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1.
Biosensors (Basel) ; 11(2)2021 Jan 27.
Article in English | MEDLINE | ID: covidwho-2215575

ABSTRACT

Worldwide infection disease due to SARS-CoV-2 is tremendously affecting our daily lives. High-throughput detection methods for nucleic acids are emergently desired. Here, we show high-sensitivity and high-throughput metasurface fluorescence biosensors that are applicable for nucleic acid targets. The all-dielectric metasurface biosensors comprise silicon-on-insulator nanorod array and have prominent electromagnetic resonances enhancing fluorescence emission. For proof-of-concept experiment on the metasurface biosensors, we have conducted fluorescence detection of single-strand oligoDNAs, which model the partial sequences of SARS-CoV-2 RNA indicated by national infection institutes, and succeeded in the high-throughput detection at low concentrations on the order of 100 amol/mL without any amplification technique. As a direct detection method, the metasurface fluorescence biosensors exhibit high performance.


Subject(s)
Biosensing Techniques/methods , COVID-19 Nucleic Acid Testing/methods , COVID-19/diagnosis , SARS-CoV-2/genetics , High-Throughput Screening Assays/methods , Humans , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/methods , RNA, Viral/analysis , Sensitivity and Specificity
2.
Parasitol Res ; 121(7): 1867-1885, 2022 Jul.
Article in English | MEDLINE | ID: covidwho-2174153

ABSTRACT

Malaria control measures have been in use for years but have not completely curbed the spread of infection. Ultimately, global elimination is the goal. A major playmaker in the various approaches to reaching the goal is the issue of proper diagnosis. Various diagnostic techniques were adopted in different regions and geographical locations over the decades, and these have invariably produced diverse outcomes. In this review, we looked at the various approaches used in malaria diagnostics with a focus on methods favorably used during pre-elimination and elimination phases as well as in endemic regions. Microscopy, rapid diagnostic testing (RDT), loop-mediated isothermal amplification (LAMP), and polymerase chain reaction (PCR) are common methods applied depending on prevailing factors, each with its strengths and limitations. As the drive toward the elimination goal intensifies, the search for ideal, simple, fast, and reliable point-of-care diagnostic tools is needed more than ever before to be used in conjunction with a functional surveillance system supported by the ideal vaccine.


Subject(s)
Malaria, Falciparum , Malaria , Diagnostic Tests, Routine/methods , Goals , Humans , Malaria/diagnosis , Malaria/prevention & control , Malaria, Falciparum/epidemiology , Microscopy/methods , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/methods , Polymerase Chain Reaction/methods , Sensitivity and Specificity
4.
Biosensors (Basel) ; 12(11)2022 Nov 08.
Article in English | MEDLINE | ID: covidwho-2109935

ABSTRACT

Worldwide infection due to SARS-CoV-2 revealed that short-time and extremely high-sensitivity detection of nucleic acids is a crucial technique for human beings. Polymerase chain reactions have been mainly used for the SARS-CoV-2 detection over the years. However, an advancement in quantification of the detection and shortening runtime is important for present and future use. Here, we report a rapid detection scheme that is a combination of nucleic acid amplification and a highly efficient fluorescence biosensor, that is, a metasurface biosensor composed of a pair of an all-dielectric metasurface and a microfluidic transparent chip. In the present scheme, we show a series of proof-of-concept experimental results that the metasurface biosensors detected amplicons originating from attomolar SARS-CoV-2 nucleic acids and that the amplification was implemented within 1 h. Furthermore, this detection capability substantially satisfies an official requirement of 100 RNA copies/140 µL, which is a criterion for the reliable infection tests.


Subject(s)
Biosensing Techniques , COVID-19 , Nucleic Acids , Humans , SARS-CoV-2 , COVID-19/diagnosis , Sensitivity and Specificity , Nucleic Acid Amplification Techniques/methods , Molecular Diagnostic Techniques/methods
5.
Nature ; 611(7936): 570-577, 2022 11.
Article in English | MEDLINE | ID: covidwho-2106425

ABSTRACT

Expanding our global testing capacity is critical to preventing and containing pandemics1-9. Accordingly, accessible and adaptable automated platforms that in decentralized settings perform nucleic acid amplification tests resource-efficiently are required10-14. Pooled testing can be extremely efficient if the pooling strategy is based on local viral prevalence15-20; however, it requires automation, small sample volume handling and feedback not available in current bulky, capital-intensive liquid handling technologies21-29. Here we use a swarm of millimetre-sized magnets as mobile robotic agents ('ferrobots') for precise and robust handling of magnetized sample droplets and high-fidelity delivery of flexible workflows based on nucleic acid amplification tests to overcome these limitations. Within a palm-sized printed circuit board-based programmable platform, we demonstrated the myriad of laboratory-equivalent operations involved in pooled testing. These operations were guided by an introduced square matrix pooled testing algorithm to identify the samples from infected patients, while maximizing the testing efficiency. We applied this automated technology for the loop-mediated isothermal amplification and detection of the SARS-CoV-2 virus in clinical samples, in which the test results completely matched those obtained off-chip. This technology is easily manufacturable and distributable, and its adoption for viral testing could lead to a 10-300-fold reduction in reagent costs (depending on the viral prevalence) and three orders of magnitude reduction in instrumentation cost. Therefore, it is a promising solution to expand our testing capacity for pandemic preparedness and to reimagine the automated clinical laboratory of the future.


Subject(s)
Automation , COVID-19 Testing , Magnets , Molecular Diagnostic Techniques , Nucleic Acid Amplification Techniques , Robotics , SARS-CoV-2 , Humans , COVID-19/diagnosis , COVID-19/virology , COVID-19 Testing/methods , Molecular Diagnostic Techniques/economics , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/economics , Nucleic Acid Amplification Techniques/methods , Pandemics/prevention & control , RNA, Viral/analysis , RNA, Viral/genetics , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification , Sensitivity and Specificity , Algorithms , Automation/economics , Automation/methods , Robotics/methods , Indicators and Reagents/economics
6.
Biotechniques ; 73(5): 247-255, 2022 Nov.
Article in English | MEDLINE | ID: covidwho-2098927

ABSTRACT

Loop-mediated isothermal amplification (LAMP) has proven a robust and reliable nucleic acid amplification method that is well suited for simplified and rapid molecular diagnostics. Various approaches have emerged for sequence-specific detection of LAMP products, but with limitations to their widespread utility or applicability for single-nucleotide polymorphism detection and multiplexing. Here we demonstrate the use of simple hybridization probes (as used for qPCR) that enable simple multiplexing and SARS-CoV-2 variant typing in reverse-transcription LAMP. This approach requires no modification to the LAMP primers and is amenable to the detection of single-nucleotide polymorphisms and small sequence changes, which is usually difficult in LAMP. By extending LAMP's ability to be utilized for multitarget and single-base change detection, we hope to increase its potential to enable more and better molecular diagnostic testing.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , SARS-CoV-2/genetics , Sensitivity and Specificity , Nucleic Acid Amplification Techniques/methods , Molecular Diagnostic Techniques/methods , RNA, Viral
7.
Methods ; 203: 431-446, 2022 07.
Article in English | MEDLINE | ID: covidwho-2096167

ABSTRACT

Infectious diseases are a global health problem affecting billions of people. Developing rapid and sensitive diagnostic tools is key for successful patient management and curbing disease spread. Currently available diagnostics are very specific and sensitive but time-consuming and require expensive laboratory settings and well-trained personnel; thus, they are not available in resource-limited areas, for the purposes of large-scale screenings and in case of outbreaks and epidemics. Developing new, rapid, and affordable point-of-care diagnostic assays is urgently needed. This review focuses on CRISPR-based technologies and their perspectives to become platforms for point-of-care nucleic acid detection methods and as deployable diagnostic platforms that could help to identify and curb outbreaks and emerging epidemics. We describe the mechanisms and function of different classes and types of CRISPR-Cas systems, including pros and cons for developing molecular diagnostic tests and applications of each type to detect a wide range of infectious agents. Many Cas proteins (Cas3, Cas9, Cas12, Cas13, Cas14 etc.) have been leveraged to create highly accurate and sensitive diagnostic tools combined with technologies of signal amplification and fluorescent, potentiometric, colorimetric, lateral flow assay detection and other. In particular, the most advanced platforms -- SHERLOCK/v2, DETECTR, CARMEN or CRISPR-Chip -- enable detection of attomolar amounts of pathogenic nucleic acids with specificity comparable to that of PCR but with minimal technical settings. Further developing CRISPR-based diagnostic tools promises to dramatically transform molecular diagnostics, making them easily affordable and accessible virtually anywhere in the world. The burden of socially significant diseases, frequent outbreaks, recent epidemics (MERS, SARS and the ongoing COVID-19) and outbreaks of zoonotic viruses (African Swine Fever Virus etc.) urgently need the developing and distribution of express-diagnostic tools. Recently devised CRISPR-technologies represent the unprecedented opportunity to reshape epidemiological surveillance and molecular diagnostics.


Subject(s)
African Swine Fever Virus , COVID-19 , Communicable Diseases , Animals , COVID-19/diagnosis , COVID-19/epidemiology , CRISPR-Cas Systems/genetics , Communicable Diseases/diagnosis , Communicable Diseases/genetics , Humans , Nucleic Acid Amplification Techniques/methods , Point-of-Care Systems , Swine
8.
Int J Mol Sci ; 23(21)2022 Oct 28.
Article in English | MEDLINE | ID: covidwho-2090209

ABSTRACT

Consistently emerging variants and the life-threatening consequences of SARS-CoV-2 have prompted worldwide concern about human health, necessitating rapid and accurate point-of-care diagnostics to limit the spread of COVID-19. Still, However, the availability of such diagnostics for COVID-19 remains a major rate-limiting factor in containing the outbreaks. Apart from the conventional reverse transcription polymerase chain reaction, loop-mediated isothermal amplification-based (LAMP) assays have emerged as rapid and efficient systems to detect COVID-19. The present study aims to develop RT-LAMP-based assay system for detecting multiple targets in N, ORF1ab, E, and S genes of the SARS-CoV-2 genome, where the end-products were quantified using spectrophotometry, paper-based lateral-flow devices, and electrochemical sensors. The spectrophotometric method shows a LOD of 10 agµL-1 for N, ORF1ab, E genes and 100 agµL-1 for S gene in SARS-CoV-2. The developed lateral-flow devices showed an LOD of 10 agµL-1 for all four gene targets in SARS-CoV-2. An electrochemical sensor developed for N-gene showed an LOD and E-strip sensitivity of log 1.79 ± 0.427 pgµL-1 and log 0.067 µA/pg µL-1/mm2, respectively. The developed assay systems were validated with the clinical samples from COVID-19 outbreaks in 2020 and 2021. This multigene target approach can effectively detect emerging COVID-19 variants using combination of various analytical techniques at testing facilities and in point-of-care settings.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , SARS-CoV-2/genetics , COVID-19/diagnosis , Sensitivity and Specificity , Nucleic Acid Amplification Techniques/methods , Molecular Diagnostic Techniques/methods , RNA, Viral/genetics
9.
ACS Appl Mater Interfaces ; 14(45): 50534-50542, 2022 Nov 16.
Article in English | MEDLINE | ID: covidwho-2087120

ABSTRACT

The CRISPR-Cas system was developed into a molecular diagnostic tool with high sensitivity, low cost, and high specificity in recent years. Colorimetric assays based on nanozymes offer an attractive point-of-care testing method for their low cost of use and user-friendly operation. Here, a MnO2 nanozyme-mediated CRISPR-Cas12a system was instituted to detect SARS-CoV-2. MnO2 nanorods linked to magnetic beads via a single-stranded DNA (ssDNA) linker used as an oxidase-like nanozyme inducing the color change of 3,3',5,5'-tetramethylbenzidine, which can be distinguished by the naked eye. The detection buffer color will change when the Cas12a is activated by SARS-CoV-2 and indiscriminately cleave the linker ssDNA. The detection limit was 10 copies per microliter and showed no cross-reaction with other coronaviruses. The nanozyme-mediated CRISPR-Cas12a system shows high selectivity and facile operation, with great potential for molecular diagnosis in point-of-care testing applications.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , SARS-CoV-2/genetics , CRISPR-Cas Systems/genetics , Manganese Compounds , COVID-19/diagnosis , Nucleic Acid Amplification Techniques/methods , Oxides , DNA, Single-Stranded
10.
Anal Chem ; 94(44): 15472-15480, 2022 Nov 08.
Article in English | MEDLINE | ID: covidwho-2087112

ABSTRACT

Integrated clustered regularly interspaced short palindromic repeat (CRISPR)-loop-mediated amplification (LAMP) technology is of great importance in CRISPR-based diagnostic systems, which urgently needs to be developed to improve diagnostic accuracy. A labor-free, contamination-free, and fully automated droplet manipulation platform for the CRISPR-LAMP technology has not been developed before. Herein, we propose a fully automated CRISPR-LAMP platform, which can precisely manipulate the CRISPR-LAMP droplet and perform combined reactions with high sensitivity and specificity. SARS-CoV-2 Spike T478K, D614G, P681R, and P681H mutations, typical point mutations of B.1.617.2 (Delta) and Omicron variants, are monitored with this platform with a detection limit of 102 copies/µL. Allele discrimination between the mutants and wild type is significant with the designed one/two-mismatch CRISPR RNA (crRNA) at a limit of 102 copies/µL. Chemically synthesized and modified crRNAs greatly increase the CRISPR-LAMP signal, which advance the wide application. Combined with the previously developed RdRp CRISPR-LAMP assay, clinical results showed that Spike T478K and P681H can discriminate the mutant type form the wild type with 70% (49.66-85.50%, 95% confidence interval) and 78% (57.27-90.62%, 95% confidence interval) sensitivity, respectively, and 100% specificity (51.68-100%, 95% confidence interval), and the RdRp target can detect SARS-CoV-2 strains with 85% sensitivity (65.39-95.14%, 95% confidence interval) and 100% specificity (51.68-100%, 95% confidence interval). We believe that this automatic digital microfluid (DMF) system can advance the integrated CRISPR-LAMP technology with higher stability, sensitivity, and practicability, also for other CRISPR-associated diagnostic platforms.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , SARS-CoV-2/genetics , COVID-19/diagnosis , Nucleic Acid Amplification Techniques/methods , RNA-Dependent RNA Polymerase , Sensitivity and Specificity
11.
Anal Biochem ; 659: 114960, 2022 Dec 15.
Article in English | MEDLINE | ID: covidwho-2085839

ABSTRACT

COVID-19 pandemic highlighted the demand for the fast and reliable detection of viral RNA. Although various methods for RNA amplification and detection have been proposed, some limitations, including those caused by reverse transcription (RT), need to be overcome. Here, we report on the direct detection of specific RNA by conventional polymerase chain reaction (PCR) requiring no prior RT step. It was found that Hemo KlenTaq (HKTaq), which is posed as DNA-dependent DNA polymerase, possesses reverse transcriptase activity and provides reproducible amplification of RNA targets with an efficiency comparable to common RT-PCR. Using nasopharyngeal swab extracts from COVID-19-positive patients, the high reliability of SARS-CoV-2 detection based on HKTaq was demonstrated. The most accurate detection of specific targets are provided by nearby primers, which allow to determine RNA in solutions affected to multiple freeze-thaw cycles. HKTaq can be used for elaboration of simplified amplification techniques intended for the analysis of any specific RNA and requiring only one DNA polymerase.


Subject(s)
COVID-19 , RNA, Viral , Humans , Clinical Laboratory Techniques/methods , COVID-19 Testing , Nucleic Acid Amplification Techniques/methods , Pandemics , Reproducibility of Results , Reverse Transcriptase Polymerase Chain Reaction , RNA, Viral/genetics , RNA, Viral/analysis , RNA-Directed DNA Polymerase/genetics , SARS-CoV-2/genetics , Sensitivity and Specificity , Taq Polymerase/metabolism
12.
Viruses ; 14(9)2022 09 17.
Article in English | MEDLINE | ID: covidwho-2043977

ABSTRACT

Rapid and accurate diagnosis of SARS-CoV-2 infection is essential for the management of the COVID-19 outbreak. RT-LAMP LoopDeetect COVID-19 (LoopDeescience, France) is a rapid molecular diagnostic tool which operates with the LoopDeelab (LoopDeescience, France) device. RAPID COVID is a prospective double-blind research protocol which was conducted to evaluate the concordance between Loopdeetect COVID-19 and RT-PCR Allplex 2019 n-Cov (Seegene, Korea). Between 11 May 2020 and 14 June 2021, a total of 1122 nasopharyngeal swab specimens were collected, of which 741 were finally analysed. There were 32 "positive" and "indeterminate" RT-PCR results. The intrinsic performances of Loopdeetect COVID-19 are equivalent to other commercial RT-LAMP PCR COVID-19 kits, with a sensitivity and specificity of 69.23% [CI 95%: 48.21-85.67] and 100% [CI 95%: 99.58-100.00], respectively. To the best of our knowledge, LoopDeelab is the only LAMP PCR diagnostic device allowing such a fast and reliable analysis with low-cost equipment; this makes it a new and innovative technology, designed for field use. This device being portable, the development of other detection kits will be useful for the management of epidemics with a high attack rate and would facilitate the rapid application of health measures.


Subject(s)
COVID-19 , COVID-19/diagnosis , COVID-19/epidemiology , COVID-19 Testing , Humans , Nucleic Acid Amplification Techniques/methods , Pandemics , Prospective Studies , SARS-CoV-2/genetics , Sensitivity and Specificity
13.
Chemosphere ; 307(Pt 1): 135645, 2022 Nov.
Article in English | MEDLINE | ID: covidwho-2035840

ABSTRACT

Respiratory viruses are a serious threat to human wellbeing that can cause pandemic disease. As a result, it is critical to identify virus in a timely, sensitive, and precise manner. The present novel coronavirus-2019 (COVID-19) disease outbreak has increased these concerns. The research of developing various methods for COVID-19 virus identification is one of the most rapidly growing research areas. This review article compares and addresses recent improvements in conventional and advanced electroanalytical approaches for detecting COVID-19 virus. The popular conventional methods such as polymerase chain reaction (PCR), loop mediated isothermal amplification (LAMP), serology test, and computed tomography (CT) scan with artificial intelligence require specialized equipment, hours of processing, and specially trained staff. Many researchers, on the other hand, focused on the invention and expansion of electrochemical and/or bio sensors to detect SARS-CoV-2, demonstrating that they could show a significant role in COVID-19 disease control. We attempted to meticulously summarize recent advancements, compare conventional and electroanalytical approaches, and ultimately discuss future prospective in the field. We hope that this review will be helpful to researchers who are interested in this interdisciplinary field and desire to develop more innovative virus detection methods.


Subject(s)
COVID-19 , SARS-CoV-2 , Artificial Intelligence , COVID-19/diagnosis , Humans , Nucleic Acid Amplification Techniques/methods , Pandemics , Sensitivity and Specificity
14.
Biosens Bioelectron ; 217: 114739, 2022 Dec 01.
Article in English | MEDLINE | ID: covidwho-2031163

ABSTRACT

The coronavirus disease 2019 (COVID-19) pandemic has created a huge demand for sensitive and rapid detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The current gold standard for SARS-CoV-2 detection is reverse transcription-polymerase chain reaction (RT-PCR)-based nucleic acid amplification. However, RT-PCR is time consuming and requires specialists and large instruments that are unattainable for point-of-care testing (POCT). To develop POCT for SARS-CoV-2, we combined recombinase polymerase amplification (RPA) and FeS2 nanozyme strips to achieve facile nucleic acid amplification and subsequent colorimetric signal enhancement based on the high peroxidase-like activity of the FeS2 nanozymes. This method showed a nucleic acid limit of detection (LOD) for SARS-CoV-2 of 200 copies/mL, close to that of RT-PCR. The unique catalytic properties of the FeS2 nanozymes enabled the nanozyme-strip to amplify colorimetric signals via the nontoxic 3,3',5,5'-tetramethylbenzidine (TMB) substrate. Importantly, the detection of clinical samples of human papilloma virus type 16 (HPV-16) showed 100% agreement with previous RT-PCR results, highlighting the versatility and reliability of this method. Our findings suggest that nanozyme-based nucleic acid detection has great potential in the development of POCT diagnosis for COVID-19 and other viral infections.


Subject(s)
Biosensing Techniques , COVID-19 , Nucleic Acids , COVID-19/diagnosis , Humans , Nucleic Acid Amplification Techniques/methods , Peroxidases , RNA, Viral/analysis , RNA, Viral/genetics , Recombinases , Reproducibility of Results , SARS-CoV-2/genetics , Sensitivity and Specificity
15.
Funct Integr Genomics ; 22(6): 1391-1401, 2022 Dec.
Article in English | MEDLINE | ID: covidwho-2014179

ABSTRACT

The coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has caused millions of infections and deaths worldwide since it infected humans almost 3 years ago. Improvements of current assays and the development of new rapid tests or to diagnose SARS-CoV-2 are urgent. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) is a rapid and propitious assay, allowing to detect both colorimetric and/or fluorometric nucleic acid amplifications. This study describes the analytical and clinical evaluation of RT-LAMP assay for detection of SARS-CoV-2, by designing LAMP primers targeting N (nucleocapsid phosphoprotein), RdRp (polyprotein), S (surface glycoprotein), and E (envelope protein) genes. The assay's performance was compared with the gold standard RT-PCR, yielding 94.6% sensitivity and 92.9% specificity. Among the tested primer sets, the ones for S and N genes had the highest analytical sensitivity, showing results in about 20 min. The colorimetric and fluorometric comparisons revealed that the latter is faster than the former. The limit of detection (LoD) of RT-LAMP reaction in both assays is 50 copies/µl of the reaction mixture. However, the simple eye-observation advantage of the colorimetric assay (with a color change from yellow to red) serves a promising on-site point-of-care testing method anywhere, including, for instance, laboratory and in-house applications.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , SARS-CoV-2/genetics , Reverse Transcription , Colorimetry/methods , COVID-19/diagnosis , COVID-19/genetics , Sensitivity and Specificity , Nucleic Acid Amplification Techniques/methods , RNA, Viral/genetics
16.
J Med Virol ; 94(12): 5858-5866, 2022 Dec.
Article in English | MEDLINE | ID: covidwho-2013628

ABSTRACT

To rapidly identify individuals infected with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and control the spread of coronavirus disease (COVID-19), there is an urgent need for highly sensitive on-site virus detection methods. A clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas)-based molecular diagnostic method was developed for this purpose. Here, a CRISPR system-mediated lateral flow assay (LFA) for SARS-CoV-2 was established based on multienzyme isothermal rapid amplification, CRISPR-Cas13a nuclease, and LFA. To improve the limit of detection (LoD), the crispr RNA, amplification primer, and probe were screened, in addition to concentrations of various components in the reaction system. The LoD of CRISPR detection was improved to 0.25 copy/µl in both fluorescence- and immunochromatography-based assays. To enhance the quality control of the CRISPR-based LFA method, glyceraldehyde-3-phosphate dehydrogenase was detected as a reference using a triple-line strip design in a lateral flow strip. In total, 52 COVID-19-positive and 101 COVID-19-negative clinical samples examined by reverse transcription polymerase chain reaction (RT-PCR) were tested using the CRISPR immunochromatographic detection technique. Results revealed 100% consistency, indicating the comparable effectiveness of our method to that of RT-PCR. In conclusion, this approach significantly improves the sensitivity and reliability of CRISPR-mediated LFA and provides a crucial tool for on-site detection of SARS-CoV-2.


Subject(s)
COVID-19 , CRISPR-Associated Proteins , COVID-19/diagnosis , CRISPR-Associated Proteins/genetics , Humans , Nucleic Acid Amplification Techniques/methods , RNA , Reproducibility of Results , SARS-CoV-2/genetics , Sensitivity and Specificity
17.
PLoS One ; 17(9): e0273912, 2022.
Article in English | MEDLINE | ID: covidwho-2009711

ABSTRACT

PURPOSE: To demonstrate the diagnostic performance of rapid SARS-CoV-2 RT-LAMP assays, comparing the performance of genomic versus sub-genomic sequence target with subsequent application in an asymptomatic screening population. METHODS: RT-LAMP diagnostic specificity (DSe) and sensitivity (DSe) was determined using 114 RT-PCR clinically positive and 88 RT-PCR clinically negative swab samples processed through the diagnostic RT-PCR service within the University Hospitals of Leicester NHS Trust. A swab-based RT-LAMP SARS-CoV-2 screening programme was subsequently made available to all staff and students at the University of Leicester (Autumn 2020), implemented to ISO 15189:2012 standards using NHS IT infrastructure and supported by University Hospital Leicester via confirmatory NHS diagnostic laboratory testing of RT-LAMP 'positive' samples. RESULTS: Validation samples reporting a Ct < 20 were detected at 100% DSe and DSp, reducing to 95% DSe (100% DSp) for all samples reporting a Ct < 30 (both genomic dual sub-genomic assays). Advisory screening identified nine positive cases in 1680 symptom free individuals (equivalent to 540 cases per 100,000) with results reported back to participants and feed into national statistics within 48 hours. CONCLUSION: This work demonstrates the utility of a rapid RT-LAMP assay for collapsing transmission of SARS-CoV-2 in an asymptomatic screening population.


Subject(s)
COVID-19 , SARS-CoV-2 , COVID-19/diagnosis , COVID-19/epidemiology , Humans , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/methods , RNA, Viral/genetics , SARS-CoV-2/genetics , Sensitivity and Specificity
18.
J Am Chem Soc ; 144(36): 16310-16315, 2022 09 14.
Article in English | MEDLINE | ID: covidwho-2008246

ABSTRACT

We introduce a new method to generate an amplified signal in CRISPR-Cas-based detection. Target recognition activates a CRISPR-Cas complex, leading to catalytic cleavage of horseradish peroxidase (HRP)-labeled oligonucleotides from the surface of microbeads. We show that the HRP released into solution can be monitored through colorimetric, fluorometric, or luminescent approaches, yielding up to ∼75-fold turn-on signal and limits of detection (LODs) as low as ∼10 fM. Compared to Cas-based detection with a conventional fluorophore/quencher reporter, this strategy improves the LOD by ∼30-fold. As a proof-of-concept, we show the rapid (<1 h), PCR-free, and room temperature (25 °C) detection of a nucleic acid marker for the SARS-CoV-2 virus with the naked eye at clinically relevant concentrations. We further show that the probe set can be programmed to be recognized and activated in the presence of non-nucleic acid targets. Specifically, we show adenosine triphosphate (ATP) binding to an aptamer can activate CRISPR-Cas and trigger a colorimetric readout, enabling the analysis of ATP in human serum samples with sensitivity on par with that of several commercially available kits. Taken together, the strategy reported herein offers a simple and sensitive platform to detect analytes where target amplification is either inconvenient (e.g., PCR under point-of-care settings) or impossible.


Subject(s)
Biosensing Techniques , COVID-19 , Nucleic Acids , Adenosine Triphosphate/analysis , COVID-19/diagnosis , CRISPR-Cas Systems , Horseradish Peroxidase , Humans , Nucleic Acid Amplification Techniques/methods , SARS-CoV-2/genetics
19.
Biosens Bioelectron ; 216: 114683, 2022 Nov 15.
Article in English | MEDLINE | ID: covidwho-2007469

ABSTRACT

Developing highly accurate and simple approaches to rapidly identify and isolate SARS-CoV-2 infected patients is important for the control of the COVID-19 pandemic. We, herein, reported the performance of a Cas12a-assisted RTF-EXPAR strategy for the identification of SARS-CoV-2 RNA. This assay combined the advantages of RTF-EXPAR with CRISPR-Cas12a can detect SARS-CoV-2 within 40 min, requiring only isothermal control. Particularly, the simultaneous use of EXPAR amplification and CRISPR improved the detection sensitivity, thereby realizing ultrasensitive SARS-CoV-2 RNA detection with a detection limit of 3.77 aM (∼2 copies/µL) in an end-point fluorescence read-out fashion, and at 4.81 aM (∼3 copies/µL) level via a smartphone-assisted analysis system (RGB analysis). Moreover, Cas12a increases the specificity by intrinsic sequence-specific template recognition. Overall, this method is fast, sensitive, and accurate, needing minimal equipment, which holds great promise to meet the requirements of point-of-care molecular detection of SARS-CoV-2.


Subject(s)
Biosensing Techniques , COVID-19 , Biosensing Techniques/methods , COVID-19/diagnosis , CRISPR-Cas Systems/genetics , Humans , Nucleic Acid Amplification Techniques/methods , Pandemics , RNA, Viral/analysis , RNA, Viral/genetics , SARS-CoV-2/genetics , Sensitivity and Specificity
20.
Anal Chim Acta ; 1229: 340343, 2022 Oct 09.
Article in English | MEDLINE | ID: covidwho-2007360

ABSTRACT

Home-testing of SARS-CoV-2 is an ideal approach for controlling the pandemic of COVID-19 and alleviating the shortage of medical resource caused by this acute infectious disease. Herein, a portable device that enables real-time monitoring of isothermal nucleic acid amplification tests (INAATs) through the electrochemistry method was fabricated for home-testing of SARS-CoV-2. First, a disposable plug-and-play pH-sensitive potentiometric sensor that matches this electrochemical INAATs (E-INAATs) device was designed to allow the label-free pH sensing detection of nucleic acid. By applying Nafion film on the polyaniline-based working electrode, this sensor exhibited an excellent linear potentiometric response to pH value in the range of 6.0-8.5 with a slope of -37.45 ± 1.96 mV/pH unit. A Bluetooth module was integrated into this device to enable the users real-time monitoring INAATs on their smartphones at home. Moreover, by presetting criteria, the detection results could be automatically judged by the device to avoid human errors. Finally, the utility of this E-INAATs device was demonstrated by detecting the presence of SARS-CoV-2 nucleocapsid protein gene in artificial samples with a sensitivity of 2 × 102 copies/test within 25 min, which was comparable with fluorescence and colorimetric assay. This portable, easy-operated, sensitive, and affordable device is particularly desirable for the full integration of household SARS-CoV-2 detection products and will open a new prospect for the control of infectious diseases via electrochemical NAATs.


Subject(s)
COVID-19 , Nucleic Acids , COVID-19/diagnosis , Humans , Nucleic Acid Amplification Techniques/methods , RNA, Viral , SARS-CoV-2/genetics , Sensitivity and Specificity , Smartphone
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