Conclusion: The reported RUNX1-RUNX1T1 and WT1 transcript levels were different among laboratories for the same sample. Most of the participated laboratories reported highly consistent result with that of PKUPH. The relationship between laboratories of the different transcript levels may not be the same.


Assuntos
Leucemia Mieloide Aguda , China , Subunidade alfa 2 de Fator de Ligação ao Core , Humanos , Proteína 1 Parceira de Translocação de RUNX1 , Reação em Cadeia da Polimerase em Tempo Real , Transcrição Genética , Proteínas WT1
12.
Zhonghua Yi Xue Za Zhi ; 99(48): 3814-3818, 2019 Dec 24.
Artigo em Chinês | MEDLINE | ID: mdl-31874520

RESUMO

Objective: To investigate the effects of miR-106a-5p on the proliferation and migration of vascular endothelial cells, and the possible target gene of miR-106a-5p in endothelial cells. Methods: Human umbilical vein endothelial cells (HUVEC) were cultured in vitro, and the expression of mir-106a-5p in HUVECs was detected by real-time fluorescent quantitative polymerase chain reaction (RT-qPCR). Cell counting kit-8 (CCK-8) and wound healing assays were used to detected the proliferation and migration of HUVECs respectively.Dual luciferase repoter assay was used to identify the possible target gene of miR-106a-5p--STAT3. Results: mir-106a-5p inhibited the proliferation function of HUVECs (F=13.62, P<0.01). And mir-106a-5p expressed the migration of HUVEC,the closure rate in the mimic group was reduced after 12 h and 24 h of scratching (49.93/31.31) (χ(2)=8.240, P<0.05), (78.87/44.80) (χ(2)=10.50, P<0.01). The direct target gene of mir-106a-5p was STAT3, and miR-106a-5p regulated the expression of STAT3 through post-transcriptionally controlling. Conclusion: mir-106a-5p could inhibit proliferation and migration of HUVEC, and the possible target gene was STAT3.


Assuntos
Células Endoteliais da Veia Umbilical Humana , Humanos , MicroRNAs , Reação em Cadeia da Polimerase em Tempo Real , Fator de Transcrição STAT3 , Veias Umbilicais
13.
Medicine (Baltimore) ; 98(50): e17814, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31852062

RESUMO

The purpose of our research was to evaluate diagnostic performance of serum microRNA-135a (miR-135a) in non-small cell lung cancer (NSCLC).Quantitative real time-polymerase chain reaction was employed to detect the expression serum of miR-135a in NSCLC patients and controls. The influence of serum miR-135a level on clinical characteristics of NSCLC patients was explored through the Chi-square test. Serum carcinoembryonic antigen (CEA) level was estimated via enzyme-linked immunosorbent assay. Receiver operating characteristic (ROC) curve was plotted to elucidate diagnostic roles of serum miR-135a and CEA in NSCLC.The expression level of serum miR-135a was significantly lower in NSCLC patients than in healthy controls (0.40 ±â€Š0.29 vs 1.00 ±â€Š0.40, P < .001). Moreover, miR-135a expression was related to lymph node metastasis (P = .021), tumor differentiation (P = .020), and tumor node metastasis stage (P = .031). ROC curve showed serum miR-135a level could discriminate NSCLC patients from healthy controls (P < .0001) with a corresponding cutoff value of 0.665, and a sensitivity and specificity of 81.3% and 83.1%, respectively. The area under the curve was 0.888. In diagnosis analysis on the combination of miR-135a and CEA, when its specificity was maintained at 90%, diagnosis cut-off point reached 0.678.Serum miR-135a level is significantly downregulated in NSCLC and serves as a potential diagnostic biomarker for the disease.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/diagnóstico , Neoplasias Pulmonares/diagnóstico , MicroRNAs/sangue , Estadiamento de Neoplasias/métodos , Idoso , Biomarcadores Tumorais/sangue , Carcinoma Pulmonar de Células não Pequenas/sangue , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Neoplasias Pulmonares/sangue , Masculino , Pessoa de Meia-Idade , RNA Neoplásico/genética , Curva ROC , Reação em Cadeia da Polimerase em Tempo Real
14.
Mikrobiyol Bul ; 53(4): 355-363, 2019 Oct.
Artigo em Turco | MEDLINE | ID: mdl-31709933

RESUMO

One of the most important steps for the control of tuberculosis is rapid and accurate detection of Mycobacterium tuberculosis in clinical samples. The early and accurate diagnosis of tuberculosis allows the initiation of the effective treatment regimen as early as possible. However the early diagnosis of tuberculosis can be achieved by the integration of molecular methods into the diagnostic algorithm of tuberculosis together with the gold standard culture methods. For this reason, molecular methods have become valuable diagnostic tools in routine diagnostic laboratories in recent years. The aim of this study was to determine the diagnostic efficacy of Anyplex MTB/NTM test (Seegene, South Korea) used for the molecular diagnosis of tuberculosis in routine molecular diagnostic laboratories. In addition to this aim, a preliminary evaluation of in-house polymerase chain reaction (PCR) primers that was designed to produce a kit as an alternative against imported commercial kits was performed. Ten thousand six hundred fifthy two clinical specimens that were collected from suspected tuberculosis cases in three years were included in the study. All samples were tested by microscopic examination after staining, culture and real-time PCR (Rt-PCR) methods. The smears were examined by microscope after staining with Kinyoun method for the existence of acid resistant bacilli. For culture, following the N-acetyl-L-sistein-sodium hydroxide homogenization and decontamination procedure, the samples were inoculated into the MGIT (Mycobacteria Growth Indicator Tube) tubes (Becton Dickinson, USA). Rt-PCR method was performed by using Anyplex MTB/NTM test. In the first stage of the study, the performance of the Anyplex MTB/NTM test was compared with the gold standard culture method. M.tuberculosis was isolated in 178 specimens out of 10.652 (1.7%). After the comparison with the gold standard culture method, the sensitivity and specificity of Anyplex MTB/NTM test was found to be 84% and 99% respectively in pulmonary samples, and 74% and 99% respectively in extrapulmonary samples. In the second stage of the study, PCR method with laboratory designed primers was applied to 100 culture positive samples. The PCR results of 98 samples were found to be in agreement with the culture, while M.tuberculosis DNA was not detected in two samples. As a result of the study it was concluded that Anyplex MTB/NTM test is a rapid, practical and reliable method that can be used in routine tuberculosis diagnosis. The high agreement between PCR method using the laboratory-designed primers and the PCR method used in routine practice will lighten the way for the development of national tuberculosis molecular diagnostic kits with a relevant cost. In this way, it will be possible to perform rapid diagnosis in a more cost-effective manner in routine diagnosis laboratories.


Assuntos
Mycobacterium tuberculosis , Tuberculose , Humanos , Reação em Cadeia da Polimerase em Tempo Real/normas , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Tuberculose/diagnóstico , Tuberculose/microbiologia
15.
Mikrobiyol Bul ; 53(4): 442-450, 2019 Oct.
Artigo em Turco | MEDLINE | ID: mdl-31709941

RESUMO

Toxoplasmosis is one of the most common parasitic infection in humans. Serological and molecular methods are used for diagnosis. Molecular methods are becoming increasingly preferred, since they lead to shortening of diagnostic time. In our study, it was aimed to determine Toxoplasma gondii by a cost-effective, quantitative, fast and reliable method without using a commercial kit, and apply method verification. T.gondii strain which was continued by mouse inoculation in our laboratory was used for method verification study. For this purpose DNA extraction was performed using a commercial kit. The limit of detection and, high and low positivity rates were determined by serial dilutions of DNA sample. Accuracy and certainty studies were performed using with TG-F, TG-R primers and TaqMan TG probe for method verification of the test. In the study with serial dilutions of DNA sample, detection limit was determined as 10-3 dilutions (0.028 copies/reaction). Furthermore 10-1 dilution (2.8 copies/reaction) was considered as high positive, 10-2 dilution (0.28 copies/reaction) was considered as low positive and method verification studies were performed. The accuracy of test was determined as 0.62 for high positive samples and 0.14 for low positive samples. CV value of intra-assay certainty was 0.62 for high positive samples and 0.14 for low positive samples, whereas, CV value of inter-assay certainty was calculated as 1.03 for high positive samples and 2.34 for low positive samples. Correlation coefficient was determined as 0.99. The coefficient of variation of inhouse realtime PCR method used in our study was found to be below 15%, and it was decided to be suitable for routine laboratory studies.


Assuntos
Reação em Cadeia da Polimerase em Tempo Real , Toxoplasma , Toxoplasmose , Animais , Primers do DNA , DNA de Protozoário , Humanos , Camundongos , Parasitologia/métodos , Reação em Cadeia da Polimerase em Tempo Real/normas , Sensibilidade e Especificidade , Toxoplasma/genética , Toxoplasmose/diagnóstico
16.
Zhonghua Bing Li Xue Za Zhi ; 48(11): 873-877, 2019 Nov 08.
Artigo em Chinês | MEDLINE | ID: mdl-31775437

RESUMO

Objective: To study common problems in BRAF gene mutation detection, and conditions for repetition testing using thyroid fine needle aspiration specimens. Methods: A total of 8 644 cases of thyroid fine-needle aspiration specimens at China-Japan Friendship Hospital were collected between February, 2012 and July, 2018. BRAF gene mutation was detected by real-time PCR. Repeat testing was performed in 237 cases when the results were inconsistent with clinical or cytological diagnosis or when uncertain results were obtained. Results: The final positive rates of BRAF mutation was 22.0% (1 897/8 625). Nineteen cases were excluded due to inadequate DNA samples. The average Ct value of internal quality control was 16.061, and the average Ct value of the positive samples was 19.147. Among 237 repeat tests, 51.4% (19/37) continued to have poor DNA quality and 48.6% (18/37) had adequate DNA resulting in 1 positive case and 17 negative cases. In 40 repetition of initial negative cases, results were unchanged. In initial positive cases, 40.4% (40/99) with a difference of Ct value (between BRAF gene and internal quality control) between 8 to 12 turned negative after repetition, 69.8% (37/53) of these cases with a difference of more than 12 turned negative after repetition. The sensitivity and specificity of BRAF mutation were 83.97% and 96.94%, respectively. Conclusions: Difference between BRAF gene Ct value and internal quality control Ct value is recommended as a reliability index for the test result. Cases with a difference greater than 8 should be subjected to repeat testing.


Assuntos
Proteínas Proto-Oncogênicas B-raf/genética , Nódulo da Glândula Tireoide/genética , Nódulo da Glândula Tireoide/patologia , Biópsia por Agulha Fina , China , Análise Mutacional de DNA , Humanos , Mutação , Reação em Cadeia da Polimerase em Tempo Real , Reprodutibilidade dos Testes
17.
Artigo em Inglês | MEDLINE | ID: mdl-31778390

RESUMO

Public parks are leisure environments widely used by both, adults and children, often accompained by their pets. Soil contamination of these environments by enteric viruses and intestinal parasites occurs through these animals feces. The aim of this work was to detect Carnivore protoparvovirus 1 (CPV-1) and different species of Mastadenovirus in soils samples from a park located in a medium-sized city in Brazil and evaluate the presence of helminth eggs and larvae in 18 points of a public park soil samples, as well as feces found on this site during six months. Parasitological analyzes were conducted through flotation and sedimentation techniques, and polymerase chain reaction (PCR) was used for viral detection. Of the 216 soil and 16 feces samples, 49% (106/216) and 12% (2/16) were positivefor nematodes larvae, respectively, through sedimentation techniques. Toxocara spp eggs were found in one soil sample and one feces sample, Trichuris spp eggs were found in only one feces sample and Hookworms eggs were found in four soil samples. After reconstruction work in the streets near the park, 30% (64/216) of the samples were positive for Human Mastadenovirus C (HAdV-C), 1.4% (3/216) for HAdV-E and 0.4% (1/216) for Canine Mastadenovirus A (CAdV-A). The parasitic forms found in this study have demonstrated that the contamination of the park's soil pose a threat to human and animal health. This was the first study to report the presence of HAdVs and CAdVs in soil samples.


Assuntos
Ancylostomatoidea/isolamento & purificação , Mastadenovirus/isolamento & purificação , Microbiologia do Solo , Solo/parasitologia , Toxocara/isolamento & purificação , Ancylostomatoidea/classificação , Ancylostomatoidea/genética , Animais , Cães , Fezes/parasitologia , Humanos , Mastadenovirus/classificação , Mastadenovirus/genética , Parques Recreativos , Reação em Cadeia da Polimerase em Tempo Real , Toxocara/classificação , Toxocara/genética
18.
Water Sci Technol ; 80(5): 817-826, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31746788

RESUMO

Accurate evaluation of viable Ascaris ova in wastewater is the key to mitigating Ascaris reinfections in endemic regions. In this study, the viability of Ascaris ova in raw wastewater was determined using three different detection methods: culture-based, BacLight Live/Dead staining and propidium monoazide-quantitative polymerase chain reaction (PMA-qPCR). Furthermore, comparative assessment of viability utilising the aforementioned detection methods was performed using seeded experiments in wastewater. The percentage of viability was: culture-based (82%), BacLight Live/Dead staining (87%) and PMA-qPCR (85%) respectively. Despite the fact that no statistical difference was shown in the viability determination among the three methods, PMA-qPCR-based viability determination would be preferable over the other two methods for evaluating potential public health risks with A. suum ova due to its accuracy, being least subjective and its rapid reaction time.


Assuntos
Ascaris , Águas Residuárias , Animais , Azidas , Viabilidade Microbiana , Propídio , Reação em Cadeia da Polimerase em Tempo Real , Coloração e Rotulagem
19.
Klin Lab Diagn ; 64(11): 700-704, 2019.
Artigo em Russo | MEDLINE | ID: mdl-31747502

RESUMO

The reagent kit AmpligenBurk-mallei/pseudomallei-RT PCR is designed for detecting in vitro diagnostics and differentiate the DNA of glanders and melioidosis pathogens by real-time multiplex PCR in biological (clinical) material and cultures of microorganisms, as well as environmental objects and solid food products (rice). During clinical testing diagnostic value of reagent kit AmpligenBurk-mallei/pseudomallei-RT PCR has been studied. Based on the results obtained, a high analytical sensitivity (1×103 microbe cells/ml) and specificity (100%) of PCR-RT with the developed reagent kit were established, regardless of the type of material being studied. The diagnostic sensitivity of PCR-RT using a set of reagents was at least 98.0% and specificity at least 99%. The stages of state examination have been completed, a registration certificate has been obtained at Roszdravnadzor, production, sale and use of reagent kit in medical laboratory practice have been permitted.


Assuntos
Mormo/diagnóstico , Melioidose/diagnóstico , Kit de Reagentes para Diagnóstico/normas , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Animais , Burkholderia mallei , Burkholderia pseudomallei , Cavalos , Sensibilidade e Especificidade
20.
Braz J Med Biol Res ; 52(10): e8385, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31618367

RESUMO

Malignant melanoma (MM) is one of the malignant tumors with highly metastatic and aggressive biological actions. Schizandrin A (SchA) is a bioactive lignin compound with strong anti-oxidant and anti-aging properties, which is stable at room temperature and is often stored in a cool dry place. Hence, we investigated the effects of SchA on MM cell line A375 and its underlying mechanism. A375 cells were used to construct an in vitro MM cell model. Cell viability, proliferation, apoptosis, and migration were detected by Cell Counting Kit-8, BrdU assay, flow cytometry, and transwell two-chamber assay, respectively. The cell cycle-related protein cyclin D1 and cell apoptotic proteins (Bcl-2, Bax, cleaved-caspase-3, and cleaved-caspase-9) were analyzed by western blot. Alteration of H19 expression was achieved by transfecting with pEX-H19. PI3K/AKT pathway was measured by detecting phosphorylation of PI3K and AKT. SchA significantly decreased cell viability in a dose-dependent manner. Furthermore, SchA inhibited cell proliferation and cyclin D1 expression. SchA increased cell apoptosis along with the up-regulation of pro-apoptotic proteins (cleaved-caspase-3, cleaved-caspase-9, and Bax) and the down-regulation of anti-apoptotic protein (Bcl-2). Besides, SchA decreased migration and down-regulated matrix metalloproteinases (MMP)-2 and MMP-9. SchA down-regulated lncRNA H19. Overexpression of H19 blockaded the inhibitory effects of SchA on A375 cells. SchA decreased the phosphorylation of PI3K and AKT while H19 overexpression promoted the phosphorylation of PI3K and AKT. SchA inhibited A375 cell growth, migration, and the PI3K/AKT pathway through down-regulating H19.


Assuntos
Apoptose/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Ciclo-Octanos/farmacologia , Regulação para Baixo/efeitos dos fármacos , Lignanas/farmacologia , Melanoma/patologia , Compostos Policíclicos/farmacologia , Western Blotting , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , MicroRNAs/metabolismo , RNA Longo não Codificante , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/efeitos dos fármacos
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