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1.
Clin Chem Lab Med ; 59(10): 1735-1744, 2021 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-34187131

RESUMO

OBJECTIVES: External quality assessment (EQA) schemes provide information on individual and general analytical performance of participating laboratories and test systems. The aim of this study was to investigate the use and performance of SARS-CoV-2 virus genome detection systems in Austrian laboratories and their preparedness to face challenges associated with the pandemic. METHODS: Seven samples were selected to evaluate performance and estimate variability of reported results. Notably, a dilution series was included in the panel as a measure of reproducibility and sensitivity. Several performance criteria were evaluated for individual participants as well as in the cohort of all participants. RESULTS: A total of 109 laboratories participated and used 134 platforms, including 67 different combinations of extraction and PCR platforms and corresponding reagents. There were no false positives and 10 (1.2%) false negative results, including nine in the weakly positive sample (Ct ∼35.9, ∼640 copies/mL). Twenty (22%) laboratories reported results of mutation detection. Twenty-five (19%) test systems included amplification of human RNA as evidence of proper sampling. The overall linearity of Ct values from individual test systems for the dilution series was good, but inter-assay variability was high. Both operator-related and systematic failures appear to have caused incorrect results. CONCLUSIONS: Beyond providing certification for participating laboratories, EQA provides the opportunity for participants to evaluate their performance against others so that they may improve operating procedures and test systems. Well-selected EQA samples offer additional inferences to be made about assay sensitivity and reproducibility, which have practical applications.


Assuntos
COVID-19/diagnóstico , Genoma Viral , Garantia da Qualidade dos Cuidados de Saúde , SARS-CoV-2/isolamento & purificação , Áustria/epidemiologia , COVID-19/virologia , Humanos , Laboratórios , Técnicas de Diagnóstico Molecular/métodos , Pandemias , SARS-CoV-2/genética , Sensibilidade e Especificidade
2.
Appl Environ Microbiol ; 81(9): 3077-85, 2015 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-25724966

RESUMO

Vibrio cholerae is a severe human pathogen and a frequent member of aquatic ecosystems. Quantification of V. cholerae in environmental water samples is therefore fundamental for ecological studies and health risk assessment. Beside time-consuming cultivation techniques, quantitative PCR (qPCR) has the potential to provide reliable quantitative data and offers the opportunity to quantify multiple targets simultaneously. A novel triplex qPCR strategy was developed in order to simultaneously quantify toxigenic and nontoxigenic V. cholerae in environmental water samples. To obtain quality-controlled PCR results, an internal amplification control was included. The qPCR assay was specific, highly sensitive, and quantitative across the tested 5-log dynamic range down to a method detection limit of 5 copies per reaction. Repeatability and reproducibility were high for all three tested target genes. For environmental application, global DNA recovery (GR) rates were assessed for drinking water, river water, and water from different lakes. GR rates ranged from 1.6% to 76.4% and were dependent on the environmental background. Uncorrected and GR-corrected V. cholerae abundances were determined in two lakes with extremely high turbidity. Uncorrected abundances ranged from 4.6×10(2) to 2.3×10(4) cell equivalents liter(-1), whereas GR-corrected abundances ranged from 4.7×10(3) to 1.6×10(6) cell equivalents liter(-1). GR-corrected qPCR results were in good agreement with an independent cell-based direct detection method but were up to 1.6 log higher than cultivation-based abundances. We recommend the newly developed triplex qPCR strategy as a powerful tool to simultaneously quantify toxigenic and nontoxigenic V. cholerae in various aquatic environments for ecological studies as well as for risk assessment programs.


Assuntos
Técnicas Bacteriológicas/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Vibrio cholerae/classificação , Vibrio cholerae/isolamento & purificação , Microbiologia da Água , Técnicas Bacteriológicas/normas , DNA Bacteriano/química , DNA Bacteriano/genética , Humanos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase em Tempo Real/normas , Padrões de Referência , Sensibilidade e Especificidade , Análise de Sequência de DNA , Vibrio cholerae/genética
3.
Clin Chem Lab Med ; 46(9): 1239-44, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18783342

RESUMO

BACKGROUND: Yersinia pestis (Y. pestis) is a zoonotic bacterium mainly circulating among rodents and their fleas. Transmission to humans can cause bubonic, pneumonic or septicemic plague with a high case-fatality rate. Therefore, rapid and reliable diagnostic tools are crucial. The objective of this study was to assess the inter-laboratory reproducibility of in-house developed real-time PCR assays for the identification of Y. pestis. METHODS: A total of four samples of quantified Y. pestis DNA and two blank samples were sent blinded to 14 laboratories. To standardize the procedures, oligonucleotides were provided and the same instrument platform and a commercial mastermix were used. The participants were requested to report their results including cycle threshold and melting temperature values. RESULTS: All participating laboratories were able to perform the real-time PCR assays according to the protocols provided and identified the samples containing Y. pestis DNA correctly. Significant differences between the reference laboratory and participating laboratories were observed in cycle threshold values and melting temperatures. This, however, did not adversely affect the interpretation of results. CONCLUSIONS: Our real-time PCR system proved to be highly reproducible and has the potential of complementing the diagnostic tools for rapid identification of Y. pestis isolates. Further steps of validation are needed to determine diagnostic accuracy and predictive values with clinical samples.


Assuntos
Reação em Cadeia da Polimerase/métodos , Reação em Cadeia da Polimerase/normas , Yersinia pestis/isolamento & purificação , Armas Biológicas , Laboratórios , Reprodutibilidade dos Testes , Fatores de Tempo , Yersinia pestis/genética
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