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1.
Anal Chim Acta ; 1177: 338758, 2021 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-34482896

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the unprecedented global pandemic of coronavirus disease-2019 (COVID-19). Efforts are needed to develop rapid and accurate diagnostic tools for extensive testing, allowing for effective containment of the infection via timely identification and isolation of SARS-CoV-2 carriers. Current gold standard nucleic acid tests require many separate steps that need trained personnel to operate specialist instrumentation in laboratory environments, hampering turnaround time and test accessibility, especially in low-resource settings. We devised an integrated on-chip platform coupling RNA extraction based on immiscible filtration assisted by surface tension (IFAST), with RNA amplification and detection via colorimetric reverse-transcription loop mediated isothermal amplification (RT-LAMP), using two sets of primers targeting open reading frame 1a (ORF1a) and nucleoprotein (N) genes of SARS-CoV-2. Results were identified visually, with a colour change from pink to yellow indicating positive amplification, and further confirmed by DNA gel electrophoresis. The specificity of the assay was tested against HCoV-OC43 and H1N1 RNAs. The assay based on use of gene N primers was 100% specific to SARS-CoV-2 with no cross-reactivity to HCoV-OC43 nor H1N1. Proof-of-concept studies on water and artificial sputum containing genomic SARS-CoV-2 RNA showed our IFAST RT-LAMP device to be capable of extracting and detecting 470 SARS-CoV-2 copies mL-1 within 1 h (from sample-in to answer-out). IFAST RT-LAMP is a simple-to-use, integrated, rapid and accurate COVID-19 diagnostic platform, which could provide an attractive means for extensive screening of SARS-CoV-2 infections at point-of-care, especially in resource-constrained settings.


Assuntos
COVID-19 , Dispositivos Lab-On-A-Chip , RNA Viral , COVID-19/diagnóstico , Humanos , Vírus da Influenza A Subtipo H1N1 , Técnicas de Diagnóstico Molecular , Técnicas de Amplificação de Ácido Nucleico , RNA Viral/isolamento & purificação , SARS-CoV-2 , Sensibilidade e Especificidade
2.
J Nanobiotechnology ; 19(1): 273, 2021 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-34496881

RESUMO

The control of contagious or refractory diseases requires early, rapid diagnostic assays that are simple, fast, and easy-to-use. Here, easy-to-implement CRISPR/Cas12a-based diagnostic platform through Raman transducer generated by Raman enhancement effect, term as SERS-CRISPR (S-CRISPR), are described. The S-CRISPR uses high-activity noble metallic nanoscopic materials to increase the sensitivity in the detection of nucleic acids, without amplification. This amplification-free platform, which can be performed within 30-40 min of incubation time, is then used for detection of SARS-CoV-2 derived nucleic acids in RNA extracts obtained from nasopharyngeal swab specimens (n = 112). Compared with the quantitative reverse transcription polymerase chain reaction (RT-qPCR), the sensitivity and specificity of S-CRISPR reaches 87.50% and 100%, respectively. In general, the S-CRISPR can rapidly identify the RNA of SARS-CoV-2 RNA without amplification and is a potential strategy for nucleic acid point of care test (POCT).


Assuntos
Sistemas CRISPR-Cas/genética , Técnicas de Amplificação de Ácido Nucleico , SARS-CoV-2/genética , SARS-CoV-2/isolamento & purificação , Análise Espectral Raman , COVID-19/diagnóstico , COVID-19/virologia , Regulação Fúngica da Expressão Gênica , Genes Virais , Humanos , RNA Viral/análise , Sensibilidade e Especificidade
3.
Zhongguo Xue Xi Chong Bing Fang Zhi Za Zhi ; 33(4): 334-338, 2021 Aug 20.
Artigo em Chinês | MEDLINE | ID: mdl-34505438

RESUMO

OBJECTIVE: To develop a rapid test for detection of Schistosoma japonicum specific gene fragments based on the recombinase-aided isothermal amplification assay (RAA) and nucleic acid dipstick test. METHODS: The S. japonicum SjG28 gene fragment was selected as the target gene fragment, and the primers and fluorescent probe were designed and synthesized. Then, a S. japonicum nucleic acid dipstick test was established. The sensitivity of this dipstick test was evaluated by detecting different copies of recombinant plasmids containing the S. japonicum SjG28 gene fragment and different concentrations of genomic DNA from adult worms of S. japonicum, and the specificity of the dipstick test was evaluated by detecting the genomic DNA from Clonorchis sinensis, S. mansoni, Ancylostoma duodenale, S. haematobium, Babesia and Paragonimus westermani. RESULTS: The S. japonicum nucleic acid dipstick test based on the S. japonicum SjG28 gene fragment showed the minimum detectable limit of 10 copies/µL of the recombinant plasmid containing the S. japonicum SjG28 gene fragment and the minimum detectable limit of 1 pg/µL of S. japonicum genomic DNA, and the dipstick assay tested negative for the genomic DNA from C. sinensis, S. mansoni, A. duodenale, S. haematobium, Babesia and P. westermani. CONCLUSIONS: A rapid, simple, and visualized assay is established for detection of S. japonicum specific gene fragments based on RAA and nucleic acid dipstick test.


Assuntos
Ácidos Nucleicos , Schistosoma japonicum , Animais , Técnicas de Amplificação de Ácido Nucleico , Recombinases , Schistosoma japonicum/genética , Sensibilidade e Especificidade
4.
Zhonghua Gan Zang Bing Za Zhi ; 29(8): 803-806, 2021 Aug 20.
Artigo em Chinês | MEDLINE | ID: mdl-34517465

RESUMO

Nucleic acid testing is the gold standard for diagnosing HCV infection, and it is of great significance to guide the treatment of HCV patients and change the follow-up strategy. In recent years, its detection technology has become increasingly mature. This article summarizes the necessity and current status of nucleic acid testing for hepatitis C through the analysis of domestic and foreign literature, in order to provide reference for the formulation of relevant policies to eliminate hepatitis C in China.


Assuntos
Hepatite C , RNA Viral , China , Hepacivirus/genética , Hepatite C/diagnóstico , Humanos , Técnicas de Amplificação de Ácido Nucleico , RNA Viral/genética
5.
Talanta ; 235: 122728, 2021 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-34517596

RESUMO

With the emergence of microRNA (miRNA) as a key player in early clinical disease diagnosis, development of rapidly sensitive and quantitative miRNA detection methods are imperative. Herein, a label-free SERS assay coupled with duplex-specific nuclease (DSN) signal amplification strategy was proposed for facilely ultrasensitive and quantitative analysis of miRNA-21. Firstly, magnetic beads assembled with excessive capture DNA were utilized to hybridize the target miRNA-21. These DNA-RNA heteroduplexes were cleaved by DSN to generate small nucleotide fragments into the supernatant and the miRNA-21 released and rehybridized another DNA, going to the next DSN cycle. Consequently, numerous of small nucleotide fragments of capture DNA were released from magnetic beads and the miRNA-21 signal was transferred and amplified by the SERS signals of total phosphate backbones which are abundant in nucleotide. Furthermore, iodide-modified Ag nanoparticles (AgINPs) was employed to generate a strong and reproducible SERS signal. The proposed method displayed excellent performance for miRNA-21 detection with the linear range from 0.33 fM to 3.3 pM, and a lower detection limit of 42 aM. Moreover, this strategy exhibited effectively base discrimination capability and was successfully applied for monitoring the expression levels of miRNA-21 in different cancer cell lines and human serum.


Assuntos
Técnicas Biossensoriais , Nanopartículas Metálicas , MicroRNAs , Ouro , Humanos , Iodetos , Limite de Detecção , MicroRNAs/genética , Técnicas de Amplificação de Ácido Nucleico , Prata
6.
Talanta ; 235: 122735, 2021 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-34517602

RESUMO

Accumulative evidences have indicated that abnormal expression of microRNAs (miRNAs) is closely associated with many health disorders, making them be regarded as potentialbiomarkers for early clinical diagnosis. Therefore, it is extremely necessary to develop a highly sensitive, specific and reliable approach for miRNA analysis. Catalytic hairpin assembly (CHA) signal amplification is an enzyme-free toehold-mediated strand displacement method, exhibiting significant potential in improving the sensitivity of miRNA detection strategies. In this review, we first describe the potential of miRNAs as disease biomarkers and therapeutics, and summarize the latest advances in CHA signal amplification-based sensing strategies for miRNA monitoring. We describe the characteristics and mechanism of CHA signal amplification and classify the CHA-based miRNA sensing strategies into several categories based on the "signal conversion substance", including fluorophores, enzymes, nanomaterials, and nucleotide sequences. Sensing performance, limit of detection, merits and disadvantages of these miRNA sensing strategies are discussed. Moreover, the current challenges and prospects are also presented.


Assuntos
Técnicas Biossensoriais , MicroRNAs , Nanoestruturas , Catálise , Corantes Fluorescentes , Limite de Detecção , MicroRNAs/genética , Técnicas de Amplificação de Ácido Nucleico
7.
Talanta ; 235: 122802, 2021 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-34517660

RESUMO

MicroRNAs (miRNAs) are physiological status-related molecules which can be used as biomarkers for diseases, such as cancers. The point-of-care testing (POCT) of miRNAs has great application potential in early diagnosis and process monitoring of diseases. In this paper, a fast and dual signal outputs detection for microRNA-21 (miRNA-21) was established by using both personal glucose meter (PGM) and fluorescence spectrometer. In such an assay protocol, a dual-functional hairpin structure was rationally designed to recognize miRNA-21 and serve as the carrier of the reporter adenosine monophosphate (AMP). The hairpin structure can be specifically degraded by exonuclease T (Exo T) after hybridization with the target miRNA-21, releasing a large amount of AMP as the reporter. Then a smart signal conversion machinery composed of four enzymes and the corresponding substrates was employed to produce dual output signals through enzymatic cascade reactions. The machinery includes two parts: an adenosine triphosphate (ATP) generation system and a glucose consumption/NADPH production system. The produced AMP in the former step triggers the production of ATP, and subsequently the consumption of glucose and the production of NADPH. The changes of both glucose and NADPH are proportional to the concentration of miRNA-21, and can be determined by PGM and fluorescence spectrometer, respectively. Besides, the build-in substrate-recycling mechanism achieves signal amplification of the cascade enzymatic reactions. Under the optimal experimental conditions, the PGM signal is linearly correlated with the concentration of miRNA-21 in the range from 5 to 150 nM, with the limit of detection (LOD) of 3.65 nM. The LOD of fluorescence detection mode is even lowered to 0.03 nM. The miRNA-21-spiked serum samples, as well as the actual serum samples from cancer patients, have been successfully detected by this detection strategy. Thus the established assay provides a POCT solution for cancer diagnosis and prognosis.


Assuntos
Técnicas Biossensoriais , MicroRNAs , Humanos , Limite de Detecção , MicroRNAs/genética , Técnicas de Amplificação de Ácido Nucleico , Hibridização de Ácido Nucleico
8.
Talanta ; 235: 122810, 2021 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-34517667

RESUMO

MicroRNAs (miRNAs) are currently recognized as novel biomarkers for cancer early diagnosis, therapy selection, and progression monitoring. Herein, we developed an ultrasensitive and label-free homogeneous colorimetric strategy for miRNA detection based on engineering entropy-driven amplification (EDA) coupled with nicking enzyme-assisted AuNP aggregation. In our design, the target miRNA could specifically trigger the EDA recycling process. One of the EDA products could open the hairpin probe and form a dual strand containing a nicking endonuclease (Nb.BbvCl) cleavage region. After adding nicking endonuclease in the sensing solution, the product DNA fragments could act as two linkers, inducing the aggregation of ssDNA-modified AuNPs. Simultaneously, the liberating complementary strands continued to cyclic hybridization with the hairpin probe. This multiple signal amplification colorimetric strategy showed a wide linear range from 10 fM to 100 pM with a much lower detection limit of 3.13 fM for miRNA let-7a, which also performed well in a complex sample matrix. Most importantly, the naked eye could clearly distinguish the 10 fM color change caused by let-7a to be measured. Moreover, this approach could easily extend to multiple miRNAs with target-specific sequence substitutions. Therefore, this ultrasensitive visual strategy for miRNA demonstrated attractive potentials for promising applications in clinical diagnosis.


Assuntos
Técnicas Biossensoriais , Nanopartículas Metálicas , MicroRNAs , Entropia , Ouro , Limite de Detecção , MicroRNAs/genética , Técnicas de Amplificação de Ácido Nucleico
9.
Talanta ; 235: 122814, 2021 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-34517670

RESUMO

Simple and sensitive detection of telomerase activity is of vital importance for both early diagnosis and therapy of malignant tumors. Inspired by DNA-biobarcode amplification reported by Chad A. Mirkin, we developed a facile DNA-biobarcode-like SERS-based copper-mediated signal amplification strategy for sensitive detection of telomerase activity. In this strategy, a duplex DNA constructed by hybridization of a copper oxide nanoparticle (CuO NP)-labeled reporting sequence (RS) with the telomerase primer sequence (TS) is ingeniously designed, and anchored on the magnetic bead (MB) to build the CuO NPs-encoded magnetic bead (MB-CuO NPs) detection probe. Upon selective sensing of telomerase, telomerase elongation reaction and structure change of TS products make the CuO NP-RS displace and separate from MB. The separated CuO NPs are dissolved into a mass of Cu2+, which prompt monodisperse dopamine-functionalized AgNPs (D-AgNPs) signal probe into aggregation, resulting in color changes and significantly enhancing of SERS signal. The SERS signal increases with the increase of Cu2+, which is directly proportional to the telomerase. Benefiting from the transformation of CuO NP to Cu2+ with a high amplification effect, this strategy could realize the telomerase activity measurement down to 3 HeLa cells and a dynamic range of 10-10000 cells. It shows a significant improvement of sensitivity without need for other enzymes and elaborate design, which escapes from the complicated manipulations and design in polymerase chain reaction (PCR) and DNA amplification techniques. Moreover, with this strategy, telomerase activities of different cell lines and telomerase inhibitors screening were successfully performed. Significantly, it can also be utilized for visual detection of telomerase, which validates the potential on-site application and its application as point-of-care testing (POCT) for efficient monitoring. Given the high-performance for telomerase analysis, the strategy has a promising application in biological detection and clinical diagnosis, as well as point-of-care tests.


Assuntos
Técnicas Biossensoriais , Telomerase , Cobre , DNA , Células HeLa , Humanos , Técnicas de Amplificação de Ácido Nucleico , Telomerase/genética , Telomerase/metabolismo
10.
Anal Chim Acta ; 1180: 338846, 2021 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-34538333

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a zoonotic RNA virus characterized by high transmission rates and pathogenicity worldwide. Continued control of the COVID-19 pandemic requires the diversification of rapid, easy to use, sensitive, and portable methods for SARS-CoV-2 sample preparation and analysis. Here, we propose a method for SARS-CoV-2 viral enrichment and enzymatic extraction of RNA from clinically relevant matrices in under 10 min. This technique utilizes affinity-capture hydrogel particles to concentrate SARS-CoV-2 from solution, and leverages existing PDQeX technology for RNA isolation. Characterization of our method is accomplished with reverse transcription real-time polymerase chain reaction (RT-PCR) for relative, comparative RNA detection. In a double-blind study analyzing viral transport media (VTM) obtained from clinical nasopharyngeal swabs, our sample preparation method demonstrated both comparable results to a routinely used commercial extraction kit and 100% concordance with laboratory diagnoses. Compatibility of eluates with alternative forms of analysis was confirmed using microfluidic RT-PCR (µRT-PCR), recombinase polymerase amplification (RPA), and loop-mediated isothermal amplification (LAMP). The alternative methods explored here conveyed successful amplification from all RNA eluates originating from positive clinical samples. Finally, this method demonstrated high performance within a saliva matrix across a broad range of viral titers and dilutions up to 90% saliva matrix, and sets the stage for miniaturization to the microscale.


Assuntos
COVID-19 , Pandemias , Teste para COVID-19 , Humanos , Técnicas de Diagnóstico Molecular , Técnicas de Amplificação de Ácido Nucleico , RNA Viral/genética , SARS-CoV-2
11.
Analyst ; 146(17): 5271-5279, 2021 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-34355716

RESUMO

The ability to visually detect low numbers of Salmonella in food samples is highly valuable but remains a challenge. Here we present a novel platform for ultrasensitive and visual detection of Salmonella Typhimurium by integrating the bio-barcode immunoassay (BCA), recombinase polymerase amplification (RPA), and CRISPR-Cas12a cleavage in a single reaction system (termed as BCA-RPA-Cas12a). In the system, the target bacteria were separated by immunomagnetic nanoparticles and labeled with numerous barcode AuNPs, which carry abundant bio-barcode DNA molecules to amplify the signal. Afterwards, the bio-barcode DNA molecules were amplified by RPA and subsequently triggered the cleavage activity of Cas12a to generate the fluorescence signal. Due to this triplex signal amplification, the BCA-RPA-Cas12a system can selectively detect Salmonella Typhimurium at the single-digit level with the naked eye under blue light within 60 min. Meanwhile, this novel platform was successfully applied to detect Salmonella Typhimurium in spiked milk samples with a similar sensitivity and satisfactory recovery, indicating its potential application in real samples. Furthermore, in virtue of the versatility of the antibody in the stage of BCA, the BCA-RPA-Cas12a system can be extended to further application in other bacteria detection and food safety monitoring.


Assuntos
Nanopartículas Metálicas , Recombinases , Sistemas CRISPR-Cas , Ouro , Imunoensaio , Técnicas de Amplificação de Ácido Nucleico , Recombinases/genética , Salmonella typhimurium/genética
12.
ACS Sens ; 6(8): 2815-2837, 2021 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-34392681

RESUMO

This review covers emerging biosensors for SARS-CoV-2 detection together with a review of the biochemical and clinical assays that are in use in hospitals and clinical laboratories. We discuss the gap in bridging the current practice of testing laboratories with nucleic acid amplification methods, and the robustness of assays the laboratories seek, and what emerging SARS-CoV-2 sensors have currently addressed in the literature. Together with the established nucleic acid and biochemical tests, we review emerging technology and antibody tests to determine the effectiveness of vaccines on individuals.


Assuntos
COVID-19 , SARS-CoV-2 , Teste para COVID-19 , Humanos , Laboratórios , Técnicas de Amplificação de Ácido Nucleico
13.
Cells ; 10(8)2021 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-34440699

RESUMO

In 1998, when the PCR technique was already popular, a Japanese company called Eiken Chemical Co., Ltd. designed a method known as the loop-mediated isothermal amplification of DNA (LAMP). The method can produce up to 109 copies of the amplified DNA within less than an hour. It is also highly specific due to the use of two to three pairs of primers (internal, external, and loop), which recognise up to eight specific locations on the DNA or RNA targets. Furthermore, the Bst DNA polymerase most used in LAMP shows a high strand displacement activity, which eliminates the DNA denaturation stage. One of the most significant advantages of LAMP is that it can be conducted at a stable temperature, for instance, in a dry block heater or an incubator. The products of LAMP can be detected much faster than in standard techniques, sometimes only requiring analysis with the naked eye. The following overview highlights the usefulness of LAMP and its effectiveness in various fields; it also considers the superiority of LAMP over PCR and presents RT-LAMP as a rapid diagnostic tool for SARS-CoV-2.


Assuntos
COVID-19/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , SARS-CoV-2/isolamento & purificação , Humanos
14.
Talanta ; 234: 122637, 2021 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-34364446

RESUMO

To pursue the sensitive and efficient detection of informative biomolecules for bioanalysis and disease diagnosis, a series of signal amplification techniques have been put forward. Among them, hybridization chain reaction (HCR) is an isothermal and enzyme-free process where the cascade reaction of hybridization events is initiated by a target analyte, yielding a long nicked dsDNA molecule analogous to alternating copolymers. Compared with conventional polymerase chain reaction (PCR) that can proceed only with the aid of polymerases and complicated thermal cycling, HCR has attracted increasing attention because it can occur under mild conditions without using enzymes. As a powerful signal amplification tool, HCR has been employed to construct various simple, sensitive and economic biosensors for detecting nucleic acids, small molecules, cells, and proteins. Moreover, HCR has also been applied to assemble complex nanostructures, some of which even act as the carriers to execute the targeted delivery of anticancer drugs. Recently, HCR has engendered tremendous progress in RNA imaging applications, which can not only achieve endogenous RNA imaging in living cells or even living animals but also implement imaging-guided photodynamic therapy, paving a promising path to promote the development of theranostics. In this review, we begin with the fundamentals of HCR and then focus on summarizing the recent advances in HCR-based biosensors for biosensing and RNA imaging strategies. Further, the challenges and future perspective of HCR-based signal amplification in biosensing and theranostic application are discussed.


Assuntos
Técnicas Biossensoriais , Nanoestruturas , Ácidos Nucleicos , DNA/genética , Técnicas de Amplificação de Ácido Nucleico , Hibridização de Ácido Nucleico
15.
Sci Rep ; 11(1): 16193, 2021 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-34376716

RESUMO

We have optimised a reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay for the detection of SARS-CoV-2 from extracted RNA for clinical application. We improved the stability and reliability of the RT-LAMP assay by the addition of a temperature-dependent switch oligonucleotide to reduce self- or off-target amplification. We then developed freeze-dried master mix for single step RT-LAMP reaction, simplifying the operation for end users and improving long-term storage and transportation. The assay can detect as low as 13 copies of SARS-CoV2 RNA per reaction (25-µL). Cross reactivity with other human coronaviruses was not observed. We have applied the new RT-LAMP assay for testing clinical extracted RNA samples extracted from swabs of 72 patients in the UK and 126 samples from Greece and demonstrated the overall sensitivity of 90.2% (95% CI 83.8-94.7%) and specificity of 92.4% (95% CI 83.2-97.5%). Among 115 positive samples which Ct values were less than 34, the RT-LAMP assay was able to detect 110 of them with 95.6% sensitivity. The specificity was 100% when RNA elution used RNase-free water. The outcome of RT-LAMP can be reported by both colorimetric detection and quantifiable fluorescent reading. Objective measures with a digitized reading data flow would allow for the sharing of results for local or national surveillance.


Assuntos
Teste de Ácido Nucleico para COVID-19/métodos , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Teste de Ácido Nucleico para COVID-19/normas , Humanos , Técnicas de Diagnóstico Molecular/normas , Técnicas de Amplificação de Ácido Nucleico/normas , Sensibilidade e Especificidade
16.
Anal Chem ; 93(33): 11617-11625, 2021 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-34375096

RESUMO

Single-cell microRNA (miRNA) analysis helps people understand the causes of diseases and formulate new disease treatment strategies. However, miRNA from a single cell is usually very rare and requires signal amplification for accurate quantification. Here, to amplify the signal, we constructed the cascaded DNA circuits consisting of catalytic hairpin assembly and hybrid chain reaction into the bead array platform, on which the uniformly distributed beads were adopted for miRNA quantification. After exponential signal amplification, a consistent linear correlation between the percentage of fluorescent beads and the copy number of miRNA was detected. The proposed bead array can achieve ultrahigh sensitivity as low as 60 copies of miR-155 and high specificity for distinguishing single nucleotide differences. This method has been successfully applied to the quantitative detection of miRNA in a single cancer cell. The high sensitivity, programmability, and simple workflow of the bead array chip will give a huge advantage in basic and clinical research.


Assuntos
MicroRNAs , Catálise , DNA/genética , Humanos , MicroRNAs/genética , Técnicas de Amplificação de Ácido Nucleico , Análise de Célula Única
17.
Anal Chem ; 93(35): 11956-11964, 2021 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-34424659

RESUMO

Coronavirus diseases such as the coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), pose serious threats. Portable and accurate nucleic acid detection is still an urgent need to achieve on-site virus screening and timely infection control. Herein, we have developed an on-site, semiautomatic detection system, aiming at simultaneously overcoming the shortcomings suffered by various commercially available assays, such as low accuracy, poor portability, instrument dependency, and labor intensity. Ultrasensitive isothermal amplification [i.e., reverse transcription loop-mediated isothermal amplification (RT-LAMP)] was applied to generate intensified SARS-CoV-2 RNA signals, which were then transduced to portable commercial pregnancy test strips (PTSs) via ultraspecific human chorionic gonadotropin (hCG)-conjugated toehold-mediated strand exchange (TMSE) probes (hCG-P). The entire detection was integrated into a four-channel, palm-size microfluidic device, named the microfluidic point-of-care (POC) diagnosis system based on the PTS (MPSP) detection system. It provides rapid, cost-effective, and sensitive detection, of which the lowest concentration of detection was 0.5 copy/µL of SARS-CoV-2 RNA, regardless of the presence of other similar viruses, even highly similar severe acute respiratory syndrome coronavirus (SARS-CoV). The successful detection of the authentic samples from different resources evaluated the practical application. The commercial PTS provides a colorimetric visible signal, which is instrument- and optimization-free. Therefore, this MPSP system can be immediately used for SARS-CoV-2 emergency detection, and it is worthy of further optimization to achieve full automation and detection for other infectious diseases.


Assuntos
COVID-19 , Testes de Gravidez , Feminino , Humanos , Dispositivos Lab-On-A-Chip , Técnicas de Diagnóstico Molecular , Técnicas de Amplificação de Ácido Nucleico , Sistemas Automatizados de Assistência Junto ao Leito , Gravidez , RNA Viral/genética , SARS-CoV-2 , Sensibilidade e Especificidade
18.
Virol J ; 18(1): 178, 2021 08 30.
Artigo em Inglês | MEDLINE | ID: mdl-34461941

RESUMO

BACKGROUND: Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19 pandemic, has infected more than 179 million people worldwide. Testing of infected individuals is crucial for identification and isolation, thereby preventing further spread of the disease. Presently, Taqman™ Reverse Transcription Real Time PCR is considered gold standard, and is the most common technique used for molecular testing of COVID-19, though it requires sophisticated equipments, expertise and is also relatively expensive. OBJECTIVE: Development and optimization of an alternate molecular testing method for the diagnosis of COVID-19, through a two step Reverse Transcription Loop-mediated isothermal AMPlification (RT-LAMP). RESULTS: Primers for LAMP were carefully designed for discrimination from other closely related human pathogenic coronaviruses. Care was also taken that primer binding sites are present in conserved regions of SARS-CoV2. Our analysis shows that the primer binding sites are well conserved in all the variants of concern (VOC) and variants of interest (VOI), notified by World Health Organization (WHO). These lineages include B.1.1.7, B.1.351, P.1, B.1.617.2, B.1.427/B.1.429, P.2, B.1.525, P.3, B.1.526 and B.1.617.1. Various DNA polymerases with strand displacement activity were evaluated and conditions were optimized for LAMP amplification and visualization. Different LAMP primer sets were also evaluated using synthetic templates as well as patient samples. CONCLUSION: In a double blind study, the RT-LAMP assay was validated on more than 150 patient samples at two different sites. The RT-LAMP assay appeared to be 89.2% accurate when compared to the Taqman™ rt-RT-PCR assay.


Assuntos
Teste para COVID-19/métodos , COVID-19/virologia , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , SARS-CoV-2/genética , COVID-19/diagnóstico , Humanos , Transcrição Reversa , SARS-CoV-2/isolamento & purificação , SARS-CoV-2/fisiologia , Sensibilidade e Especificidade
19.
Nat Commun ; 12(1): 5089, 2021 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-34429424

RESUMO

The current nucleic acid signal amplification methods for SARS-CoV-2 RNA detection heavily rely on the functions of biological enzymes which imposes stringent transportation and storage conditions, high cost and global supply shortages. Here, a non-enzymatic whole genome detection method based on a simple isothermal signal amplification approach is developed for rapid detection of SARS-CoV-2 RNA and potentially any types of nucleic acids regardless of their size. The assay, termed non-enzymatic isothermal strand displacement and amplification (NISDA), is able to quantify 10 RNA copies.µL-1. In 164 clinical oropharyngeal RNA samples, NISDA assay is 100 % specific, and it is 96.77% and 100% sensitive when setting up in the laboratory and hospital, respectively. The NISDA assay does not require RNA reverse-transcription step and is fast (<30 min), affordable, highly robust at room temperature (>1 month), isothermal (42 °C) and user-friendly, making it an excellent assay for broad-based testing.


Assuntos
Teste de Ácido Nucleico para COVID-19/métodos , COVID-19/diagnóstico , Técnicas de Amplificação de Ácido Nucleico/métodos , RNA Viral/isolamento & purificação , SARS-CoV-2/genética , SARS-CoV-2/isolamento & purificação , COVID-19/virologia , Teste para COVID-19 , Humanos , RNA Viral/genética , Recombinação Genética
20.
J Biomed Nanotechnol ; 17(7): 1364-1370, 2021 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-34446139

RESUMO

Researchers have conducted in-depth research on DNA methylation mechanism, which is related to various diseases such as deficiency of imprinted gene and occurrence of tumors. This study provides a novel rapid quantitative detection assay and real-time fluorescence recombinase-aided amplification assay (RAA) for DNA methylation. Firstly, specific sequence of methylation genes was chosen and primers and fluorogenic probe for RAA experiment were designed and synthesized. Lastly, these amplification products were proven by sequencing and analysis. Results showed that the amplification efficiency and template concentration of RAA had linear dependent (R² > 95%) when the concentration range was 4.64×108 copies/µL˜4.64×104 copies/µL. The test assay can also detect positive samples when the template concentration is below 4.64×104 copies/µL. Remarkably, the entire experiment process only takes 15-20 minutes, so it is beneficial for rapid bedside simple screening of some special DNA methylation sites, such as detection of resistance genes. In a word, this method has very great potential for diseases with DNA methylation in clinical settings, especially if methylation analysis needs to be done quickly and easily.


Assuntos
Metilação de DNA , Recombinases , Primers do DNA , Técnicas de Amplificação de Ácido Nucleico , Recombinases/genética , Recombinases/metabolismo , Sensibilidade e Especificidade
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