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1.
Front Cell Infect Microbiol ; 11: 613304, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33598439

RESUMO

Background: The emerging Coronavirus Disease-2019 (COVID-19) has challenged the public health globally. With the increasing requirement of detection for SARS-CoV-2 outside of the laboratory setting, a rapid and precise Point of Care Test (POCT) is urgently needed. Methods: Targeting the nucleocapsid (N) gene of SARS-CoV-2, specific primers, and probes for reverse transcription recombinase-aided amplification coupled with lateral flow dipstick (RT-RAA/LFD) platform were designed. For specificity evaluation, it was tested with human coronaviruses, human influenza A virus, influenza B viruses, respiratory syncytial virus, and hepatitis B virus, respectively. For sensitivity assay, it was estimated by templates of recombinant plasmid and pseudovirus of SARS-CoV-2 RNA. For clinical assessment, 100 clinical samples (13 positive and 87 negatives for SARS-CoV-2) were tested via quantitative reverse transcription PCR (RT-qPCR) and RT-RAA/LFD, respectively. Results: The limit of detection was 1 copies/µl in RT-RAA/LFD assay, which could be conducted within 30 min at 39°C, without any cross-reaction with other human coronaviruses and clinical respiratory pathogens. Compared with RT-qPCR, the established POCT assay offered 100% specificity and 100% sensitivity in the detection of clinical samples. Conclusion: This work provides a convenient POCT tool for rapid screening, diagnosis, and monitoring of suspected patients in SARS-CoV-2 endemic areas.


Assuntos
/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , /genética , /virologia , /genética , Primers do DNA/genética , Humanos , Fosfoproteínas/genética , Testes Imediatos , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real/instrumentação , Recombinases/metabolismo , Transcrição Reversa , Sensibilidade e Especificidade
2.
Talanta ; 224: 121850, 2021 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-33379066

RESUMO

In detecting infectious diseases, such as coronavirus 2019 (COVID-19), real-time reverse-transcription polymerase chain reaction (RT-PCR) is one of the most important technologies for RNA detection and disease diagnosis. To achieve high quality assurance, appropriate positive and negative controls are critical for disease detection using RT-PCR kits. In this study, we have found that commercial kits often adopt DNAs instead of RNAs as the positive controls, which can't report the kit problems in reverse transcription, thereby increasing risk of the false negative results when testing patient samples. To face the challenge, we have proposed and developed the chemically modified RNAs, such as phosphoroselenaote and phosphorothioate RNAs (Se-RNA and S-RNA), as the controls. We have found that while demonstrating the high thermostability, biostability, chemostability and exclusivity (or specificity), both Se-RNA and S-RNA can be fine templates for reverse transcription, indicating their potentials as both positive and negative controls for RT-PCR kits.


Assuntos
/métodos , RNA Viral/análise , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , /instrumentação , DNA Viral/análise , Reações Falso-Negativas , Humanos , Estabilidade de RNA , RNA Viral/química , Kit de Reagentes para Diagnóstico , Reação em Cadeia da Polimerase em Tempo Real/instrumentação , Reação em Cadeia da Polimerase Via Transcriptase Reversa/instrumentação , /genética
3.
Food Chem ; 339: 127891, 2021 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-32861930

RESUMO

We propose a visual strategy for simultaneous detection of multiple adulterated components in beef by integration of multiple polymerase chain reaction (mPCR) with the lateral flow strip (LFS). The primer sets for adulterated components are uniquely designed with different nucleic acid tags (NAT), enabling the amplicons with specific wobbled sequences at two opposite ends. The wobbled sequences will precisely hybridize with the pre-immobilized capture probes on T lines (T1, T2 and T3) and C line, contributing to the coloration of LFS. Taking advantages of extraordinary amplification efficiency of PCR and simplicity of LFS, common adulterated components including chicken, duck and pork can be easily detected with LOD as low as 0.01% (wt%), which is comparable to that of quantitative real-time polymerase chain reaction (qPCR) but with more simplified operations and reduced costs. The method can be extended to identification of other components by replacing the functional primer set. This method can be a useful candidate for meat quality control at the resource-limited setups.


Assuntos
Galinhas/genética , Patos/genética , Carne/análise , Reação em Cadeia da Polimerase em Tempo Real/métodos , Animais , Bovinos , DNA/isolamento & purificação , DNA/metabolismo , Ouro/química , Limite de Detecção , Nanopartículas Metálicas/química , Reação em Cadeia da Polimerase em Tempo Real/instrumentação , Suínos
4.
PLoS One ; 15(12): e0242408, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33315885

RESUMO

We sought to develop a smooth and low cost sample preparation and DNA extraction protocol, streamlined with a ready-to-use qPCR in a portable instrument to overcome some of the existing hurdles. Several solutions were evaluated as to their ability to liquefy a mucin-based matrix. Each liquefied matrix, supplemented with either Mycobacterium tuberculosis (MTB) H37Rv strain DNA or intact cells, was aliquoted onto a filter paper embedded with solubilizing agents, and was subsequently dried up. Most of the nucleic acids, including genomic DNA from the bacilli and the host, binds to the filter paper. Next, several protocols were evaluated to elute the DNA from the paper, using qPCR to detect the insertion sequence IS6110, a M. tuberculosis complex genomic marker. The limit of detection (LOD) of the best protocol was then evaluated using parallel seeding and colony counting. The protocol was also evaluated using seventeen sputum samples, previously characterized by the GeneXpert or culture. Two instruments (the ABI7500 Standard and the Q3-Plus system) and two reagents storage formats (frozen or ready-to-use) were evaluated. Solutions containing guanidine isothiocyanate exerted the best liquefying effect on the mucin-based matrix extracted from one 6-mm punches, followed by a brief incubation at 95°C. The resulting DNA contained impurities, but a simple 1:10 dilution elicited the detection of MTB and human genomic targets. The described protocol presented an apparent LOD of 02 CFU/mL of MTB. Challenging the protocol with previously characterized samples showed substantial agreement with GeneXpert MTB/RIF results (sensitivity of 90%, agreement of 88.9%, kappa coefficient of 0.77), and moderate agreement with culture results (sensitivity of 100%, agreement of 78.9%, kappa coefficient of 0.58). This work presents a sensitive proof-of-concept protocol for sputum liquefaction and decontamination followed by a simple DNA extraction procedure, in which the extraction steps are streamlined with a ready-to-use qPCR in a portable instrument that can be employed in low infrastructure settings.


Assuntos
Programas de Rastreamento/métodos , Mycobacterium tuberculosis/isolamento & purificação , Sistemas Automatizados de Assistência Junto ao Leito , Manejo de Espécimes/métodos , Tuberculose Pulmonar/diagnóstico , Brasil , DNA Bacteriano/isolamento & purificação , Humanos , Limite de Detecção , Programas de Rastreamento/economia , Programas de Rastreamento/instrumentação , Mycobacterium tuberculosis/genética , Estudo de Prova de Conceito , Reação em Cadeia da Polimerase em Tempo Real/economia , Reação em Cadeia da Polimerase em Tempo Real/instrumentação , Reação em Cadeia da Polimerase em Tempo Real/métodos , Manejo de Espécimes/economia , Escarro/microbiologia , Tuberculose Pulmonar/microbiologia
5.
Medicine (Baltimore) ; 99(50): e22964, 2020 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-33327229

RESUMO

Long non-coding RNAs (lncRNAs) have been evidenced to be associated with the development of multiple diseases. However, the expression pattern and function of lncRNAs in acute ischemic stroke remain unclear. To determine the differential expression of lncRNAs in acute ischemic stroke, we analyzed the expression profile of lncRNAs by high-throughput sequencing analysis. Gene Ontology (GO) and pathway analyses were employed to analyze the gene function and identify enriched pathways of the differentially expressed lncRNAs. We also built an lncRNA-mRNA expression correlation network and verified the interactions of selected lncRNAs in acute ischemic stroke. To further confirm the results of the expression profile, 6 differentially expressed lncRNAs were randomly selected and quantitative RT-PCR (qRT-PCR) performed. We identified 44,578 aberrantly expressed lncRNAs, including 228 upregulated and 16 downregulated lncRNAs. The qRT-PCR results showed that ENSG00000269900, ENSG00000196559, ENSG00000202198, ENSG00000226482, ENSG00000260539 (up), and XLOC_013994_2 (down) were abnormally expressed, which was consistent with the sequencing results. The upregulated expression of lncRNA ENSG00000226482 may activate the adipocytokine signaling pathway, resulting in acute ischemia stroke. In summary, we analyzed the lncRNAs expression profile in acute ischemic stroke patients and identified the functions and enriched metabolic pathways, proposing new insights into the diagnostic and therapeutic biomarkers for this disease.


Assuntos
Biomarcadores/metabolismo , RNA Longo não Codificante/genética , Adipocinas/metabolismo , Idoso , Estudos de Casos e Controles , China/epidemiologia , Regulação para Baixo , Feminino , Ontologia Genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Imagem por Ressonância Magnética/métodos , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real/instrumentação , Tomografia Computadorizada por Raios X/métodos , Regulação para Cima
6.
J Appl Lab Med ; 5(6): 1307-1312, 2020 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-32761092

RESUMO

BACKGROUND: Numerous nucleic acid amplification assays utilizing different target genes of the SARS-CoV-2 genome have received emergency use authorization (EUA) by the United States Food and Drug Administration (FDA). Limited data are available comparing the test performance characteristics of these assays. METHODS: A diagnostic comparison study was performed to evaluate the performance of the Cepheid Xpert Xpress SARS-CoV-2 assay compared to the Hologic Panther Fusion SARS-CoV-2 assay using clinical nasopharyngeal specimens. Agreement between the two assays was assessed by overall, positive, and negative percent agreement and Cohen's kappa coefficient. RESULTS: A total of 104 (54 positive and 50 negative) clinical nasopharyngeal samples were tested by both assays. Using the Panther Fusion as a reference standard, the Xpert demonstrated an overall agreement of 99.0% [95% confidence interval (CI): 94.8-100], positive percent agreement of 98.1% (95% CI: 90.1-100), and a negative percent agreement of 100% (95% CI: 94.2-100). The kappa coefficient was 0.98 (95% CI: 0.94-1.0). One sample positive by the Panther Fusion with a cycle threshold (Ct) of 38.6 was found to be reproducibly negative by the Xpert assay. CONCLUSIONS: The Cepheid Xpert Xpress SARS-CoV-2 assay provides test performance comparable to the Hologic Panther Fusion SARS-CoV-2 assay while offering laboratories rapid, on-demand testing capacity.


Assuntos
Betacoronavirus/isolamento & purificação , Técnicas de Laboratório Clínico/instrumentação , Infecções por Coronavirus/diagnóstico , Técnicas de Diagnóstico Molecular/instrumentação , Pneumonia Viral/diagnóstico , RNA Viral/isolamento & purificação , Automação Laboratorial/instrumentação , Betacoronavirus/genética , Técnicas de Laboratório Clínico/estatística & dados numéricos , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/virologia , Humanos , Técnicas de Diagnóstico Molecular/estatística & dados numéricos , Reação em Cadeia da Polimerase Multiplex/instrumentação , Pandemias , Pneumonia Viral/epidemiologia , Pneumonia Viral/virologia , Sistemas Automatizados de Assistência Junto ao Leito/estatística & dados numéricos , Kit de Reagentes para Diagnóstico/estatística & dados numéricos , Reação em Cadeia da Polimerase em Tempo Real/instrumentação , Reprodutibilidade dos Testes , Fatores de Tempo , Estados Unidos/epidemiologia
7.
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
8.
Artigo em Inglês | MEDLINE | ID: mdl-32656101

RESUMO

CDC and WHO guidelines for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) diagnosis only recommend synthetic fiber swabs for nasopharyngeal (NP) sampling. We show that cotton-tipped plastic swabs do not inhibit PCR and have equivalent performance to rayon swabs. Cotton-tipped plastic swabs are massively produced worldwide and would prevent swab supply shortages under the current high SARS-CoV-2 testing demands, particularly in developing countries.


Assuntos
Betacoronavirus/isolamento & purificação , Técnicas de Laboratório Clínico/instrumentação , Infecções por Coronavirus/diagnóstico , Equipamentos para Diagnóstico/provisão & distribução , Equipamentos Descartáveis/provisão & distribução , Pneumonia Viral/diagnóstico , Reação em Cadeia da Polimerase em Tempo Real/instrumentação , Betacoronavirus/genética , Celulose/provisão & distribução , Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/virologia , Fibra de Algodão/provisão & distribução , Humanos , Nasofaringe , Pandemias , Plásticos/provisão & distribução , Pneumonia Viral/virologia , RNA Viral/análise , Reação em Cadeia da Polimerase em Tempo Real/métodos , Manejo de Espécimes/instrumentação , Manejo de Espécimes/métodos
9.
PLoS One ; 15(6): e0234682, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32530929

RESUMO

Novel Corona virus/Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2 or 2019-nCoV), and the subsequent disease caused by the virus (coronavirus disease 2019 or COVID-19), is an emerging global health concern that requires a rapid diagnostic test. Quantitative reverse transcription PCR (qRT-PCR) is currently the standard for SARS-CoV-2 detection; however, Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) may allow for faster and cheaper field based testing at point-of-risk. The objective of this study was to develop a rapid screening diagnostic test that could be completed in 30-45 minutes. Simulated patient samples were generated by spiking serum, urine, saliva, oropharyngeal swabs, and nasopharyngeal swabs with a portion of the SARS-CoV-2 nucleic sequence. RNA isolated from nasopharyngeal swabs collected from actual COVID-19 patients was also tested. The samples were tested using RT-LAMP as well as by conventional qRT-PCR. Specificity of the RT-LAMP was evaluated by also testing against other related coronaviruses. RT-LAMP specifically detected SARS-CoV-2 in both simulated patient samples and clinical specimens. This test was performed in 30-45 minutes. This approach could be used for monitoring of exposed individuals or potentially aid with screening efforts in the field and potential ports of entry.


Assuntos
Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Pneumonia Viral/diagnóstico , Testes Imediatos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Betacoronavirus/genética , Infecções por Coronavirus/virologia , Primers do DNA , Humanos , Técnicas de Diagnóstico Molecular/economia , Técnicas de Diagnóstico Molecular/instrumentação , Técnicas de Amplificação de Ácido Nucleico/economia , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Pandemias , Pneumonia Viral/virologia , Reação em Cadeia da Polimerase em Tempo Real/economia , Reação em Cadeia da Polimerase em Tempo Real/instrumentação , Sensibilidade e Especificidade , Fatores de Tempo
10.
Euro Surveill ; 25(24)2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32583765

RESUMO

Containment strategies and clinical management of coronavirus disease (COVID-19) patients during the current pandemic depend on reliable diagnostic PCR assays for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we compare 11 different RT-PCR test systems used in seven diagnostic laboratories in Germany in March 2020. While most assays performed well, we identified detection problems in a commonly used assay that may have resulted in false-negative test results during the first weeks of the pandemic.


Assuntos
Betacoronavirus/genética , Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/diagnóstico , Equipamentos para Diagnóstico , Pneumonia Viral/diagnóstico , Técnicas de Laboratório Clínico/instrumentação , Fezes/virologia , Alemanha , Humanos , Laboratórios , Reação em Cadeia da Polimerase Multiplex/instrumentação , Reação em Cadeia da Polimerase Multiplex/métodos , Pandemias , Reação em Cadeia da Polimerase em Tempo Real/instrumentação , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/instrumentação , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Sensibilidade e Especificidade
11.
Clin Microbiol Infect ; 26(8): 1076-1081, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32422410

RESUMO

OBJECTIVE: To evaluate the performance of an ultrafast single-tube nucleic acid isothermal amplification detection assay for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA using clinical samples from multiple centres. METHODS: A reverse transcription recombinase-aided amplification (RT-RAA) assay for SARS-CoV-2 was conducted within 15 minutes at 39°C with portable instruments after addition of extracted RNA. The clinical performance of RT-RAA assay was evaluated using 947 clinical samples from five institutions in four regions of China; approved commercial fluorescence quantitative real-time PCR (qRT-PCR) kits were used for parallel detection. The sensitivity and specificity of RT-RAA were compared and analysed. RESULTS: The RT-RAA test results of 926 samples were consistent with those of qRT-PCR (330 were positive, 596 negative); 21 results were inconsistent. The sensitivity and specificity of RT-RAA was 97.63% (330/338, 95% confidence interval (CI) 95.21 to 98.90) and 97.87% (596/609, 95% CI 96.28 to 98.81) respectively. The positive and negative predictive values were 96.21% (330/343, 95% CI 93.45 to 97.88) and 98.68% (596/604, 95% CI 97.30 to 99.38) respectively. The total coincidence rate was 97.78% (926/947, 95% CI 96.80 to 98.70), and the kappa was 0.952 (p < 0.05). CONCLUSIONS: With comparable sensitivity and specificity to the commercial qRT-PCR kits, RT-RAA assay for SARS-CoV-2 exhibited the distinctive advantages of simplicity and rapidity in terms of operation and turnaround time.


Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/diagnóstico , Pneumonia Viral/diagnóstico , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real/instrumentação , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , China , Testes Diagnósticos de Rotina/instrumentação , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Pandemias , Sensibilidade e Especificidade , Temperatura , Adulto Jovem
12.
RNA ; 26(7): 771-783, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32358057

RESUMO

The current COVID-19 pandemic presents a serious public health crisis, and a better understanding of the scope and spread of the virus would be aided by more widespread testing. Nucleic-acid-based tests currently offer the most sensitive and early detection of COVID-19. However, the "gold standard" test pioneered by the U.S. Centers for Disease Control and Prevention takes several hours to complete and requires extensive human labor, materials such as RNA extraction kits that could become in short supply, and relatively scarce qPCR machines. It is clear that a huge effort needs to be made to scale up current COVID-19 testing by orders of magnitude. There is thus a pressing need to evaluate alternative protocols, reagents, and approaches to allow nucleic-acid testing to continue in the face of these potential shortages. There has been a tremendous explosion in the number of papers written within the first weeks of the pandemic evaluating potential advances, comparable reagents, and alternatives to the "gold-standard" CDC RT-PCR test. Here we present a collection of these recent advances in COVID-19 nucleic acid testing, including both peer-reviewed and preprint articles. Due to the rapid developments during this crisis, we have included as many publications as possible, but many of the cited sources have not yet been peer-reviewed, so we urge researchers to further validate results in their own laboratories. We hope that this review can urgently consolidate and disseminate information to aid researchers in designing and implementing optimized COVID-19 testing protocols to increase the availability, accuracy, and speed of widespread COVID-19 testing.


Assuntos
Betacoronavirus/isolamento & purificação , Técnicas de Laboratório Clínico , Técnicas de Amplificação de Ácido Nucleico/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Betacoronavirus/genética , Sistemas CRISPR-Cas , Centers for Disease Control and Prevention, U.S. , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Infecções por Coronavirus/diagnóstico , Humanos , Nasofaringe/virologia , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Sistemas Automatizados de Assistência Junto ao Leito , RNA Viral/genética , RNA Viral/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/instrumentação , Fatores de Tempo , Estados Unidos , Fluxo de Trabalho
13.
Food Chem ; 324: 126821, 2020 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-32361093

RESUMO

As large-scale planting of genetically modified (GM) crops increases, the development of a rapid and convenient method for on-site detection of GM crops is important. We combined the advantages of recombinase polymerase amplification (RPA) and fluorescence detection to establish a rapid, sensitive, specific, and simple detection platform for on-site detection of MON863 maize. Test samples were added directly to the platform after simple pre-treatment with a DNA extraction-free method. Results were obtained through real-time monitoring with a portable instrument, which facilitated sample-in/answer-out on-site detection. The entire detection process, including sample preparation, RPA and identification of amplification results, was accomplished in approximately 10 min. Furthermore, the detection was achieved with a simple and inexpensive portable device. This method has high potential for application in other fields requiring rapid detection of DNA targets, such as in field research, resource-limited areas, and science education.


Assuntos
Produtos Agrícolas/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Plantas Geneticamente Modificadas/genética , Zea mays/genética , Primers do DNA/genética , Fluorescência , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Reação em Cadeia da Polimerase em Tempo Real/instrumentação , Reação em Cadeia da Polimerase em Tempo Real/métodos , Recombinases/genética
14.
J Clin Virol ; 128: 104390, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32388471

RESUMO

BACKGROUND: The ongoing SARS-CoV-2 pandemic presents a unique challenge for diagnostic laboratories around the world. Automation of workflows in molecular diagnostics is instrumental for coping with the large number of tests ordered by clinicians, as well as providing fast-tracked rapid testing for highly urgent cases. In this study we evaluated a SARS-CoV-2 LDT for the NeuMoDx 96 system, a fully automated device performing extraction and real-time PCR. METHODS: A publicly available SARS-CoV-2 RT-PCR assay was adapted for the automated system. Analytical performance was evaluated using in-vitro transcribed RNA and clinical performance was compared to the cobas 6800-based reference assay within the lab. RESULTS: The Envelope (E) Gene-LDT displayed good analytical performance with an LoD of 95.55 cp/mL and no false positives during evaluation of cross-reactivity. A total of 176 patient samples were tested with both the E-Gene-LDT and the reference assay. Positive and negative agreement were 100 % and 99.2 % respectively. Invalid-rate was 6.3 %. CONCLUSION: The E-Gene-LDT showed analytical and clinical performance comparable to the cobas6800-based reference assay. Due to its random-access workflow concept and rapid time-to-result of about 80 min, the system is very well suited for providing fast-tracked SARS-CoV-2 diagnostics for urgent clinical samples in the hospital setting.


Assuntos
Técnicas de Laboratório Clínico/métodos , Pandemias , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Betacoronavirus , Técnicas de Laboratório Clínico/instrumentação , Infecções por Coronavirus/diagnóstico , Hospitais , Humanos , Pneumonia Viral , Reação em Cadeia da Polimerase em Tempo Real/instrumentação , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/instrumentação , Fatores de Tempo , Fluxo de Trabalho
15.
PLoS One ; 15(4): e0232044, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32320441

RESUMO

Canine distemper virus (CDV) is a multi-host pathogen that can cause significant mortality in domestic, wild terrestrial and marine mammals. It is a major conservation threat in some endangered species. Infection can result in severe respiratory disease and fatal encephalitis. Diagnosis and disease monitoring in wildlife, and differentiation of CDV from rabies (a life-threatening zoonotic disease that can produce similar neurologic signs), would benefit from the availability of a portable, point-of-care (POC) diagnostic test. We therefore developed a quantitative RT-PCR assay for CDV using shelf-stable, lyophilized reagents and target-specific primers and probes for use with the handheld Biomeme two3™ qPCR thermocycler. Biomeme's extraction methodology, lyophilized reagents, and thermocycler were compared to our standard laboratory-based methods to assess sensitivity, efficiency and overall test performance. Results using a positive control plasmid for CDV showed comparable sensitivity (detection of 50 copies) and PCR efficiency between the two platforms, and CDV detection was similar between platforms when tested using a modified live CDV vaccine. Significantly higher Ct values (average Ct = 5.1 cycles) were observed using the Biomeme platform on known CDV positive animal samples. CDV detection using the Biomeme platform was similar in 25 of 26 samples from suspect CDV cases when compared to standard virology laboratory testing. One false positive was observed that was negative upon retest. The Biomeme methodology can be adapted for detection of specific targets, and this portable technology saves time by eliminating the need for local or international sample transport for laboratory-based diagnostics. However, results of our testing suggest that decreased diagnostic sensitivity (higher Ct values) relative to laboratory-based methods was observed using animal samples, so careful validation and optimization are essential. Portable qPCR platforms can empower biologists and wildlife health professionals in remote and low-resource settings, which will greatly improve our understanding of CDV disease ecology and associated conservation threats in wildlife.


Assuntos
Vírus da Cinomose Canina/genética , Cinomose/virologia , Reação em Cadeia da Polimerase em Tempo Real/instrumentação , Reação em Cadeia da Polimerase em Tempo Real/métodos , Animais , Animais Selvagens , Áustria , Vírus da Cinomose Canina/imunologia , Congelamento , Cabelo/virologia , Nariz/virologia , Sistemas Automatizados de Assistência Junto ao Leito , RNA Viral/isolamento & purificação , Guaxinins/virologia , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Pele/virologia , Estados Unidos , Vacinas Atenuadas
16.
Lab Anim ; 54(3): 239-250, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31195883

RESUMO

Hygienic monitoring of laboratory rodents has focused more and more on the analysis of environmental sample material by quantitative polymerase chain reaction (qPCR) assays. This approach requires profound knowledge of specific genetic sequences of the agents to be monitored and the assays need to be permanently adapted to take the latest research into account. [Pasteurella] pneumotropica was recently reclassified into the new genus Rodentibacter, with Rodentibacter (R.) pneumotropicus and R. heylii as the most commonly detected species in laboratory mouse colonies. This study aimed at the development of a specific qPCR assay for the simultaneous detection of both agents. A novel primer probe set, based on detection of the specific virulence factor' 'inclusion body protein A' gene (ibpA), was confirmed by testing the assay on currently described Rodentibacter type species and other Pasteurellaceae. Furthermore, it was validated within four different barrier units and results were compared with the cultural analysis of sentinel mice. The assay was suitable to specifically detect R. pneumotropicus and R. heylii and discriminate them from other murine Rodentibacter spp. In addition, it revealed high sensitivity for the detection of both agents in environmental sampling material including exhaust air dust in individually ventilated cage systems. Altogether, higher pathogen prevalence was detected via qPCR of environmental samples compared with cultural diagnostics of sentinel mice. This study describes a qPCR assay for the simultaneous detection of R. pneumotropicus and R. heylii. This assay was demonstrated to be beneficial during routine health monitoring, especially with regard to environmental sampling strategies.


Assuntos
Camundongos/microbiologia , Pasteurellaceae/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/métodos , Fatores de Virulência/isolamento & purificação , Animais , Animais de Laboratório/microbiologia , Feminino , Reação em Cadeia da Polimerase em Tempo Real/instrumentação
17.
Food Chem ; 309: 125654, 2020 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-31678669

RESUMO

A specialized DNA extraction method and a SYBR Green quantitative polymerase chain reaction (SyG-qPCR) assay were combined to generate a ready-to-use kit for rapid detection of porcine admixtures in processed meat products. Our qPCR assay utilized repetitive LINE-1 elements specific to the genome of Sus scrofa domesticus (pig) as a target and incorporated internal controls. We improved the genomic DNA extraction method, and reduced extraction times to the minimum. The method was validated for specificity, sensitivity (0.001% w/w) and robustness, and values were compared with those of a commercially available kit. We also tested our method using 121 processed food products and consistently detected amplification only in samples containing pork. Due to its efficiency and cost-effectiveness, our method represents a valuable new method for detecting food adulteration with pork that is superior to existing quality control approaches.


Assuntos
DNA/análise , Contaminação de Alimentos/análise , Compostos Orgânicos/química , Reação em Cadeia da Polimerase em Tempo Real/métodos , Animais , DNA/isolamento & purificação , DNA/normas , Elementos Nucleotídeos Longos e Dispersos/genética , Produtos da Carne/análise , Controle de Qualidade , Reação em Cadeia da Polimerase em Tempo Real/instrumentação , Sus scrofa/genética , Suínos
18.
PLoS One ; 14(11): e0224751, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31738773

RESUMO

BACKGROUND: The COBAS AmpliPrep/COBAS TaqMan assay HCV (CAP/CTM) is widely used in clinical routine for HCV testing. Recently, the new cobas HCV test was established for high throughput testing with minimal operator intervention. As different assays may yield different quantitative/qualitative results that possibly impact treatment decisions, the aim of this study was to externally evaluate the cobas HCV test performance in comparison to CAP/CTM in a clinically relevant setting. METHODS: Serum samples were obtained from 270 patients who received direct acting antiviral therapy with different treatment regimens at two study sites (Hannover and Frankfurt) in 2016. Overall, 1545 samples (baseline, on-treatment and follow-up) were tested in parallel by both assays. RESULTS: The mean difference between cobas HCV and CAP/CTM for the quantification of HCV RNA was 0.008 log10 IU/ml HCV RNA (95% limits of agreement: -0.02-0.036) showing excellent agreement of both assays. With respect to clinical cut offs (HCV RNA detectable vs. target not detected and HCV RNA above the lower limit of quantification (LLOQ) vs.

Assuntos
Antivirais/administração & dosagem , Monitoramento de Medicamentos/instrumentação , Hepacivirus/isolamento & purificação , Hepatite C Crônica/tratamento farmacológico , Carga Viral/efeitos dos fármacos , Adulto , Hepacivirus/genética , Hepatite C Crônica/sangue , Hepatite C Crônica/virologia , Ensaios de Triagem em Larga Escala/instrumentação , Humanos , Técnicas de Diagnóstico Molecular/instrumentação , RNA Viral/genética , RNA Viral/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/instrumentação
19.
Biol Pharm Bull ; 42(10): 1761-1765, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31582664

RESUMO

The CYP2D6 gene is the most well characterized gene involved in drug metabolism and is known to have both gene duplication and deletion variants. We report an optimized method for the determination of copy number variation (CNV) in the CYP2D6 gene by a novel purification process for a real-time quantitative PCR. This high-throughput low-cost method accurately determines CNV in the CYP2D6 gene enabling reliable estimates of disease prediction in large epidemiological samples.


Assuntos
Citocromo P-450 CYP2D6/genética , Ensaios de Triagem em Larga Escala/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Variações do Número de Cópias de DNA , Feminino , Genótipo , Ensaios de Triagem em Larga Escala/instrumentação , Humanos , Reação em Cadeia da Polimerase em Tempo Real/instrumentação , Saliva/química
20.
Methods Mol Biol ; 2054: 147-155, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31482454

RESUMO

The introduction of tyrosine-kinase inhibitors (TKI) targeting specific EGFR mutations for the treatment non-small cell lung cancer patients (NSCLC) dramatically increased the clinical outcome in a subset of patients harboring specific activating EGFR mutations. Three different generations of TKI have been developed until now, demonstrating increasing progression-free survival as well as overall survival. However, to benefit of the treatment, the analysis of the genomic content of each patient is mandatory. Additionally, resistance mutations are prevalent and occur frequently and rapidly during treatment. Therefore, tests to detect EGFR mutations at initial diagnosis as well as during treatment, e.g., from liquid biopsies, have been developed and implemented in clinical daily practice for theranostic purpose.As EGFR mutation testing has to be highly reliable, fast, and easy to perform, the automatic qPCR system Idylla™ has been developed and implemented for clinical mutation testing from tissue samples and soon from circulating free DNA. Therefore, we here describe how the Idylla™ system can be used for the analysis of EGFR mutations in NSCLC patients. Importantly, as the results are massively influenced by the preanalytical steps, we also provide information on the correct sample selection to avoid nonconclusive results.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Análise Mutacional de DNA/instrumentação , Neoplasias Pulmonares/genética , Reação em Cadeia da Polimerase em Tempo Real/instrumentação , Líquido da Lavagem Broncoalveolar , Carcinoma Pulmonar de Células não Pequenas/patologia , Análise Mutacional de DNA/métodos , Receptores ErbB/genética , Fixadores/química , Formaldeído/química , Humanos , Pulmão/patologia , Neoplasias Pulmonares/patologia , Mutação , Inclusão em Parafina/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reprodutibilidade dos Testes , Fixação de Tecidos/métodos , Fluxo de Trabalho
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