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1.
Viruses ; 13(4)2021 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-33810324

RESUMO

Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) to detect SARS-CoV-2 RNA is an essential test to monitor the occurrence of COVID-19. A methodology is proposed for the determination of maximum pool size and adjustments of cut-off values of cycle threshold (Ct in RT-qPCR pool testing, to compensate for the dilution caused by pooling. The trade-off between pool size and test sensitivity is stated explicitly. The procedure was designed to ensure that samples that would be detectable in individual testing remain detectable in pool testing. The proposed relaxation in cut-off is dependent on the pool size, allowing a relatively tight correction to avoid loss of detection of positive samples. The methodology was evaluated in a study of pool testing of adults attending a public emergency care unit, reference for COVID-19 in Belo Horizonte, Brazil, and presenting flu-like symptoms. Even samples on the edge of detectability in individual testing were detected correctly. The proposed procedure enhances the consistency of RT-qPCR pool testing by enforcing that the scales of detectability in pool processing and in individual sample processing are compatible. This may enhance the contribution of pool testing to large-scale testing for COVID-19.


Assuntos
/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , /genética , Adulto , Idoso , Idoso de 80 Anos ou mais , /normas , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase em Tempo Real/normas , /fisiologia , Adulto Jovem
2.
Methods Mol Biol ; 2238: 293-312, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33471340

RESUMO

With a widely established use of quantitative real-time PCR (qRT-PCR) for gene expression analysis, reliable and stable expression of reference genes is often discussed. Suitable reference genes should show less variation of expression across the target samples and allow for error minimization by normalization of qRT-PCR data. Therefore, selection of reliable reference genes is essential for accurate results and to support the conclusions drawn on expression levels of genes under study. In this chapter, we describe the workflow for selection and evaluation of reference genes in rice, including identification of candidate genes by using Genevestigator® and evaluation of expression stability using various algorithms. The ranking of the genes guides qRT-PCR performance and data analysis. This protocol used rice as an example but is not limited to rice, and could be applied to other species as well.


Assuntos
Algoritmos , Oryza/metabolismo , Proteínas de Plantas/metabolismo , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase em Tempo Real/normas , Técnicas Genéticas , Oryza/crescimento & desenvolvimento , Padrões de Referência
3.
Methods Mol Biol ; 2195: 225-243, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32852767

RESUMO

Reverse transcription (RT) based quantitative PCR (qPCR) for quantifying microRNAs (miRNAs) in in the circulation presents specific challenges. Here, we describe an optimized research protocol to assess serum sample quality and quantify levels of a panel of four test miRNAs (miR-371a-3p, miR-372-3p, miR-373-3p, and miR-367-3p) that enables highly sensitive and specific malignant germ cell tumor (GCT) diagnosis and monitoring. This protocol utilizes a multiplex RT step using Taqman miRNA stem-loop primers. A multiplexed preamplification stage is then employed to increase the sensitivity of the final quantification step, which is performed using standard singleplex Taqman qPCR methodology.


Assuntos
Biomarcadores Tumorais , MicroRNA Circulante , Neoplasias Embrionárias de Células Germinativas/diagnóstico , Neoplasias Embrionárias de Células Germinativas/genética , Interpretação Estatística de Dados , Humanos , Reação em Cadeia da Polimerase Multiplex , Neoplasias Embrionárias de Células Germinativas/sangue , Técnicas de Amplificação de Ácido Nucleico , Prognóstico , Controle de Qualidade , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase em Tempo Real/normas , Fluxo de Trabalho
4.
PLoS One ; 15(12): e0244023, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33347478

RESUMO

BACKGROUND: PCR is more sensitive than immunofluorescence assay (IFA) for detection of Pneumocystis jirovecii. However, PCR cannot always distinguish infection from colonization. This study aimed to compare the performance of real-time PCR and IFA for diagnosis of P. jirovecii pneumonia (PJP) in a real-world clinical setting. METHODS: A retrospective cohort study was conducted at a 1,300-bed hospital between April 2017 and December 2018. Patients whose respiratory sample (bronchoalveolar lavage or sputum) were tested by both Pneumocystis PCR and IFA were included. Diagnosis of PJP was classified based on multicomponent criteria. Sensitivity, specificity, 95% confidence intervals (CI), and Cohen's kappa coefficient were calculated. RESULTS: There were 222 eligible patients. The sensitivity and specificity of PCR was 91.9% (95% CI, 84.0%-96.7%) and 89.7% (95% CI, 83.3%-94.3%), respectively. The sensitivity and specificity of IFA was 7.0% (95% CI, 2.6%-14.6%) and 99.2% (95% CI, 95.6%-100.0%), respectively. The percent agreement between PCR and IFA was 56.7% (Cohen's kappa -0.02). Among discordant PCR-positive and IFA-negative samples, 78% were collected after PJP treatment. Clinical management would have changed in 14% of patients using diagnostic information, mainly based on PCR results. CONCLUSIONS: PCR is highly sensitive compared with IFA for detection of PJP. Combining clinical, and radiological features with PCR is useful for diagnosis of PJP, particularly when respiratory specimens cannot be promptly collected before initiation of PJP treatment.


Assuntos
Imunofluorescência/métodos , Técnicas de Diagnóstico Molecular/métodos , Pneumonia por Pneumocystis/diagnóstico , Reação em Cadeia da Polimerase em Tempo Real/métodos , Adulto , Idoso , Líquido da Lavagem Broncoalveolar/microbiologia , Feminino , Imunofluorescência/normas , Humanos , Masculino , Pessoa de Meia-Idade , Técnicas de Diagnóstico Molecular/normas , Pneumocystis/genética , Pneumocystis/isolamento & purificação , Pneumocystis/patogenicidade , Pneumonia por Pneumocystis/microbiologia , Reação em Cadeia da Polimerase em Tempo Real/normas , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Escarro/microbiologia
5.
EBioMedicine ; 61: 103036, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-33045467

RESUMO

BACKGROUND: Real-time reverse transcription-PCR (rRT-PCR) has been the most effective and widely implemented diagnostic technology since the beginning of the COVID-19 pandemic. However, fuzzy rRT-PCR readouts with high Ct values are frequently encountered, resulting in uncertainty in diagnosis. METHODS: A Specific Enhancer for PCR-amplified Nucleic Acid (SENA) was developed based on the Cas12a trans-cleavage activity, which is specifically triggered by the rRT-PCR amplicons of the SARS-CoV-2 Orf1ab (O) and N fragments. SENA was first characterized to determine its sensitivity and specificity, using a systematic titration experiment with pure SARS-CoV-2 RNA standards, and was then verified in several hospitals, employing a couple of commercial rRT-PCR kits and testing various clinical specimens under different scenarios. FINDINGS: The ratio (10 min/5 min) of fluorescence change (FC) with mixed SENA reaction (mix-FCratio) was defined for quantitative analysis of target O and N genes, and the Limit of Detection (LoD) of mix-FCratio with 95% confidence interval was 1.2≤1.6≤2.1. Totally, 295 clinical specimens were analyzed, among which 21 uncertain rRT-PCR cases as well as 4 false negative and 2 false positive samples were characterized by SENA and further verified by next-generation sequencing (NGS). The cut-off values for mix-FCratio were determined as 1.145 for positive and 1.068 for negative. INTERPRETATION: SENA increases both the sensitivity and the specificity of rRT-PCR, solving the uncertainty problem in COVID-19 diagnosis and thus providing a simple and low-cost companion diagnosis for combating the pandemic. FUNDING: Detailed funding information is available at the end of the manuscript.


Assuntos
Proteínas de Bactérias/metabolismo , Betacoronavirus/genética , Proteínas Associadas a CRISPR/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Endodesoxirribonucleases/metabolismo , RNA Viral/metabolismo , Reação em Cadeia da Polimerase em Tempo Real/métodos , Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Humanos , Limite de Detecção , Cavidade Nasal/virologia , Técnicas de Amplificação de Ácido Nucleico/métodos , Técnicas de Amplificação de Ácido Nucleico/normas , Proteínas do Nucleocapsídeo/genética , Proteínas do Nucleocapsídeo/metabolismo , Pandemias , Fosfoproteínas , Pneumonia Viral/diagnóstico , Pneumonia Viral/patologia , Pneumonia Viral/virologia , Poliproteínas , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real/normas , Padrões de Referência , Proteínas Virais/genética , Proteínas Virais/metabolismo
6.
Sci Rep ; 10(1): 17258, 2020 10 14.
Artigo em Inglês | MEDLINE | ID: mdl-33057113

RESUMO

Quantitative real time polymerase chain reaction (qPCR) data are normalised using endogenous control genes. We aimed to: (1) demonstrate a pathway to identify endogenous control genes for qPCR analysis of formalin-fixed paraffin-embedded (FFPE) tissue using bladder cancer as an exemplar; and (2) examine the influence of probe length and sample age on PCR amplification and co-expression of candidate genes on apparent expression stability. RNA was extracted from prospective and retrospective samples and subject to qPCR using TaqMan human endogenous control arrays or single tube assays. Gene stability ranking was assessed using coefficient of variation (CoV), GeNorm and NormFinder. Co-expressed genes were identified from The Cancer Genome Atlas (TCGA) using the on-line gene regression analysis tool GRACE. Cycle threshold (Ct) values were lower for prospective (19.49 ± 2.53) vs retrospective (23.8 ± 3.32) tissues (p < 0.001) and shorter vs longer probes. Co-expressed genes ranked as the most stable genes in the TCGA cohort by GeNorm when analysed together but ranked lower when analysed individually omitting co-expressed genes indicating bias. Stability values were < 1.5 for the 20 candidate genes in the prospective cohort. As they consistently ranked in the top ten by CoV, GeNorm and Normfinder, UBC, RPLP0, HMBS, GUSB, and TBP are the most suitable endogenous control genes for bladder cancer qPCR.


Assuntos
Regulação Neoplásica da Expressão Gênica , Neoplasias/patologia , Humanos , Neoplasias/metabolismo , Inclusão em Parafina , Estudos Prospectivos , RNA/metabolismo , RNA/normas , Reação em Cadeia da Polimerase em Tempo Real/normas , Padrões de Referência , Análise de Regressão , Estudos Retrospectivos , Proteínas Ribossômicas/genética , Proteína de Ligação a TATA-Box/genética
7.
Genes (Basel) ; 11(10)2020 10 12.
Artigo em Inglês | MEDLINE | ID: mdl-33053675

RESUMO

WHO declared the novel coronavirus (COVID-19) outbreak a global pandemic on 11 March 2020. The establishment of standardized RT-qPCR protocols for respiratory secretions testing, as well as sharing of specimens, data, and information became critical. Here, we investigate the analytical performance of two interim RT-qPCR protocols (Charité and Centers for Disease Control (CDC)) for the qualitative detection of SARS-CoV-2 executed in a fully automated platform. Analytical specificity, PCR amplification efficiency, analytical sensitivity (limit of detection), and cross-reactivity were evaluated using contrived samples. The on-going accuracy was evaluated by retrospective analysis of our test results database (real clinical samples). N1, E, and a modified version of RdRP assays presented adequate analytical specificity, amplification efficiency, and analytical sensitivity using contrived samples. The three assays were applied to all individuals who requested the SARS-CoV-2 molecular test assay in our laboratory and it was observed that N1 gave more positive results than E, and E gave more positive results than RdRP (modified). The RdRP and E were removed from the test and its final version, based on N1 assay only, was applied to 30,699 Brazilian individuals (from 19 February 2020 to 8 May 2020). The aggregated test results available in the database were also presented.


Assuntos
Automação Laboratorial/normas , Técnicas de Laboratório Clínico/normas , Reação em Cadeia da Polimerase em Tempo Real/normas , Reação em Cadeia da Polimerase Via Transcriptase Reversa/normas , Automação Laboratorial/métodos , Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/diagnóstico , Humanos , Limite de Detecção , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos
8.
PLoS One ; 15(9): e0239161, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32915926

RESUMO

The middle ear is a small and hard to reach compartment, limiting the amount of tissue that can be extracted and the possibilities for studying the molecular mechanisms behind diseases like cholesteatoma. In this paper 14 reference gene candidates were evaluated in the middle ear mucosa of cholesteatoma patients and two different control tissues. ACTB and GAPDH were shown to be the optimal genes for the normalisation of target gene expression when investigating middle ear mucosa in multiplex qPCR analysis. Validation of reference genes using c-MYC expression confirmed the suitability of ACTB and GAPDH as reference genes and showed an upregulation of c-MYC in middle ear mucosa during cholesteatoma. The occurrence of participants of the innate immunity, TLR2 and TLR4, were analysed in order to compare healthy middle ear mucosa to cholesteatoma. Analysis of TLR2 and TLR4 showed variable results depending on control tissue used, highlighting the importance of selecting relevant control tissue when investigating causes for disease. It is our belief that a consensus regarding reference genes and control tissue will contribute to the comparability and reproducibility of studies within the field.


Assuntos
Colesteatoma da Orelha Média/genética , Orelha Média/patologia , Membrana Mucosa/patologia , Reação em Cadeia da Polimerase em Tempo Real/normas , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Colesteatoma da Orelha Média/imunologia , Colesteatoma da Orelha Média/patologia , Colesteatoma da Orelha Média/cirurgia , Orelha Média/imunologia , Orelha Média/cirurgia , Feminino , Regulação da Expressão Gênica/imunologia , Humanos , Masculino , Pessoa de Meia-Idade , Membrana Mucosa/imunologia , Membrana Mucosa/cirurgia , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/imunologia , Padrões de Referência , Reprodutibilidade dos Testes , Receptor 2 Toll-Like/genética , Receptor 2 Toll-Like/imunologia , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/imunologia , Adulto Jovem
9.
J Clin Lab Anal ; 34(10): e23554, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32977349

RESUMO

BACKGROUND: To compare the diagnostic efficacy between two different real-time reverse transcription polymerase chain reaction (RT-PCR) test kits for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleic acid detection and provide references for laboratories. METHODS: Throat swab samples from 18 hospitalized patients were clinically diagnosed with coronavirus disease 2019 (COVID-19) and 100 hospitalized patients without COVID-19 were collected. SARS-CoV-2 nucleic acid was detected in throat swab samples with RT-PCR test kits from Sansure Biotech Inc (Hunan, China) and Shanghai BioGerm Medical Biotechnology Co., Ltd.(Shanghai, China). The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and kappa value were analyzed, and three parallel tests were performed with three weakly positive samples. RESULTS: The sensitivity, specificity, PPV, NPV, and kappa value of the Sansure PCR kit were 0.833, 1.000, 1.000, 0.971, and 0.894, respectively, and the sensitivity, specificity, PPV, NPV, and kappa value of the BioGerm PCR kit were 0.944, 1.000, 1.000, 0.990, and 0.966, respectively. For the three parallel tests, the coefficient of variation value of the BioGerm PCR kit in all three samples was the smallest for both the ORF1ab and N gene. CONCLUSION: The detection efficacy of the BioGerm PCR kit for SARS-CoV-2 nucleic acid detection was relatively higher than that of the Sansure PCR kit.


Assuntos
Técnicas de Laboratório Clínico , Infecções por Coronavirus/diagnóstico , Pneumonia Viral/diagnóstico , Reação em Cadeia da Polimerase em Tempo Real , Betacoronavirus/genética , Técnicas de Laboratório Clínico/métodos , Técnicas de Laboratório Clínico/normas , Humanos , Pandemias , Faringe/virologia , Valor Preditivo dos Testes , RNA Viral/análise , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase em Tempo Real/normas
10.
J Bras Nefrol ; 42(2 suppl 1): 4-8, 2020 Aug 26.
Artigo em Inglês, Português | MEDLINE | ID: mdl-32877490

RESUMO

The Covid-19 pandemic brought several challenges to the healthcare system: diagnosis, treatment and measures to prevent the spread of the disease. With the greater availability and variety of diagnostic tests, it is essential to properly interpret them. This paper intends to help dialysis units concerning the use of clinical criteria and diagnostic tests for decision making regarding the discontinuation of isolation of patients with suspected or confirmed Covid-19, as well as the return to work activities for employees with suspected or confirmed Covid-19.


Assuntos
Betacoronavirus , Técnicas de Laboratório Clínico/normas , Infecções por Coronavirus/diagnóstico , Nefrologia/normas , Pneumonia Viral/diagnóstico , Diálise Renal , Retorno ao Trabalho , Algoritmos , Brasil , Lista de Checagem , Tomada de Decisão Clínica , Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/epidemiologia , Humanos , Doenças Profissionais/diagnóstico , Pandemias , Isolamento de Pacientes , Pneumonia Viral/epidemiologia , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase em Tempo Real/normas , Sociedades Médicas/normas , Unidade Hospitalar de Urologia/normas
11.
Ann Oncol ; 31(10): 1320-1335, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32745693

RESUMO

We established an international consortium to review and discuss relevant clinical evidence in order to develop expert consensus statements related to cancer management during the severe acute respiratory syndrome coronavirus 2-related disease (COVID-19) pandemic. The steering committee prepared 10 working packages addressing significant clinical questions from diagnosis to surgery. During a virtual consensus meeting of 62 global experts and one patient advocate, led by the European Society for Medical Oncology, statements were discussed, amended and voted upon. When consensus could not be reached, the panel revised statements until a consensus was reached. Overall, the expert panel agreed on 28 consensus statements that can be used to overcome many of the clinical and technical areas of uncertainty ranging from diagnosis to therapeutic planning and treatment during the COVID-19 pandemic.


Assuntos
Betacoronavirus , Consenso , Infecções por Coronavirus/terapia , Oncologia/normas , Neoplasias/terapia , Pneumonia Viral/terapia , Sociedades Médicas/normas , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/imunologia , Gerenciamento Clínico , Europa (Continente)/epidemiologia , Fator Estimulador de Colônias de Granulócitos/farmacologia , Fator Estimulador de Colônias de Granulócitos/uso terapêutico , Humanos , Oncologia/métodos , Neoplasias/epidemiologia , Neoplasias/imunologia , Pandemias/prevenção & controle , Pneumonia Viral/epidemiologia , Pneumonia Viral/imunologia , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase em Tempo Real/normas , Linfócitos T Citotóxicos/efeitos dos fármacos , Linfócitos T Citotóxicos/imunologia , Telemedicina/métodos , Telemedicina/normas
12.
Int J Mol Sci ; 21(16)2020 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-32784770

RESUMO

Sensitive molecular assays are critical for coronavirus disease 2019 (COVID-19) diagnosis. Here, we designed and evaluated two single-tube nested (STN) real-time RT-PCR assays, targeting SARS-CoV-2 RdRp/Hel and N genes. Both STN assays had a low limit of detection and did not cross react with other human coronaviruses and respiratory viruses. Using 213 initial respiratory specimens from suspected COVID-19 patients, the sensitivity of both the STN COVID-19-RdRp/Hel and the STN COVID-19-N assays was 100% (99/99), while that of the comparator non-nested N assay was 95% (94/99). Among 108 follow-up specimens from confirmed COVID-19 patients who tested negative by the non-nested COVID-19-RdRp/Hel assay, 28 (25.9%) were positive for SARS-CoV-2 by the STN COVID-19-RdRp/Hel or the STN COVID-19-N assay. To evaluate the performance of our novel STN assays in pooled specimens, we created four sample pools, with each pool consisting of one low positive specimen and 49 negative specimens. While the non-nested COVID-19-RdRp/Hel assay was positive in only one of four sample pools (25%), both of the STN assays were positive in two of four samples pools (50%). In conclusion, the STN assays are highly sensitive and specific for SARS-CoV-2 detection. Their boosted sensitivity offers advantages in non-traditional COVID-19 testing algorithms such as saliva screening and pooled sample screening during massive screening.


Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/virologia , Técnicas de Diagnóstico Molecular/métodos , Pneumonia Viral/virologia , Reação em Cadeia da Polimerase em Tempo Real/métodos , Betacoronavirus/patogenicidade , Infecções por Coronavirus/diagnóstico , Humanos , Técnicas de Diagnóstico Molecular/normas , Proteínas do Nucleocapsídeo/genética , Pandemias , Fosfoproteínas , Pneumonia Viral/diagnóstico , Reação em Cadeia da Polimerase em Tempo Real/normas , Mucosa Respiratória/virologia , Sensibilidade e Especificidade , Proteínas não Estruturais Virais/genética
13.
PLoS One ; 15(8): e0236338, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32785215

RESUMO

Dysregulation of BCL2 is a pathophysiology observed in haematological malignancies. For implementation of available treatment-options it is preferred to know the relative quantification of BCL2 mRNA with appropriate reference genes. For the choice of reference genes-(i) Reference Genes were selected by assessing variation of >60,000 genes from 4 RNA-seq datasets of haematological malignancies followed by filtering based on their GO biological process annotations and proximity of their chromosomal locations to known disease translocations. Selected genes were experimentally validated across various haematological malignancy samples followed by stability comparison using geNorm, NormFinder, BestKeeper and RefFinder. (ii) 43 commonly used Reference Genes were obtained from literature through extensive systematic review. Levels of BCL2 mRNA was assessed by qPCR normalized either by novel reference genes from this study or GAPDH, the most cited reference gene in literature and compared. The analysis showed PTCD2, PPP1R3B and FBXW9 to be the most unregulated genes across lymph-nodes, bone marrow and PBMC samples unlike the Reference Genes used in literature. BCL2 mRNA level shows a consistent higher expression in haematological malignancy patients when normalized by these novel Reference Genes as opposed to GAPDH, the most cited Reference Gene. These reference genes should also be applicable in qPCR platforms using Taqman probes and other model systems including cell lines and rodent models. Absence of sample from healthy-normal individual in diagnostic cases call for careful selection of Reference Genes for relative quantification of a biomarker by qPCR.BCL2 can be used as molecular diagnostics only if normalized with a set of reference genes with stable yet low levels of expression across different types of haematological malignancies.


Assuntos
Biomarcadores Tumorais/isolamento & purificação , Neoplasias Hematológicas/diagnóstico , Proteínas Proto-Oncogênicas c-bcl-2/isolamento & purificação , RNA Mensageiro/isolamento & purificação , RNA-Seq/normas , Animais , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/genética , Medula Óssea/patologia , Linhagem Celular Tumoral , Conjuntos de Dados como Assunto , Modelos Animais de Doenças , Estudos de Viabilidade , Regulação Neoplásica da Expressão Gênica , Genes Essenciais , Neoplasias Hematológicas/sangue , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/patologia , Humanos , Leucócitos Mononucleares , Proteínas Proto-Oncogênicas c-bcl-2/sangue , Proteínas Proto-Oncogênicas c-bcl-2/genética , RNA Mensageiro/sangue , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real/normas , Padrões de Referência
14.
Mikrobiyol Bul ; 54(3): 418-428, 2020 Jul.
Artigo em Turco | MEDLINE | ID: mdl-32755518

RESUMO

Pneumocystis jirovecii is a human-specific species and causes fatal infections like P.jirovecii pneumonia (PCP) in immunocompromised persons. Although direct microscopy is the gold standard in the diagnosis of the microorganism, molecular methods such as polymerase chain reaction (PCR) are needed in non-human immune deficiency virus (HIV) immunosuppresive patients with low P.jirovecii burden. In this study, we aimed to evaluate the value of real-time PCR (Rt-PCR) in the laboratory diagnosis of P.jirovecii. Bronchoalveolar lavage (BAL) specimens of 658 patients sent to Dokuz Eylul University Hospital Central Medical Parasitology Laboratory on suspicion of PCP were included in the study. BAL fluids were evaluated for identification of P.jirovecii mitochondrial gene coding ribosomal large subunit (mtLSUrRNA) using Rt-PCR. In addition, Giemsa and Gomori's methenamine silver (GMG) staining assays were applied to all samples and nested PCR (n-PCR) assay was applied to positive samples detected by real time PCR. Ninety-two (14.3%) of these samples were positive by Rt-PCR. Of these 92 patients, 85 (92.4%) were positive with n-PCR. Only seven of the specimens had P.jirovecii cysts and trophozoites with microscopic examination. The mean cycle threshold (CT ) value of Rt-PCR positive patients was 29.7 (18.17 ≤ CT ≤ 37.96). P.jirovecii load in these patients was calculated as 2.6 x 101-6.15 x 107 copies/ml. The difference between the mean CT values of n-PCR positive and negative results was statistically significant (p< 0.01). The CT values of Rt-PCR of the samples with positive microscopy were; 18.2, 20.9, 22.2, 24.3, 24.7, 26.5, 29.7. The difference between the CT means of the samples with positive and negative microscopy was statistically significant (p< 0.05). When positive patients were grouped according to their diagnosis; the lowest mean CT value (CTmean= 24.8) was found in HIV-positive patients. On the other hand, CT values were found to be significantly lower in the organ transplantation patients (CTmean= 26.15) and in the collagen-vascular-inflammatory patient group (CTmean= 27.8). This study demonstrated that Rt-PCR was the effective method in the diagnosis of P.jirovecii in the laboratory. Conventional n-PCR method was found to be more unsuccessful than Rt-PCR in the presence of very low density organism; direct microscopy is generally found to be positive in samples with a higher burden of P.jirovecii.


Assuntos
Pneumocystis carinii , Pneumonia por Pneumocystis , Reação em Cadeia da Polimerase em Tempo Real , Líquido da Lavagem Broncoalveolar/microbiologia , Técnicas de Laboratório Clínico/normas , Humanos , Pneumocystis carinii/genética , Pneumonia por Pneumocystis/diagnóstico , Pneumonia por Pneumocystis/microbiologia , Reação em Cadeia da Polimerase em Tempo Real/normas , Sensibilidade e Especificidade
15.
Virulence ; 11(1): 964-967, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32726172

RESUMO

Currently, testing for coronavirus is performed with time and personnel consuming PCR assays. The aim of this study was to evaluate the sensitivity, specificity and capacity of a fully automated, random access high-throughput real-time PCR-based diagnostic platform for the detection of SARS-CoV-2. The NeuMoDx N96 system displayed an equal or better detection rate for SARS-CoV-2 compared with the LightCycler 480II system and showed a specificity of 100%. The median PCR run time for all 28 PCR runs was 91 (IQR 84-97) minutes. The capacity of the NeuMoDx N96 could easily surpass the capacity of most currently used molecular test systems and significantly reduce the turn-around time.


Assuntos
Betacoronavirus/isolamento & purificação , Sequenciamento de Nucleotídeos em Larga Escala/métodos , RNA Viral/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/métodos , Betacoronavirus/genética , Sequenciamento de Nucleotídeos em Larga Escala/instrumentação , Sequenciamento de Nucleotídeos em Larga Escala/normas , Humanos , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real/instrumentação , Reação em Cadeia da Polimerase em Tempo Real/normas , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Fatores de Tempo
16.
Mikrobiyol Bul ; 54(2): 306-317, 2020 Apr.
Artigo em Turco | MEDLINE | ID: mdl-32723285

RESUMO

Malaria is a life-threatening parasitic disease caused by the parasites belonging to Plasmodium genus. Microscopic examination of Giemsa stained blood smears is accepted as the gold standard diagnostic method. It is recommended to use more than one method in order to strengthen the laboratory diagnosis of malaria which is an important health problem in our country as in the whole world. In this study, it was aimed to compare the results of three different molecular methods and determine which molecular method could be used in the diagnostic algorithm to be applied. DNA was extracted from 280 whole blood sample stored in EDTA tubes using a commercial kit. Three different polymerase chain reaction (PCR) methods were used for the detection of Plasmodium spp. in DNA samples obtained and the results were compared. First, multiplex nested PCR was applied and then in-house real-time PCR (Rt-PCR) which was validated in our laboratory and a commercial Rt-PCR kit were applied. Multiplex nested PCR was accepted as the gold standard and 182 samples that were evaluated as Plasmodium spp. positive and 98 samples that were evaluated as negative were also studied by in-house and commercial Rt-PCR methods. In multiplex nested PCR's first step reaction 1670 base pairs (bp) band was observed in Plasmodium spp. positive samples and 117 bp band was observed in Plasmodium vivax positive samples in the second step reaction. Tm values of P.vivax positive samples were determined as 78-79 in the melting analysis of the in-house Rt-PCR. CT values of the positive samples in in-house Rt-PCR were between 20.03-31.71 and were between 17.26-34.94 in the commercial Rt-PCR. With the in-house Rt-PCR method 180 cases were determined as positive, while with the commercial Rt-PCR method 178 cases were determined as positive. Two samples with the in-house Rt-PCR and 4 samples with the commercial Rt-PCR were considered as false negative. When the sensitivity and specificity of the both methods were calculated, the sensitivity of the in-house Rt-PCR method was 0.98, the specificity was 0.97, the positive predictive value (PPV) was 98%, the negative predictive value (NPV) was 97%, the sensitivity of the commercial Rt-PCR was 0.97, the specificity was 0.95, the PPV was 97%, the NPV was 95%. A high level of agreement (κ: 0.953) was determined between the in-house and the commercial Rt-PCR methods. In order for a test to be accepted as a confirmatory test, its specificity must be high. It was decided that sensitivity and specificity of the in-house Rt-PCR were suitable for using this method in the laboratory diagnosis of Plasmodium species.


Assuntos
Malária , Reação em Cadeia da Polimerase Multiplex , Plasmodium , Reação em Cadeia da Polimerase em Tempo Real , DNA de Protozoário/genética , Humanos , Malária/sangue , Malária/diagnóstico , Malária/parasitologia , Reação em Cadeia da Polimerase Multiplex/normas , Plasmodium/classificação , Plasmodium/genética , Plasmodium falciparum/genética , Plasmodium vivax/genética , Reação em Cadeia da Polimerase em Tempo Real/normas , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
17.
Emerg Infect Dis ; 26(10): 2353-2360, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32723432

RESUMO

External quality assessment (EQA) is essential for ensuring reliable test results, especially when laboratories are using assays authorized for emergency use for newly emerging pathogens. We developed an EQA panel to assess the quality of real-time reverse transcription PCR assays being used in South Korea to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). With the participation of 23 public health organization laboratories and 95 nongovernmental laboratories involved in SARS-CoV-2 testing, we conducted qualitative and semiquantitative performance assessments by using pooled respiratory samples containing different viral loads of SARS-CoV-2 or human coronavirus OC43. A total of 110 (93.2%) laboratories reported correct results for all qualitative tests; 29 (24.6%) laboratories had >1 outliers according to cycle threshold values. Our EQA panel identified the potential weaknesses of currently available commercial reagent kits. The methodology we used can provide practical experience for those planning to conduct evaluations for testing of SARS-CoV-2 and other emerging pathogens in the future.


Assuntos
Betacoronavirus/isolamento & purificação , Técnicas de Laboratório Clínico/normas , Infecções por Coronavirus/diagnóstico , Pneumonia Viral/diagnóstico , RNA Viral/análise , Reação em Cadeia da Polimerase em Tempo Real/normas , Reação em Cadeia da Polimerase Via Transcriptase Reversa/normas , Humanos , Ensaio de Proficiência Laboratorial , Pandemias , Garantia da Qualidade dos Cuidados de Saúde , Kit de Reagentes para Diagnóstico/normas , Reação em Cadeia da Polimerase em Tempo Real/métodos , República da Coreia , Sistema Respiratório/virologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos
18.
Gene ; 757: 144948, 2020 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-32652106

RESUMO

Pseudogenes are duplicated or retrotransposed DNA sequences of native functional genes. Amplification of pseudogenes along with gene of interest often produces false positive results, which is an innate problem in Reverse Transcription-quantitative PCR (RT-qPCR). Selecting a reference gene without any interference from pseudogene amplification is therefore a challenge to overcome. Among the common reference genes used for normalization (ACTB, GAPDH, HPRT1, TUBB, RNA18SN1 and B2M), B2M was found to have no pseudogenes in silico, which has also been confirmed by PCR and RT-qPCR. We also assessed the effect of pseudogenes on the determination of the stability of reference genes through data mining. The phylogenetic analysis of pseudogenes and functional genes revealed high sequence similarity among mammals. In addition, we demonstrated the deduction of pseudogene amplification signal using ValidPrime Assay (VPA) under conditions where genomic DNA contamination could not be avoided. Hence, we recommend the use of pseudo-free reference gene with consistent expression in the samples of interest or use VPA normalization method, where genomic DNA or pseudogene amplification is inevitable.


Assuntos
Perfilação da Expressão Gênica/normas , Pseudogenes , Reação em Cadeia da Polimerase em Tempo Real/normas , Microglobulina beta-2/genética , Animais , Sequência Conservada , Evolução Molecular , Humanos , Padrões de Referência
19.
Euro Surveill ; 25(27)2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32672149

RESUMO

Laboratory preparedness with quality-assured diagnostic assays is essential for controlling the current coronavirus disease (COVID-19) outbreak. We conducted an external quality assessment study with inactivated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) samples to support clinical laboratories with a proficiency testing option for molecular assays. To analyse SARS-CoV-2 testing performance, we used an online questionnaire developed for the European Union project RECOVER to assess molecular testing capacities in clinical diagnostic laboratories.


Assuntos
Técnicas de Laboratório Clínico/métodos , Técnicas de Laboratório Clínico/normas , Infecções por Coronavirus/diagnóstico , Coronavirus/isolamento & purificação , Técnicas de Diagnóstico Molecular/métodos , Pandemias , Pneumonia Viral/diagnóstico , Betacoronavirus , Serviços de Laboratório Clínico , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/virologia , Surtos de Doenças , Europa (Continente) , Humanos , Pandemias/prevenção & controle , Pneumonia Viral/epidemiologia , Pneumonia Viral/virologia , Reação em Cadeia da Polimerase em Tempo Real/normas , Reação em Cadeia da Polimerase Via Transcriptase Reversa/normas , Sensibilidade e Especificidade , Inquéritos e Questionários
20.
J Clin Virol ; 129: 104537, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32659712

RESUMO

BACKGROUND: Broad and decentralised testing of SARS-CoV-2 RNA genomes is a WHO-recommended strategy to contain the SARS-CoV-2 pandemic by identifying infected cases in order to minimize onward transmission. With the need to increase the test capacities in Austria, nation-wide numerous laboratories rapidly implemented assays for molecular detection of SARS-CoV-2 based on real-time RT-PCR assays. The objective of this study was to monitor reliability of the laboratory results for SARS-CoV-2 RNA detection through an external quality assessment (EQA) scheme. METHODS: For this, the Center for Virology, Medical University of Vienna was tasked by the Federal Ministry of Social Affairs, Health, Care and Consumer Protection to perform the first Austrian EQA on SARS-CoV-2 which was organised in cooperation with the Austrian Association for Quality Assurance and Standardization of Medical and Diagnostic Tests (ÖQUASTA). Data were analysed on the basis of qualitative outcome of testing in relation to the nucleic acid (NA) extraction and detection methods used. RESULTS AND CONCLUSION: A total of 52 laboratories participated, contributing results from 67 test panels comprising 42 distinct combinations of NA extraction and PCR reagents. By testing 3 positive (CT values: S1, 28.4; S2, 33.6; S3, 38.5) and 1 negative sample, no false-positive results were obtained by any of the laboratories. Otherwise, 40/67 tests (60 %) detected all positive samples correctly as positive, but 25/67 tests (37 %) did not detect the weakest positive sample (S3), and 3 % reported S2 and S3 as false-negative. Improvement in test sensitivity by focusing on NA extraction and/or PCR-based detection is recommended.


Assuntos
Betacoronavirus/isolamento & purificação , Técnicas de Laboratório Clínico/métodos , Técnicas de Laboratório Clínico/normas , Infecções por Coronavirus/diagnóstico , Ensaio de Proficiência Laboratorial/organização & administração , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Diagnóstico Molecular/normas , Pneumonia Viral/diagnóstico , Áustria , Erros de Diagnóstico/estatística & dados numéricos , Humanos , Pandemias , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase em Tempo Real/normas , Sensibilidade e Especificidade
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