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1.
Biosens Bioelectron ; 172: 112766, 2021 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-33126177

RESUMO

The 2019 novel coronavirus disease (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has affected all aspects of human life. Rapid, accurate, sensitive and user friendly detection method is urgently needed to facilitate early intervention and control the spread of SARS-CoV-2. Here, we propose a one-pot visual SARS-CoV-2 detection system named "opvCRISPR" by integrating reverse transcription loop-mediated isothermal amplification (RT-LAMP) and Cas12a cleavage in a single reaction system. We demonstrate that the collateral activity against single-stranded DNA (ssDNA) reporters of activated Cas12a triggered by RT-LAMP amplicon increases detection sensitivity and makes detection results observable with naked eye. The opvCRISPR enables detection at nearly single molecule level in 45 min. We validate this method with 50 SARS-CoV-2 potentially infected clinical samples. The opvCRISPR diagnostic results provide 100% agreement with the Centers for Disease Control and Prevention (CDC)-approved quantitative RT-PCR assay. The opvCRISPR holds great potential for SARS-CoV-2 detection in next-generation point-of-care molecular diagnostics.


Assuntos
/métodos , Sistemas CRISPR-Cas , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , /genética , Sequência de Bases , /instrumentação , Humanos , Técnicas de Diagnóstico Molecular/instrumentação , Técnicas de Diagnóstico Molecular/estatística & dados numéricos , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Técnicas de Amplificação de Ácido Nucleico/estatística & dados numéricos , Pandemias , RNA Viral/genética , Sensibilidade e Especificidade
2.
Nat Protoc ; 15(11): 3663-3677, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-33005038

RESUMO

The complexity of current nucleic acid isolation methods limits their use outside of the modern laboratory environment. Here, we describe a fast and affordable method to purify nucleic acids from animal, plant, viral and microbial samples using a cellulose-based dipstick. Nucleic acids can be purified by dipping in-house-made dipsticks into just three solutions: the extract (to bind the nucleic acids), a wash buffer (to remove impurities) and the amplification reaction (to elute the nucleic acids). The speed and simplicity of this method make it ideally suited for molecular applications, both within and outside the laboratory, including limited-resource settings such as remote field sites and teaching institutions. Detailed instructions for how to easily manufacture large numbers of dipsticks in house are provided. Using the instructions, readers can create more than 200 dipsticks in <30 min and perform dipstick-based nucleic acid purifications in 30 s.


Assuntos
Celulose/química , Ácidos Nucleicos/isolamento & purificação , Animais , Bactérias/química , Humanos , Técnicas de Amplificação de Ácido Nucleico/economia , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Técnicas de Amplificação de Ácido Nucleico/métodos , Ácidos Nucleicos/genética , Plantas/química , Fatores de Tempo , Vírus/química
3.
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
4.
Biosens Bioelectron ; 169: 112592, 2020 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-32942143

RESUMO

Global health and food security constantly face the challenge of emerging human and plant diseases caused by bacteria, viruses, fungi, and other pathogens. Disease outbreaks such as SARS, MERS, Swine Flu, Ebola, and COVID-19 (on-going) have caused suffering, death, and economic losses worldwide. To prevent the spread of disease and protect human populations, rapid point-of-care (POC) molecular diagnosis of human and plant diseases play an increasingly crucial role. Nucleic acid-based molecular diagnosis reveals valuable information at the genomic level about the identity of the disease-causing pathogens and their pathogenesis, which help researchers, healthcare professionals, and patients to detect the presence of pathogens, track the spread of disease, and guide treatment more efficiently. A typical nucleic acid-based diagnostic test consists of three major steps: nucleic acid extraction, amplification, and amplicon detection. Among these steps, nucleic acid extraction is the first step of sample preparation, which remains one of the main challenges when converting laboratory molecular assays into POC tests. Sample preparation from human and plant specimens is a time-consuming and multi-step process, which requires well-equipped laboratories and skilled lab personnel. To perform rapid molecular diagnosis in resource-limited settings, simpler and instrument-free nucleic acid extraction techniques are required to improve the speed of field detection with minimal human intervention. This review summarizes the recent advances in POC nucleic acid extraction technologies. In particular, this review focuses on novel devices or methods that have demonstrated applicability and robustness for the isolation of high-quality nucleic acid from complex raw samples, such as human blood, saliva, sputum, nasal swabs, urine, and plant tissues. The integration of these rapid nucleic acid preparation methods with miniaturized assay and sensor technologies would pave the road for the "sample-in-result-out" diagnosis of human and plant diseases, especially in remote or resource-limited settings.


Assuntos
Doenças Transmissíveis/diagnóstico , Dispositivos Lab-On-A-Chip , Ácidos Nucleicos/isolamento & purificação , Doenças das Plantas , Sistemas Automatizados de Assistência Junto ao Leito , Betacoronavirus/isolamento & purificação , Fracionamento Químico/instrumentação , Fracionamento Químico/métodos , Doenças Transmissíveis/microbiologia , Doenças Transmissíveis/parasitologia , Doenças Transmissíveis/virologia , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/virologia , Desenho de Equipamento , Humanos , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Técnicas de Amplificação de Ácido Nucleico/métodos , Ácidos Nucleicos/sangue , Ácidos Nucleicos/urina , Pandemias , Doenças das Plantas/microbiologia , Doenças das Plantas/parasitologia , Doenças das Plantas/virologia , Pneumonia Viral/diagnóstico , Pneumonia Viral/virologia
5.
Virus Res ; 288: 198129, 2020 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-32822689

RESUMO

The COVID-19 pandemic caused by SARS-CoV-2 affects all aspects of human life. Detection platforms that are efficient, rapid, accurate, specific, sensitive, and user friendly are urgently needed to manage and control the spread of SARS-CoV-2. RT-qPCR based methods are the gold standard for SARS-CoV-2 detection. However, these methods require trained personnel, sophisticated infrastructure, and a long turnaround time, thereby limiting their usefulness. Reverse transcription-loop-mediated isothermal amplification (RT-LAMP), a one-step nucleic acid amplification method conducted at a single temperature, has been used for colorimetric virus detection. CRISPR-Cas12 and CRISPR-Cas13 systems, which possess collateral activity against ssDNA and RNA, respectively, have also been harnessed for virus detection. Here, we built an efficient, rapid, specific, sensitive, user-friendly SARS-CoV-2 detection module that combines the robust virus amplification of RT-LAMP with the specific detection ability of SARS-CoV-2 by CRISPR-Cas12. Furthermore, we combined the RT-LAMP-CRISPR-Cas12 module with lateral flow cells to enable highly efficient point-of-care SARS-CoV-2 detection. Our iSCAN SARS-CoV-2 detection module, which exhibits the critical features of a robust molecular diagnostic device, should facilitate the effective management and control of COVID-19.


Assuntos
Betacoronavirus/genética , Sistemas CRISPR-Cas , Técnicas de Laboratório Clínico/métodos , Colorimetria/métodos , Infecções por Coronavirus/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Pneumonia Viral/diagnóstico , Técnicas de Laboratório Clínico/instrumentação , Colorimetria/instrumentação , Infecções por Coronavirus/virologia , Endodesoxirribonucleases/química , Humanos , Técnicas de Diagnóstico Molecular/instrumentação , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Pandemias , Pneumonia Viral/virologia , Sistemas Automatizados de Assistência Junto ao Leito , Reologia , Sensibilidade e Especificidade
6.
Anal Chim Acta ; 1125: 57-65, 2020 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-32674781

RESUMO

Porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), and swine acute diarrhea syndrome-coronavirus (SADS-CoV) are three emerging and re-emerging coronaviruses (CoVs). Symptoms caused by these three viruses are extremely similar, including acute diarrhea, vomiting and even death in piglets. To date, strict biosecurity is still the most effective disease prevention and control measures, and the early detection of pathogens is the most important link. Here, we developed a microfluidic-RT-LAMP chip detection system for the first time, which could detected PEDV, PDCoV and SADS-CoV simultaneously, and had advantages of rapid, simple, sensitive, high-throughput, and accurate at point-of-care settings. The lowest detection limits of the microfluidic-RT-LAMP chip method are 101 copies/µL, 102 copies/µL and 102 copies/µL for PEDV, PDCoV and SADS-CoV, respectively. The whole detection procedure can be finished rapidly in 40 min without any cross-reaction with other common swine viruses. A total of 173 clinical swine fecal samples characterized with diarrheal symptoms were used to evaluate the performance of the newly developed system, which presented good stabilities (C.V.s<5%) and specificities (100%), and possessed sensitivities of 92.24%, 92.19% and 91.23% for PEDV, PDCoV and SADS-CoV respectively. In summary, the established microfluidic-RT-LAMP chip detection system could satisfy the demanding in field diagnoses, which was suitable for promotion in remote areas due to its fast, portable and cost-effective characters.


Assuntos
Coronavirus/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , RNA Viral/análise , Alphacoronavirus/genética , Alphacoronavirus/isolamento & purificação , Animais , Coronavirus/isolamento & purificação , Diarreia/diagnóstico , Diarreia/veterinária , Diarreia/virologia , Fezes/virologia , Dispositivos Lab-On-A-Chip , Limite de Detecção , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Sistemas Automatizados de Assistência Junto ao Leito , Vírus da Diarreia Epidêmica Suína/genética , Vírus da Diarreia Epidêmica Suína/isolamento & purificação , RNA Viral/metabolismo , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Suínos
7.
J Biomed Mater Res A ; 108(10): 1974-1990, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32662571

RESUMO

The coronavirus disease 2019 (COVID-19) pandemic has revealed major shortcomings in our ability to mitigate transmission of infectious viral disease and provide treatment to patients, resulting in a public health crisis. Within months of the first reported case in China, the virus has spread worldwide at an unprecedented rate. COVID-19 illustrates that the biomaterials community was engaged in significant research efforts against bacteria and fungi with relatively little effort devoted to viruses. Accordingly, biomaterials scientists and engineers will have to participate in multidisciplinary antiviral research over the coming years. Although tissue engineering and regenerative medicine have historically dominated the field of biomaterials, current research holds promise for providing transformative solutions to viral outbreaks. To facilitate collaboration, it is imperative to establish a mutual language and adequate understanding between clinicians, industry partners, and research scientists. In this article, clinical perspectives are shared to clearly define emerging healthcare needs that can be met by biomaterials solutions. Strategies and opportunities for novel biomaterials intervention spanning diagnostics, treatment strategies, vaccines, and virus-deactivating surface coatings are discussed. Ultimately this review serves as a call for the biomaterials community to become a leading contributor to the prevention and management of the current and future viral outbreaks.


Assuntos
Betacoronavirus , Materiais Biocompatíveis , Infecções por Coronavirus , Pandemias , Pneumonia Viral , Betacoronavirus/genética , Betacoronavirus/patogenicidade , Betacoronavirus/fisiologia , Técnicas Biossensoriais , Técnicas de Laboratório Clínico , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/prevenção & controle , Infecções por Coronavirus/terapia , Infecções por Coronavirus/transmissão , Desinfecção/métodos , Sistemas de Liberação de Medicamentos , Circulação Extracorpórea , Filtração , Humanos , Testes Imunológicos/instrumentação , Testes Imunológicos/métodos , Metais , Nanoestruturas , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Técnicas de Amplificação de Ácido Nucleico/métodos , Pandemias/prevenção & controle , Pneumonia Viral/diagnóstico , Pneumonia Viral/prevenção & controle , Pneumonia Viral/terapia , Pneumonia Viral/transmissão , Equipamentos de Proteção , RNA Viral/análise , Síndrome Respiratória Aguda Grave/epidemiologia , Síndrome Respiratória Aguda Grave/transmissão , Tensoativos , Engenharia Tecidual , Vacinas Virais
8.
PLoS One ; 15(6): e0234682, 2020.
Artigo em Inglês | MEDLINE | ID: covidwho-595220

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
9.
Biosens Bioelectron ; 165: 112356, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32510339

RESUMO

Circle-to-circle amplification (C2CA) is a specific and precise cascade nucleic acid amplification method consisting of more than one round of padlock probe ligation and rolling circle amplification (RCA). Although C2CA provides a high amplification efficiency with a negligible increase of false-positive risk, it contains several step-by-step operation processes. We herein demonstrate a homogeneous and isothermal nucleic acid quantification strategy based on C2CA and optomagnetic analysis of magnetic nanoparticle (MNP) assembly. The proposed homogeneous circle-to-circle amplification eliminates the need for additional monomerization and ligation steps after the first round of RCA, and combines two amplification rounds in a one-pot reaction. The second round of RCA produces amplicon coils that anneal to detection probes grafted onto MNPs, resulting in MNP assembly that can be detected in real-time using an optomagnetic sensor. The proposed methodology was applied for the detection of a synthetic complementary DNA of SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2, also known as 2019-nCoV) RdRp (RNA-dependent RNA polymerase) coding sequence, achieving a detection limit of 0.4 fM with a dynamic detection range of 3 orders of magnitude and a total assay time of ca. 100 min. A mathematical model was set up and validated to predict the assay performance. Moreover, the proposed method was specific to distinguish SARS-CoV and SARS-CoV-2 sequences with high similarity.


Assuntos
Betacoronavirus/isolamento & purificação , Técnicas Biossensoriais/instrumentação , Infecções por Coronavirus/diagnóstico , DNA Complementar/análise , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Pneumonia Viral/diagnóstico , Técnicas Biossensoriais/métodos , Desenho de Equipamento , Estudos de Viabilidade , Humanos , Limite de Detecção , Magnetismo/instrumentação , Magnetismo/métodos , Nanopartículas de Magnetita/química , Técnicas de Amplificação de Ácido Nucleico/métodos , Pandemias
10.
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
11.
RNA ; 26(7): 771-783, 2020 07.
Artigo em Inglês | MEDLINE | ID: covidwho-154676

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
12.
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
13.
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
14.
Int J Mol Sci ; 21(5)2020 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-32143404

RESUMO

A loop-mediated isothermal amplification (LAMP) assay was developed for simple, rapid and efficient detection of Cucurbit leaf crumple virus (CuLCrV), one of the most important begomoviruses that infects cucurbits worldwide. A set of six specific primers targeting a total 240 nt sequence regions in the DNA A of CuLCrV were designed and synthesized for detection of CuLCrV from infected leaf tissues using real-time LAMP amplification with the Genie® III system, which was further confirmed by gel electrophoresis and SYBR™ Green I DNA staining for visual observation. The optimum reaction temperature and time were determined, and no cross-reactivity was seen with other begomoviruses. The LAMP assay could amplify CuLCrV from a mixed virus assay. The sensitivity assay demonstrated that the LAMP reaction was more sensitive than conventional PCR, but less sensitive than qPCR. However, it was simpler and faster than the other assays evaluated. The LAMP assay also amplified CuLCrV-infected symptomatic and asymptomatic samples more efficiently than PCR. Successful LAMP amplification was observed in mixed virus-infected field samples. This simple, rapid, and sensitive method has the capacity to detect CuLCrV in samples collected in the field and is therefore suitable for early detection of the disease to reduce the risk of epidemics.


Assuntos
Begomovirus/isolamento & purificação , DNA Viral/análise , Técnicas de Diagnóstico Molecular/instrumentação , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Técnicas de Amplificação de Ácido Nucleico/métodos , Doenças das Plantas/virologia , Begomovirus/genética , Cucurbitaceae/virologia , Primers do DNA/genética , Folhas de Planta/virologia , Reação em Cadeia da Polimerase , Reprodutibilidade dos Testes , Risco , Sensibilidade e Especificidade
15.
Sci Rep ; 10(1): 4553, 2020 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-32165708

RESUMO

Breast cancer (BC) is a common cancer in women worldwide. Despite advances in treatment, up to 30% of women eventually relapse and die of metastatic breast cancer. Liquid biopsy analysis of circulating cell-free DNA fragments in the patients' blood can monitor clonality and evolving mutations as a surrogate for tumour biopsy. Next generation sequencing platforms and digital droplet PCR can be used to profile circulating tumour DNA from liquid biopsies; however, they are expensive and time consuming for clinical use. Here, we report a novel strategy with proof-of-concept data that supports the usage of loop-mediated isothermal amplification (LAMP) to detect PIK3CA c.3140 A > G (H1047R), a prevalent BC missense mutation that is attributed to BC tumour growth. Allele-specific primers were designed and optimized to detect the p.H1047R variant following the USS-sbLAMP method. The assay was developed with synthetic DNA templates and validated with DNA from two breast cancer cell-lines and two patient tumour tissue samples through a qPCR instrument and finally piloted on an ISFET enabled microchip. This work sets a foundation for BC mutational profiling on a Lab-on-Chip device, to help the early detection of patient relapse and to monitor efficacy of systemic therapies for personalised cancer patient management.


Assuntos
Neoplasias da Mama/diagnóstico , Classe I de Fosfatidilinositol 3-Quinases/genética , Técnicas de Diagnóstico Molecular/instrumentação , Mutação de Sentido Incorreto , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Primers do DNA/genética , Detecção Precoce de Câncer , Feminino , Humanos , Dispositivos Lab-On-A-Chip , Biópsia Líquida , Células MCF-7 , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Projetos Piloto , Estudo de Prova de Conceito
16.
Analyst ; 145(6): 2405-2411, 2020 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-32053125

RESUMO

Owing to the frequent outbreak of dengue fever worldwide, a highly sensitive but in situ simple process diagnostic device is required to detect the dengue virus. However, the current immune affinity-based methods have sensitivity issues and nucleic acid-based diagnostic devices have not been suitable for field diagnosis due to the complexity in sample preparation. Here, a simple and fast nucleic acid-based diagnostic tool to directly detect dengue viruses in whole blood is demonstrated using a microbead-assisted direct sample preparation buffer (MB-buffer) and isothermal amplification (loop-mediated isothermal amplification, LAMP). To maximize the performance of the sample preparation process in the microfluidic chip platform, the chemical composition of the sample preparation buffer is simplified and combined with physical tools (heating and bead beating). The entire serial processes consisted of only (1) sample (whole blood) loading, (2) stirring for 90 s, (3) heating at 70 °C for 10 min, and (4) LAMP amplification in the simply designed microfluidic chip cartridge. A single syringe was utilized for sample loading and microfluidic solution transfer. Consequently, dengue viruses were qualitatively detected and discriminated with high sensitivity (LOD: 102 PFU per 200 µL of whole blood) in less than 1 hour without the use of any sophisticated system.


Assuntos
Vírus da Dengue/genética , Dengue/diagnóstico , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas de Diagnóstico Molecular/instrumentação , Técnicas de Amplificação de Ácido Nucleico/instrumentação , RNA Viral/genética , Dengue/sangue , Vírus da Dengue/isolamento & purificação , Desenho de Equipamento , Feminino , Humanos , Limite de Detecção , Masculino , Técnicas Analíticas Microfluídicas/métodos , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , RNA Viral/análise
17.
PLoS One ; 15(1): e0227476, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31935232

RESUMO

The pinewood nematode (PWN), Bursaphelenchus xylophilus, is a causative agent of pine wilt disease (PWD). To date, although several molecular diagnostic methods have been developed, rapid on-site diagnostic tools for detecting PWN in pinewood are limited. In this study, a point of care diagnostic (POCD) method for detecting PWN in pinewood using recombinase polymerase amplification (RPA) assay was developed. This method comprises quick gDNA extraction buffer (DAP buffer) for the direct extraction of gDNA of PWN from pinewood and a battery-mounted portable optical isothermal device (POID) for the detection of PWD in the field. The RPA assay can distinguish between the PWN and its conspecies which exist in pinewood and can complete diagnostic procedures within 25 min in the field. Moreover, the RPA assay can detect PWN in old wood samples in both natural and stored conditions. The POCD-RPA assay to detect PWN will be useful for epidemiological investigations in the field as well as for quarantine processes in the wood trade.


Assuntos
Técnicas de Amplificação de Ácido Nucleico/métodos , Pinus/parasitologia , Doenças das Plantas/parasitologia , Tylenchida/genética , Animais , Sequência de Bases , DNA de Helmintos/metabolismo , Genoma Helmíntico , Limite de Detecção , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Sistemas Automatizados de Assistência Junto ao Leito , Recombinases/metabolismo , Alinhamento de Sequência , Tylenchida/isolamento & purificação
18.
J Agric Food Chem ; 68(1): 369-375, 2020 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-31829586

RESUMO

A sensitive fluorescent DNA hydrogel aptasensor based on the self-assembly of rolling circle amplification (RCA) products was developed for ochratoxin A (OTA) detection in beer. A competitive binding mode of aptamer, complementary sequence, and target was integrated into the DNA hydrogel for OTA detection. The OTA aptamer first combined with the primer to form the hybridized product. Then, in the presence of OTA, the aptamer combined with OTA, which released the primer. The released primer hybridized with the padlock probe to form a circular template, and the RCA reaction was initiated by adding ligase, polymerase, and dNTPs. The fluorescent DNA hydrogel was obtained by adding Cy3-dUTP together with dNTPs, and the fluorescence (FL) intensity of the DNA hydrogel was positively correlated with OTA concentration. Under the optimal experimental conditions, the linear range of the relationship varied from 0.05 ng/mL to 100 ng/mL with a detection limit for OTA of 0.01 ng/mL. The fluorescent DNA hydrogel aptasensor showed good specificity and stability in beer samples. Therefore, the fabricated DNA hydrogel aptasensor shows considerable potential applications in detecting OTA for food safety.


Assuntos
Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais/métodos , DNA/química , Hidrogéis/química , Técnicas de Amplificação de Ácido Nucleico/métodos , Ocratoxinas/análise , Aptâmeros de Nucleotídeos/genética , Cerveja/análise , Técnicas Biossensoriais/instrumentação , DNA/genética , Fluorescência , Contaminação de Alimentos/análise , Limite de Detecção , Técnicas de Amplificação de Ácido Nucleico/instrumentação
19.
Artigo em Inglês | MEDLINE | ID: mdl-31812649

RESUMO

Listeria spp. are a group of gram-positive bacteria consisting of 20 species. Among them, Listeria monocytogenes is one of the major species that infects humans since it contaminates raw fruits, vegetables, and many others food products. The conventional methods for the detection of Listeria spp. and L. monocytogenes are time-consuming, taking 5-7 days. Herein, a duplex lateral flow dipstick (DLFD) test combined with loop-mediated isothermal amplification (LAMP) was developed for the identification of Listeria spp. and L. monocytogenes within approximately 45 min with the optimized LAMP reaction times at 63 °C. Under the optimized conditions, the method detection limits (MDL) with reference to genomic DNA and pure culture were 900 femtograms (fg) and 20 cfu/mL, respectively. The LAMP-DLFD showed no cross-reactivity with eighteen - other pathogenic bacteria such as Salmonella spp., Staphylococcus aureus, Escherichia coli, Campylobacter coli, C. jejuni, Enterococcus faecalis, Vibrio cholerae, V. parahaemolyticus, Pseudomonas aeruginosa, Shigella dysenteriae, S. flexneri, Bacillus cereus, Lactobacillus acidophilus, L. casei and Pediococcus pentosaceus. Among 100 samples of food products, LAMP-DLFD demonstrated 100% accuracy when compared to other standard detection methods, such as ISO11290-1, enzyme-linked fluorescent assay (ELFA) technology (VIDAS) and PCR. In conclusion, LAMP-DLFD proved to be highly specific and sensitive assays for screening detection of Listeria spp. and L. monocytogenes.


Assuntos
Listeria monocytogenes/genética , Produtos da Carne/microbiologia , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Técnicas de Amplificação de Ácido Nucleico/métodos , DNA Bacteriano/genética , Desenho de Equipamento , Genes Bacterianos/genética , Limite de Detecção , Listeria/genética , Listeria/isolamento & purificação , Listeria/patogenicidade , Listeria monocytogenes/isolamento & purificação , Listeria monocytogenes/patogenicidade
20.
Parasitology ; 147(4): 383-392, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31840627

RESUMO

Pathogenic helminth infections are responsible for severe health problems and economic losses worldwide. Timely and accurate diagnosis of helminth infections is critical for adopting suitable strategies for pathogen control. Here, we review recent advances in nucleic acid-based diagnostic methods, including polymerase chain reaction, quantitative qPCR, loop-mediated isothermal amplification and recombinase polymerase amplification, and discuss their advantages and disadvantages for diagnosing helminth infections. In addition, we highlight recent advances in biosensors for the detection of nucleic acid biomarkers that can potentially be used for the diagnosis of helminth infection.


Assuntos
Biomarcadores/análise , Técnicas Biossensoriais/métodos , Helmintíase/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Ácidos Nucleicos/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Técnicas Biossensoriais/instrumentação , Técnicas de Diagnóstico Molecular/instrumentação , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Reação em Cadeia da Polimerase/instrumentação
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