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1.
Proc Natl Acad Sci U S A ; 117(37): 22727-22735, 2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32868442

RESUMO

The COVID-19 pandemic provides an urgent example where a gap exists between availability of state-of-the-art diagnostics and current needs. As assay protocols and primer sequences become widely known, many laboratories perform diagnostic tests using methods such as RT-PCR or reverse transcription loop mediated isothermal amplification (RT-LAMP). Here, we report an RT-LAMP isothermal assay for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus and demonstrate the assay on clinical samples using a simple and accessible point-of-care (POC) instrument. We characterized the assay by dipping swabs into synthetic nasal fluid spiked with the virus, moving the swab to viral transport medium (VTM), and sampling a volume of the VTM to perform the RT-LAMP assay without an RNA extraction kit. The assay has a limit of detection (LOD) of 50 RNA copies per µL in the VTM solution within 30 min. We further demonstrate our assay by detecting SARS-CoV-2 viruses from 20 clinical samples. Finally, we demonstrate a portable and real-time POC device to detect SARS-CoV-2 from VTM samples using an additively manufactured three-dimensional cartridge and a smartphone-based reader. The POC system was tested using 10 clinical samples, and was able to detect SARS-CoV-2 from these clinical samples by distinguishing positive samples from negative samples after 30 min. The POC tests are in complete agreement with RT-PCR controls. This work demonstrates an alternative pathway for SARS-CoV-2 diagnostics that does not require conventional laboratory infrastructure, in settings where diagnosis is required at the point of sample collection.


Assuntos
Infecções por Coronavirus/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Pneumonia Viral/diagnóstico , Testes Imediatos/normas , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Betacoronavirus/genética , Betacoronavirus/patogenicidade , Humanos , Limite de Detecção , Técnicas de Diagnóstico Molecular/instrumentação , Técnicas de Diagnóstico Molecular/normas , Mucosa Nasal/virologia , Pandemias , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa/instrumentação , Reação em Cadeia da Polimerase Via Transcriptase Reversa/normas , Smartphone
2.
Viruses ; 12(9)2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32883050

RESUMO

Until vaccines and effective therapeutics become available, the practical solution to transit safely out of the current coronavirus disease 19 (CoVID-19) lockdown may include the implementation of an effective testing, tracing and tracking system. However, this requires a reliable and clinically validated diagnostic platform for the sensitive and specific identification of SARS-CoV-2. Here, we report on the development of a de novo, high-resolution and comparative genomics guided reverse-transcribed loop-mediated isothermal amplification (LAMP) assay. To further enhance the assay performance and to remove any subjectivity associated with operator interpretation of results, we engineered a novel hand-held smart diagnostic device. The robust diagnostic device was further furnished with automated image acquisition and processing algorithms and the collated data was processed through artificial intelligence (AI) pipelines to further reduce the assay run time and the subjectivity of the colorimetric LAMP detection. This advanced AI algorithm-implemented LAMP (ai-LAMP) assay, targeting the RNA-dependent RNA polymerase gene, showed high analytical sensitivity and specificity for SARS-CoV-2. A total of ~200 coronavirus disease (CoVID-19)-suspected NHS patient samples were tested using the platform and it was shown to be reliable, highly specific and significantly more sensitive than the current gold standard qRT-PCR. Therefore, this system could provide an efficient and cost-effective platform to detect SARS-CoV-2 in resource-limited laboratories.


Assuntos
Inteligência Artificial , Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/virologia , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Pneumonia Viral/virologia , Animais , Chlorocebus aethiops , Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/diagnóstico , Cães , Humanos , Células Madin Darby de Rim Canino , Pandemias , Pneumonia Viral/diagnóstico , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real , Sensibilidade e Especificidade , Células Vero
3.
Proc Natl Acad Sci U S A ; 117(39): 24450-24458, 2020 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-32900935

RESUMO

The current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has had an enormous impact on society worldwide, threatening the lives and livelihoods of many. The effects will continue to grow and worsen if economies begin to open without the proper precautions, including expanded diagnostic capabilities. To address this need for increased testing, we have developed a sensitive reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay compatible with current reagents, which utilizes a colorimetric readout in as little as 30 min. A rapid inactivation protocol capable of inactivating virions, as well as endogenous nucleases, was optimized to increase sensitivity and sample stability. This protocol, combined with the RT-LAMP assay, has a sensitivity of at least 50 viral RNA copies per microliter in a sample. To further increase the sensitivity, a purification protocol compatible with this inactivation method was developed. The inactivation and purification protocol, combined with the RT-LAMP assay, brings the sensitivity to at least 1 viral RNA copy per microliter in a sample. This simple inactivation and purification pipeline is inexpensive and compatible with other downstream RNA detection platforms and uses readily available reagents. It should increase the availability of SARS-CoV-2 testing as well as expand the settings in which this testing can be performed.


Assuntos
Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Pneumonia Viral/diagnóstico , Betacoronavirus/genética , Técnicas de Laboratório Clínico/economia , Colorimetria , Infecções por Coronavirus/economia , Infecções por Coronavirus/virologia , Genoma Viral/genética , Humanos , Concentração de Íons de Hidrogênio , Técnicas de Diagnóstico Molecular/economia , Técnicas de Amplificação de Ácido Nucleico/economia , Pandemias , Pneumonia Viral/virologia , Estabilidade de RNA , RNA Viral/química , RNA Viral/genética , RNA Viral/isolamento & purificação , Sensibilidade e Especificidade , Fatores de Tempo , Proteínas Virais/genética , Inativação de Vírus
4.
Nat Commun ; 11(1): 4464, 2020 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-32900994

RESUMO

The SARS-CoV-2 pandemic has shown how a rapid rise in demand for patient and community sample testing can quickly overwhelm testing capability globally. With most diagnostic infrastructure dependent on specialized instruments, their exclusive reagent supplies quickly become bottlenecks, creating an urgent need for approaches to boost testing capacity. We address this challenge by refocusing the London Biofoundry onto the development of alternative testing pipelines. Here, we present a reagent-agnostic automated SARS-CoV-2 testing platform that can be quickly deployed and scaled. Using an in-house-generated, open-source, MS2-virus-like particle (VLP) SARS-CoV-2 standard, we validate RNA extraction and RT-qPCR workflows as well as two detection assays based on CRISPR-Cas13a and RT-loop-mediated isothermal amplification (RT-LAMP). In collaboration with an NHS diagnostic testing lab, we report the performance of the overall workflow and detection of SARS-CoV-2 in patient samples using RT-qPCR, CRISPR-Cas13a, and RT-LAMP. The validated RNA extraction and RT-qPCR platform has been installed in NHS diagnostic labs, increasing testing capacity by 1000 samples per day.


Assuntos
Betacoronavirus/genética , Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/virologia , Pneumonia Viral/diagnóstico , Pneumonia Viral/virologia , Betacoronavirus/isolamento & purificação , Bioensaio , Sistemas CRISPR-Cas , Técnicas de Laboratório Clínico/instrumentação , Técnicas de Laboratório Clínico/normas , Humanos , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Pandemias , RNA Viral/análise , RNA Viral/genética , RNA Viral/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real , Sensibilidade e Especificidade
5.
Nat Commun ; 11(1): 4774, 2020 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-32963224

RESUMO

Detection of microbial nucleic acids in body fluids has become the preferred method for rapid diagnosis of many infectious diseases. However, culture-based diagnostics that are time-consuming remain the gold standard approach in certain cases, such as sepsis. New culture-free methods are urgently needed. Here, we describe Single MOLecule Tethering or SMOLT, an amplification-free and purification-free molecular assay that can detect microorganisms in body fluids with high sensitivity without the need of culturing. The signal of SMOLT is generated by the displacement of micron-size beads tethered by DNA probes that are between 1 and 7 microns long. The molecular extension of thousands of DNA probes is determined with sub-micron precision using a robust and rapid optical approach. We demonstrate that SMOLT can detect nucleic acids directly in blood, urine and sputum at sub-femtomolar concentrations, and microorganisms in blood at 1 CFU mL-1 (colony forming unit per milliliter) threefold faster, with higher multiplexing capacity and with a more straight-forward protocol than amplified methodologies. SMOLT's clinical utility is further demonstrated by developing a multiplex assay for simultaneous detection of sepsis-causing Candida species directly in whole blood.


Assuntos
Líquidos Corporais/química , Técnicas de Diagnóstico Molecular/métodos , Ácidos Nucleicos/isolamento & purificação , Sepse/diagnóstico , Candida/genética , Candida/isolamento & purificação , Candidíase/diagnóstico , Contagem de Colônia Microbiana , Doenças Transmissíveis/diagnóstico , DNA/isolamento & purificação , Humanos , Ácidos Nucleicos/sangue , Ácidos Nucleicos/urina , Reação em Cadeia da Polimerase/métodos , RNA/isolamento & purificação , Sensibilidade e Especificidade , Sepse/microbiologia , Infecções Estafilocócicas/diagnóstico , Staphylococcus aureus/genética , Staphylococcus aureus/isolamento & purificação , Urina
6.
J Virol Methods ; 285: 113970, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32920028

RESUMO

The global COVID-19 pandemic has led to the rapid development of tests for detection of SARS-CoV-2. Studies are required to assess the relative performance of different assays. Here, we compared the performance of two commercial assays, the cobas® SARS-CoV-2 (Roche Diagnostics) and Xpert® Xpress SARS-CoV-2 (Cepheid®) tests, and a laboratory developed RT-PCR test adapted for use on the Hologic® Panther Fusion® (Hologic®) instrument as well as Bio-Rad and QIAGEN real-time PCR detection systems. Performance characteristics for each test were determined by testing clinical specimens and reference material. All assays detect the pan-Sarbecovirus E (envelope structural protein) gene plus a SARS-CoV-2-specific target. The limit of detection for the E gene target varied from ∼2 copies/reaction to >30 copies/reaction. Due to assay-specific differences in sample processing and nucleic acid extraction, the overall analytical sensitivity ranged from 24 copies/mL specimen to 574 copies/mL specimen. Despite these differences, there was 100 % agreement between the commercial and laboratory developed tests. No false-negative or false-positive SARS-CoV-2 results were observed and there was no cross-reactivity with common respiratory viruses, including endemic coronaviruses.


Assuntos
Betacoronavirus , Técnicas de Laboratório Clínico , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/virologia , Técnicas de Diagnóstico Molecular , Pneumonia Viral/diagnóstico , Pneumonia Viral/virologia , Kit de Reagentes para Diagnóstico , Betacoronavirus/genética , Betacoronavirus/imunologia , Técnicas de Laboratório Clínico/métodos , Técnicas de Laboratório Clínico/normas , Humanos , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Diagnóstico Molecular/normas , Pandemias , Kit de Reagentes para Diagnóstico/normas , Sensibilidade e Especificidade
7.
Nat Commun ; 11(1): 4711, 2020 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-32948757

RESUMO

The recent outbreak of novel coronavirus (SARS-CoV-2) causing COVID-19 disease spreads rapidly in the world. Rapid and early detection of SARS-CoV-2 facilitates early intervention and prevents the disease spread. Here, we present an All-In-One Dual CRISPR-Cas12a (AIOD-CRISPR) assay for one-pot, ultrasensitive, and visual SARS-CoV-2 detection. By targeting SARS-CoV-2's nucleoprotein gene, two CRISPR RNAs without protospacer adjacent motif (PAM) site limitation are introduced to develop the AIOD-CRISPR assay and detect the nucleic acids with a sensitivity of few copies. We validate the assay by using COVID-19 clinical swab samples and obtain consistent results with RT-PCR assay. Furthermore, a low-cost hand warmer (~$0.3) is used as an incubator of the AIOD-CRISPR assay to detect clinical samples within 20 min, enabling an instrument-free, visual SARS-CoV-2 detection at the point of care. Thus, our method has the significant potential to provide a rapid, sensitive, one-pot point-of-care assay for SARS-CoV-2.


Assuntos
Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/virologia , Pneumonia Viral/virologia , Betacoronavirus/genética , Sistemas CRISPR-Cas , Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/diagnóstico , Genes Virais , Humanos , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Pandemias , Pneumonia Viral/diagnóstico , Sistemas Automatizados de Assistência Junto ao Leito , RNA Viral/análise , RNA Viral/genética , Sensibilidade e Especificidade , Proteínas Virais/análise , Proteínas Virais/genética
8.
Biosens Bioelectron ; 169: 112642, 2020 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-32979593

RESUMO

The outbreaks of the infectious disease COVID-19 caused by SARS-CoV-2 seriously threatened the life of humans. A rapid, reliable and specific detection method was urgently needed. Herein, we reported a contamination-free visual detection method for SARS-CoV-2 with LAMP and CRISPR/Cas12a technology. CRISPR/Cas12a reagents were pre-added on the inner wall of the tube lid. After LAMP reaction, CRISPR/Cas12a reagents were flowed into the tube and mixed with amplicon solution by hand shaking, which can effectively avoid possible amplicon formed aerosol contamination caused by re-opening the lid after amplification. CRISPR/Cas12a can highly specific recognize target sequence and discriminately cleave single strand DNA probes (5'-6FAM 3'-BHQ1). With smart phone and portable 3D printing instrument, the produced fluorescence can be seen by naked eyes without any dedicated instruments, which is promising in the point-of-care detection. The whole amplification and detection process could be completed within 40 min with high sensitivity of 20 copies RNA of SARS-CoV-2. This reaction had high specificity and could avoid cross-reactivity with other common viruses such as influenza virus. For 7 positive and 3 negative respiratory swab samples provided by Zhejiang Provincial Center for Disease Control and Prevention, our detection results had 100% positive agreement and 100% negative agreement, which demonstrated the accuracy and application prospect of this method.


Assuntos
Betacoronavirus/isolamento & purificação , Técnicas Biossensoriais/métodos , Infecções por Coronavirus/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Pneumonia Viral/diagnóstico , Sistemas Automatizados de Assistência Junto ao Leito , Betacoronavirus/genética , Técnicas Biossensoriais/instrumentação , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Infecções por Coronavirus/virologia , Desenho de Equipamento , Fluorescência , Humanos , Técnicas de Diagnóstico Molecular/instrumentação , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Pandemias , Pneumonia Viral/virologia , Sensibilidade e Especificidade , Smartphone
9.
mSphere ; 5(4)2020 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-32848011

RESUMO

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreak urgently necessitates sensitive and convenient COVID-19 diagnostics for the containment and timely treatment of patients. We aimed to develop and validate a novel reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay to detect SARS-CoV-2. Patients with suspected COVID-19 and close contacts were recruited from two hospitals between 26 January and 8 April 2020. Respiratory samples were collected and tested using RT-LAMP, and the results were compared with those obtained by reverse transcription-quantitative PCR (RT-qPCR). Samples yielding inconsistent results between these two methods were subjected to next-generation sequencing for confirmation. RT-LAMP was also applied to an asymptomatic COVID-19 carrier and patients with other respiratory viral infections. Samples were collected from a cohort of 129 cases (329 nasopharyngeal swabs) and an independent cohort of 76 patients (152 nasopharyngeal swabs and sputum samples). The RT-LAMP assay was validated to be accurate (overall sensitivity and specificity of 88.89% and 99.00%, respectively) and diagnostically useful (positive and negative likelihood ratios of 88.89 and 0.11, respectively). RT-LAMP showed increased sensitivity (88.89% versus 81.48%) and high consistency (kappa, 0.92) compared to those of RT-qPCR for SARS-CoV-2 screening while requiring only constant-temperature heating and visual inspection. The time required for RT-LAMP was less than 1 h from sample preparation to the result. In addition, RT-LAMP was feasible for use with asymptomatic patients and did not cross-react with other respiratory pathogens. The developed RT-LAMP assay offers rapid, sensitive, and straightforward detection of SARS-CoV-2 infection and may aid the expansion of COVID-19 testing in the public domain and hospitals.IMPORTANCE We developed a visual and rapid reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay targeting the S gene for SARS-CoV-2 infection. The strength of our study was that we validated the RT-LAMP assay using 481 clinical respiratory samples from two prospective cohorts of suspected COVID-19 patients and on the serial samples from an asymptomatic carrier. The developed RT-LAMP approach showed an increased sensitivity (88.89%) and high consistency (kappa, 0.92) compared with those of reverse transcription-quantitative PCR (RT-qPCR) for SARS-CoV-2 screening while requiring only constant-temperature heating and visual inspection, facilitating SARS-CoV-2 screening in well-equipped labs as well as in the field. The time required for RT-LAMP was less than 1 h from sample preparation to the result (more than 2 h for RT-qPCR). This study showed that the RT-LAMP assay was a simple, rapid, and sensitive approach for SARS-CoV-2 infection and can facilitate COVID-19 diagnosis, especially in resource-poor settings.


Assuntos
Betacoronavirus/genética , Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Pneumonia Viral/diagnóstico , Adulto , Doenças Assintomáticas , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Pandemias , Estudos Prospectivos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Sensibilidade e Especificidade
10.
J Med Microbiol ; 69(9): 1169-1178, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32755529

RESUMO

Introduction. The SARS-CoV-2 pandemic of 2020 has resulted in unparalleled requirements for RNA extraction kits and enzymes required for virus detection, leading to global shortages. This has necessitated the exploration of alternative diagnostic options to alleviate supply chain issues.Aim. To establish and validate a reverse transcription loop-mediated isothermal amplification (RT- LAMP) assay for the detection of SARS-CoV-2 from nasopharyngeal swabs.Methodology. We used a commercial RT-LAMP mastermix from OptiGene in combination with a primer set designed to detect the CDC N1 region of the SARS-CoV-2 nucleocapsid (N) gene. A single-tube, single-step fluorescence assay was implemented whereby 1 µl of universal transport medium (UTM) directly from a nasopharyngeal swab could be used as template, bypassing the requirement for RNA purification. Amplification and detection could be conducted in any thermocycler capable of holding 65 °C for 30 min and measure fluorescence in the FAM channel at 1 min intervals.Results. Assay evaluation by assessment of 157 clinical specimens previously screened by E-gene RT-qPCR revealed assay sensitivity and specificity of 87 and 100%, respectively. Results were fast, with an average time-to-positive (Tp) for 93 clinical samples of 14 min (sd±7 min). Using dilutions of SARS-CoV-2 virus spiked into UTM, we also evaluated assay performance against FDA guidelines for implementation of emergency-use diagnostics and established a limit-of-detection of 54 Tissue Culture Infectious Dose 50 per ml (TCID50 ml-1), with satisfactory assay sensitivity and specificity. A comparison of 20 clinical specimens between four laboratories showed excellent interlaboratory concordance; performing equally well on three different, commonly used thermocyclers, pointing to the robustness of the assay.Conclusion. With a simplified workflow, The N1 gene Single Tube Optigene LAMP assay (N1-STOP-LAMP) is a powerful, scalable option for specific and rapid detection of SARS-CoV-2 and an additional resource in the diagnostic armamentarium against COVID-19.


Assuntos
Infecções por Coronavirus/diagnóstico , Técnicas de Amplificação de Ácido Nucleico/métodos , Pneumonia Viral/diagnóstico , Betacoronavirus , Técnicas de Laboratório Clínico , Humanos , Técnicas de Diagnóstico Molecular/métodos , Nasofaringe/virologia , Pandemias , RNA Viral , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição Reversa , Sensibilidade e Especificidade
11.
Genes (Basel) ; 11(8)2020 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-32824573

RESUMO

The COVID-19 pandemic has spread very fast around the world. A few days after the first detected case in South Africa, an infection started in a large hospital outbreak in Durban, KwaZulu-Natal (KZN). Phylogenetic analysis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomes can be used to trace the path of transmission within a hospital. It can also identify the source of the outbreak and provide lessons to improve infection prevention and control strategies. This manuscript outlines the obstacles encountered in order to genotype SARS-CoV-2 in near-real time during an urgent outbreak investigation. This included problems with the length of the original genotyping protocol, unavailability of reagents, and sample degradation and storage. Despite this, three different library preparation methods for Illumina sequencing were set up, and the hands-on library preparation time was decreased from twelve to three hours, which enabled the outbreak investigation to be completed in just a few weeks. Furthermore, the new protocols increased the success rate of sequencing whole viral genomes. A simple bioinformatics workflow for the assembly of high-quality genomes in near-real time was also fine-tuned. In order to allow other laboratories to learn from our experience, all of the library preparation and bioinformatics protocols are publicly available at protocols.io and distributed to other laboratories of the Network for Genomics Surveillance in South Africa (NGS-SA) consortium.


Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/diagnóstico , Genoma Viral , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Técnicas de Diagnóstico Molecular/métodos , Pneumonia Viral/diagnóstico , Sequenciamento Completo do Genoma/métodos , Betacoronavirus/patogenicidade , Infecções por Coronavirus/virologia , Sequenciamento de Nucleotídeos em Larga Escala/normas , Humanos , Técnicas de Diagnóstico Molecular/normas , Pandemias , Pneumonia Viral/virologia , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Sequenciamento Completo do Genoma/normas
12.
J Int Med Res ; 48(8): 300060520949067, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32840148

RESUMO

The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleic acid test is currently the gold standard for diagnosing coronavirus disease 2019 (COVID-19). This disease requires high-quality viral nucleic acid tests, and selecting the type of specimen from patients, who are at different disease stages, to use in the nucleic acid test is challenging. This article reports in detail the diagnosis and treatment process for two patients with confirmed COVID-19 and analyzes the results of the SARS-CoV-2 nucleic acid tests that were used for different types of specimens (sputum from deep cough, nasopharyngeal swab, and feces). The nucleic acid testing results of sputum from deep cough showed the best performance for positive detection. Our findings provide a reference for selecting the most suitable specimen for the clinical diagnosis of COVID-19 and improving the positive detection rate.


Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Pneumonia Viral/diagnóstico , Idoso , Humanos , Masculino , Pessoa de Meia-Idade , Pandemias , RNA Viral/análise
13.
Curr Med Sci ; 40(4): 614-617, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32767258

RESUMO

The novel Coronavirus SARS-CoV-2 caused an outbreak of pneumonia in Wuhan, Hubei province of China in January 2020. This study aims to investigate the effects of different temperature and time durations of virus inactivation on the results of PCR testing for SARS-CoV-2. Twelve patients at the Renmin Hospital of Wuhan University suspected of being infected with SARS-CoV-2 were selected on February 13, 2020 and throat swabs were taken. The swabs were stored at room temperature (20-25°C), then divided into aliquots and subjected to different temperature for different periods in order to inactivate the viruses (56°C for 30, 45, 60 min; 65, 70, 80°C for 10, 15, 20 min). Control aliquots were stored at room temperature for 60 min. Then all aliquots were tested in a real-time fluorescence PCR using primers against SARS-CoV-2. Regardless of inactivation temperature and time, 7 of 12 cases (58.3%) tested were positive for SARS-CoV-2 by PCR, and cycle threshold values were similar. These results suggest that virus inactivation parameters exert minimal influence on PCR test results. Inactivation at 65°C for 10 min may be sufficient to ensure safe, reliable testing.


Assuntos
Betacoronavirus/genética , Betacoronavirus/isolamento & purificação , Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/virologia , Pneumonia Viral/diagnóstico , Pneumonia Viral/virologia , Reação em Cadeia da Polimerase em Tempo Real/métodos , Inativação de Vírus , Adulto , Idoso , China/epidemiologia , Infecções por Coronavirus/epidemiologia , Humanos , Controle de Infecções/métodos , Pessoal de Laboratório Médico , Pessoa de Meia-Idade , Técnicas de Diagnóstico Molecular/métodos , Exposição Ocupacional/prevenção & controle , Pandemias , Pneumonia Viral/epidemiologia , RNA Viral/análise , RNA Viral/genética , Temperatura , Fatores de Tempo
15.
J Clin Virol ; 130: 104573, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32769025

RESUMO

BACKGROUND: RT-PCR testing is crucial in the diagnostic of SARS-CoV-2 infection. The use of reliable and comparable PCR assays is a cornerstone to allow use of different PCR assays depending on the local equipment. In this work, we provide a comparison of the Cobas® (Roche) and the RealStar® assay (Altona). METHODS: Assessment of the two assays was performed prospectively in three reference Parisians hospitals, using 170 clinical samples. They were tested with the Cobas® assay, selected to obtain a distribution of cycle threshold (Ct) as large as possible, and tested with the RealStar assay with three largely available extraction platforms: QIAsymphony (Qiagen), MagNAPure (Roche) and NucliSENS-easyMag (BioMérieux). RESULTS: Overall, the agreement (positive for at least one gene) was 76 %. This rate differed considerably depending on the Cobas Ct values for gene E: below 35 (n = 91), the concordance was 99 %. Regarding the positive Ct values, linear regression analysis showed a coefficient of determination (R2) of 0.88 and the Deming regression line revealed a strong correlation with a slope of 1.023 and an intercept of -3.9. Bland-Altman analysis showed that the mean difference (Cobas® minus RealStar®) was + 3.3 Ct, with a SD of + 2.3 Ct. CONCLUSIONS: In this comparison, both RealStar® and Cobas® assays provided comparable qualitative results and a high correlation when both tests were positive. Discrepancies exist after 35 Ct and varied depending on the extraction system used for the RealStar® assay, probably due to a low viral load close to the detection limit of both assays.


Assuntos
Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Pneumonia Viral/diagnóstico , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Betacoronavirus , Humanos , Limite de Detecção , Pandemias , Estudos Prospectivos , Kit de Reagentes para Diagnóstico , Sensibilidade e Especificidade , Carga Viral , Proteínas Virais/genética
16.
J Clin Virol ; 130: 104578, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32777761

RESUMO

The SARS-CoV-2 pandemic has challenged molecular microbiology laboratories to quickly implement and validate diagnostic assays and to expand testing capacity in a short timeframe. Multiple molecular diagnostic methods received FDA emergency use authorization (EUA) and were promptly validated for use nationwide. Several studies reported the analytical and/ or clinical evaluation of these molecular assays, however differences in the viral materials used for these evaluations complicated direct comparison of their analytical performance. In this study, we compared the analytical sensitivity (lower limit of detection, LOD) of seven commonly used qualitative SARS-CoV-2 molecular assays: the Abbott Molecular RealTime SARS-CoV-2 assay, the NeuMoDx™ SARS-CoV-2 assay, the Roche Cobas®SARS-CoV-2 assay, the BD SARS-CoV-2 reagents for BD MAX™ system, the Hologic Aptima® SARS-CoV-2 assay, the Xpert Xpress SARS-CoV-2 test, and the GenMark ePlex SARS-CoV-2 test. The comparison was performed utilizing a single positive clinical specimen that was serially diluted in viral transport media and quantified by the EUA approved SARS-CoV-2 droplet digital PCR (ddPCR) assay. Replicate samples were prepared and evaluated for reproducibility across different molecular assays with multiple replicates per assay. Our data demonstrated that the seven assays could detect 100 % of replicates at a nucleocapsid gene concentration of (N1 = 1,267 and N2 = 1,392) copies/mL. At a one log less concentration, the Abbott, the Roche, and the Xpert Xpress assays detected 100 % of the tested replicates.


Assuntos
Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/diagnóstico , Pneumonia Viral/diagnóstico , Automação Laboratorial , Betacoronavirus , Humanos , Limite de Detecção , Técnicas de Diagnóstico Molecular/métodos , Pandemias , Kit de Reagentes para Diagnóstico , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
17.
Hell J Nucl Med ; 23 Suppl: 8-14, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32860390

RESUMO

On December 2019, a new coronavirus disease (COVID-19) emerged in China and spread worldwide, causing acute severe respiratory syndrome. Due to the increased transmission rate of the virus, it became of great importance the early diagnosis of the disease. The coronavirus pandemic led to the development of numerous tests in order to mass screening population for active viral load and for the identification of antibodies for epidemiological purposes. This review summarizes the different diagnostic tests available to the clinicians for the diagnosis and follow up of the SARS COV-2 infections.


Assuntos
Infecções por Coronavirus/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Pneumonia Viral/diagnóstico , Radiografia Torácica/métodos , Técnicas de Cultura de Células/métodos , Infecções por Coronavirus/sangue , Infecções por Coronavirus/diagnóstico por imagem , Humanos , Imunoensaio/métodos , Imunoensaio/normas , Técnicas de Diagnóstico Molecular/normas , Pandemias , Pneumonia Viral/sangue , Pneumonia Viral/diagnóstico por imagem , Radiografia Torácica/normas , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/normas , Sensibilidade e Especificidade , Testes Sorológicos/métodos , Testes Sorológicos/normas
18.
PLoS Pathog ; 16(8): e1008705, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32853291

RESUMO

The recent outbreak of human infections caused by SARS-CoV-2, the third zoonotic coronavirus has raised great public health concern globally. Rapid and accurate diagnosis of this novel pathogen posts great challenges not only clinically but also technologically. Metagenomic next-generation sequencing (mNGS) and reverse-transcription PCR (RT-PCR) have been the most commonly used molecular methodologies. However, each has their own limitations. In this study, we developed an isothermal, CRISPR-based diagnostic for COVID-19 with near single-copy sensitivity. The diagnostic performances of all three technology platforms were also compared. Our study aimed to provide more insights into the molecular detection of SARS-CoV-2, and also to present a novel diagnostic option for this new emerging virus.


Assuntos
Betacoronavirus/genética , Sistemas CRISPR-Cas/genética , Técnicas de Laboratório Clínico , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/genética , Pneumonia Viral/diagnóstico , Pneumonia Viral/genética , Bactérias/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Genes Virais/genética , Genoma Viral/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Técnicas de Diagnóstico Molecular/economia , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/economia , Técnicas de Amplificação de Ácido Nucleico/métodos , Pandemias , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Sensibilidade e Especificidade
19.
Biosens Bioelectron ; 167: 112479, 2020 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-32763826

RESUMO

COVID-19 pandemic outbreak is the most astounding scene ever experienced in the 21st century. It has been determined to be caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). With the global pandemic, the lack of efficient rapid and accurate molecular diagnostic testing tools has hindered the public opportunely response to the emerging viral threat. Herein, a DNA nanoscaffold hybrid chain reaction (DNHCR)-based nucleic acid assay strategy is reported for rapid detection of SARS-CoV-2 RNA. In this method, the DNA nanoscaffolds have been first constructed by the self-assembly of long DNA strands and self-quenching probes (H1). Then, the SARS-CoV-2 RNA will initiate the hybridization of H1 and free H2 DNA probes along the nanoscaffold, and an illuminated DNA nanostring is instantly obtained. By taking advantages of the localization design of the H1 probes and the temperature tolerance of the isothermal amplification, the proposed DNHCR method can detect target at short responding time (within 10 min) and mild condition (15 °C-35 °C). Moreover, the reliability of DNHCR method in serum and saliva samples have also been validated. Therefore, DNHCR-based method is expected to provide a simple and faster alternative to the traditional SARS-CoV-2 qRT-PCR assay.


Assuntos
Betacoronavirus , Técnicas Biossensoriais/métodos , Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/virologia , Pneumonia Viral/diagnóstico , Pneumonia Viral/virologia , Betacoronavirus/genética , Betacoronavirus/isolamento & purificação , Técnicas de Laboratório Clínico/estatística & dados numéricos , Infecções por Coronavirus/epidemiologia , DNA/síntese química , DNA/química , DNA/genética , Estudos de Viabilidade , Humanos , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Diagnóstico Molecular/estatística & dados numéricos , Nanoestruturas/química , Nanotecnologia , Técnicas de Amplificação de Ácido Nucleico/métodos , Pandemias , Pneumonia Viral/epidemiologia , RNA Viral/análise , RNA Viral/genética , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
20.
Viruses ; 12(8)2020 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-32784757

RESUMO

Rapid large-scale testing is essential for controlling the ongoing pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The standard diagnostic pipeline for testing SARS-CoV-2 presence in patients with an ongoing infection is predominantly based on pharyngeal swabs, from which the viral RNA is extracted using commercial kits, followed by reverse transcription and quantitative PCR detection. As a result of the large demand for testing, commercial RNA extraction kits may be limited and, alternatively, non-commercial protocols are needed. Here, we provide a magnetic bead RNA extraction protocol that is predominantly based on in-house made reagents and is performed in 96-well plates supporting large-scale testing. Magnetic bead RNA extraction was benchmarked against the commercial QIAcube extraction platform. Comparable viral RNA detection sensitivity and specificity were obtained by fluorescent and colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) using a primer set targeting the N gene, as well as RT-qPCR using a primer set targeting the E gene, showing that the RNA extraction protocol presented here can be combined with a variety of detection methods at high throughput. Importantly, the presented diagnostic workflow can be quickly set up in a laboratory without access to an automated pipetting robot.


Assuntos
Betacoronavirus/química , Betacoronavirus/genética , Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/virologia , Pneumonia Viral/virologia , RNA Viral/isolamento & purificação , Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/diagnóstico , Humanos , Fenômenos Magnéticos , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Pandemias , Pneumonia Viral/diagnóstico , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Transcrição Reversa , Sensibilidade e Especificidade
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