Your browser doesn't support javascript.
Show: 20 | 50 | 100
Results 1 - 20 de 25
Filter
1.
Mikrochim Acta ; 188(10): 335, 2021 Sep 09.
Article in English | MEDLINE | ID: covidwho-1411927

ABSTRACT

A practical colorimetric assay was developed for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). For this purpose, magnetic γ Fe2O3 nanoparticles were synthesized and used as a peroxidase-like mimic activity molecule. In the presence of γ Fe2O3 nanoparticles, the color change of H2O2 included 3,3',5,5'-tetramethylbenzidine was monitored at the wavelength of 654 nm when spike protein interacted with angiotensin-converting enzyme 2 receptor. This oxidation-reduction reaction was examined both spectroscopically and by using electrochemical techniques. The experimental parameters were optimized and the analytical characteristics investigated. The developed assay was applied to real SARS-CoV-2 samples, and very good results that were in accordance with the real time polymerase chain reaction were obtained.


Subject(s)
COVID-19 Testing/methods , COVID-19/diagnosis , Colorimetry/methods , Magnetic Iron Oxide Nanoparticles/chemistry , SARS-CoV-2/chemistry , Spike Glycoprotein, Coronavirus/chemistry , Angiotensin-Converting Enzyme 2/metabolism , Benzidines/chemistry , Biosensing Techniques/methods , COVID-19 Testing/instrumentation , Catalysis , Chromogenic Compounds/chemistry , Cysteine/chemistry , Humans , Hydrogen Peroxide/chemistry , Limit of Detection , Nasopharynx/virology , Oropharynx/virology , Oxidation-Reduction , Peroxidase/chemistry , Spike Glycoprotein, Coronavirus/metabolism
2.
ACS Appl Mater Interfaces ; 13(35): 41445-41453, 2021 Sep 08.
Article in English | MEDLINE | ID: covidwho-1371587

ABSTRACT

Airborne transmission of exhaled virus can rapidly spread, thereby increasing disease progression from local incidents to pandemics. Due to the COVID-19 pandemic, states and local governments have enforced the use of protective masks in public and work areas to minimize the disease spread. Here, we have leveraged the function of protective face coverings toward COVID-19 diagnosis. We developed a user-friendly, affordable, and wearable collector. This noninvasive platform is integrated into protective masks toward collecting airborne virus in the exhaled breath over the wearing period. A viral sample was sprayed into the collector to model airborne dispersion, and then the enriched pathogen was extracted from the collector for further analytical evaluation. To validate this design, qualitative colorimetric loop-mediated isothermal amplification, quantitative reverse transcription polymerase chain reaction, and antibody-based dot blot assays were performed to detect the presence of SARS-CoV-2. We envision that this platform will facilitate sampling of current SARS-CoV-2 and is potentially broadly applicable to other airborne diseases for future emerging pandemics.


Subject(s)
Breath Tests/instrumentation , COVID-19 Testing/instrumentation , Masks , SARS-CoV-2/isolation & purification , Air Microbiology , Antibodies, Viral/immunology , Breath Tests/methods , COVID-19 Testing/methods , Collodion/chemistry , Colorimetry/methods , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/methods , Polycarboxylate Cement/chemistry , Porosity , Proof of Concept Study , RNA, Viral/analysis , Real-Time Polymerase Chain Reaction/methods , SARS-CoV-2/chemistry , Viral Proteins/analysis , Viral Proteins/immunology
3.
Sci Rep ; 11(1): 16430, 2021 08 12.
Article in English | MEDLINE | ID: covidwho-1356582

ABSTRACT

Until there is an effective implementation of COVID-19 vaccination program, a robust testing strategy, along with prevention measures, will continue to be the most viable way to control disease spread. Such a strategy should rely on disparate diagnostic tests to prevent a slowdown in testing due to lack of materials and reagents imposed by supply chain problems, which happened at the beginning of the pandemic. In this study, we have established a single-tube test based on RT-LAMP that enables the visual detection of less than 100 viral genome copies of SARS-CoV-2 within 30 min. We benchmarked the assay against the gold standard test for COVID-19 diagnosis, RT-PCR, using 177 nasopharyngeal RNA samples. For viral loads above 100 copies, the RT-LAMP assay had a sensitivity of 100% and a specificity of 96.1%. Additionally, we set up a RNA extraction-free RT-LAMP test capable of detecting SARS-CoV-2 directly from saliva samples, albeit with lower sensitivity. The saliva was self-collected and the collection tube remained closed until inactivation, thereby ensuring the protection of the testing personnel. As expected, RNA extraction from saliva samples increased the sensitivity of the test. To lower the costs associated with RNA extraction, we performed this step using an alternative protocol that uses plasmid DNA extraction columns. We also produced the enzymes needed for the assay and established an in-house-made RT-LAMP test independent of specific distribution channels. Finally, we developed a new colorimetric method that allowed the detection of LAMP products by the visualization of an evident color shift, regardless of the reaction pH.


Subject(s)
COVID-19 Testing/methods , COVID-19/virology , Colorimetry/methods , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/methods , SARS-CoV-2/isolation & purification , COVID-19/diagnosis , Humans , Pandemics , Portugal/epidemiology , RNA, Viral/genetics , SARS-CoV-2/genetics , Saliva/chemistry , Saliva/virology , Sensitivity and Specificity
4.
Nucleic Acids Res ; 49(13): 7267-7279, 2021 07 21.
Article in English | MEDLINE | ID: covidwho-1298981

ABSTRACT

We performed in vitro selection experiments to identify DNA aptamers for the S1 subunit of the SARS-CoV-2 spike protein (S1 protein). Using a pool of pre-structured random DNA sequences, we obtained over 100 candidate aptamers after 13 cycles of enrichment under progressively more stringent selection pressure. The top 10 sequences all exhibited strong binding to the S1 protein. Two aptamers, named MSA1 (Kd = 1.8 nM) and MSA5 (Kd = 2.7 nM), were assessed for binding to the heat-treated S1 protein, untreated S1 protein spiked into 50% human saliva and the trimeric spike protein of both the wildtype and the B.1.1.7 variant, demonstrating comparable affinities in all cases. MSA1 and MSA5 also recognized the pseudotyped lentivirus of SARS-CoV-2 with respective Kd values of 22.7 pM and 11.8 pM. Secondary structure prediction and sequence truncation experiments revealed that both MSA1 and MSA5 adopted a hairpin structure, which was the motif pre-designed into the original library. A colorimetric sandwich assay was developed using MSA1 as both the recognition element and detection element, which was capable of detecting the pseudotyped lentivirus in 50% saliva with a limit of detection of 400 fM, confirming the potential of these aptamers as diagnostic tools for COVID-19 detection.


Subject(s)
Aptamers, Nucleotide , COVID-19/virology , Gene Library , Mutation , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics , Aptamers, Nucleotide/chemistry , Aptamers, Nucleotide/genetics , Base Pairing , Base Sequence , COVID-19/diagnosis , Colorimetry/methods , Humans , Nucleic Acid Conformation , SELEX Aptamer Technique
5.
Biosensors (Basel) ; 11(6)2021 May 24.
Article in English | MEDLINE | ID: covidwho-1243953

ABSTRACT

Despite collaborative efforts from all countries, coronavirus disease 2019 (COVID-19) pandemic has been continuing to spread globally, forcing the world into social distancing period, making a special challenge for public healthcare system. Before vaccine widely available, the best approach to manage severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is to achieve highest diagnostic accuracy by improving biosensor efficacy. For SARS-CoV-2 diagnostics, intensive attempts have been made by many scientists to ameliorate the drawback of current biosensors of SARS-CoV-2 in clinical diagnosis to offer benefits related to platform proposal, systematic analytical methods, system combination, and miniaturization. This review assesses ongoing research efforts aimed at developing integrated diagnostic tools to detect RNA viruses and their biomarkers for clinical diagnostics of SARS-CoV-2 infection and further highlights promising technology for SARS-CoV-2 specific diagnosis. The comparisons of SARS-CoV-2 biomarkers as well as their applicable biosensors in the field of clinical diagnosis were summarized to give scientists an advantage to develop superior diagnostic platforms. Furthermore, this review describes the prospects for this rapidly growing field of diagnostic research, raising further interest in analytical technology and strategic plan for future pandemics.


Subject(s)
Biosensing Techniques/instrumentation , COVID-19 Testing/instrumentation , SARS-CoV-2/isolation & purification , Animals , Biosensing Techniques/methods , COVID-19 Testing/methods , Colorimetry/instrumentation , Colorimetry/methods , Electrochemical Techniques/instrumentation , Electrochemical Techniques/methods , Enzyme-Linked Immunosorbent Assay/instrumentation , Enzyme-Linked Immunosorbent Assay/methods , Equipment Design , Humans , Nucleic Acid Amplification Techniques/instrumentation , Nucleic Acid Amplification Techniques/methods , Point-of-Care Testing
6.
Nat Protoc ; 16(6): 3141-3162, 2021 06.
Article in English | MEDLINE | ID: covidwho-1209962

ABSTRACT

The global pandemic of coronavirus disease 2019 (COVID-19) highlights the shortcomings of the current testing paradigm for viral disease diagnostics. Here, we report a stepwise protocol for an RNA-extraction-free nano-amplified colorimetric test for rapid and naked-eye molecular diagnosis of COVID-19. The test employs a unique dual-prong approach that integrates nucleic acid (NA) amplification and plasmonic sensing for point-of-care detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), with a sample-to-assay response time of <1 h. The RNA-extraction-free nano-amplified colorimetric test utilizes plasmonic gold nanoparticles capped with antisense oligonucleotides (ASOs) as a colorimetric reporter to detect the amplified nucleic acid from the COVID-19 causative virus, SARS-CoV-2. The ASOs are specific for the SARS-CoV-2 N-gene, and binding of the ASOs to their target sequence results in the aggregation of the plasmonic gold nanoparticles. This highly specific agglomeration step leads to a change in the plasmonic response of the nanoparticles. Furthermore, when tested using clinical samples, the accuracy, sensitivity and specificity of the test were found to be >98.4%, >96.6% and 100%, respectively, with a detection limit of 10 copies/µL. The test can easily be adapted to diagnose other viral infections with a simple modification of the ASOs and primer sequences. It also provides a low-cost, rapid approach requiring minimal instrumentation that can be used as a screening tool for the diagnosis of COVID-19 at point-of-care settings in resource-poor situations. The colorimetric readout of the test can even be monitored using a handheld optical reader to obtain a quantitative response. Therefore, we anticipate that this protocol will be widely useful for the development of biosensors for the molecular diagnostics of COVID-19 and other infectious diseases.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19/diagnosis , Gold/chemistry , Metal Nanoparticles/chemistry , Oligonucleotides, Antisense/chemistry , RNA, Viral/analysis , SARS-CoV-2/isolation & purification , COVID-19/virology , COVID-19 Nucleic Acid Testing/instrumentation , Colorimetry/instrumentation , Colorimetry/methods , Humans , Limit of Detection , Oligonucleotides, Antisense/genetics , Point-of-Care Testing , RNA, Viral/genetics , SARS-CoV-2/genetics
7.
Chem Asian J ; 16(11): 1298-1306, 2021 Jun 01.
Article in English | MEDLINE | ID: covidwho-1182102

ABSTRACT

Aptamers are short single-stranded DNA or RNA oligonucleotides selected by the technique of systematic evolution of ligands by exponential enrichment (SELEX). Aptamers have been demonstrated to bind various targets from small-molecule to cells or even tissues in the way of antibodies. Thus, they are called chemical antibodies. We summarize and evaluate recent developments in aptamer-based sensors (for short aptasensors) for virus detection in this review. These aptasensors are mainly classified into optical and electronic aptasensors based on the type of transducer. Nowadays, the smartphone has become the most widely used mobile device with billions of users worldwide. Considering the ongoing COVID-19 outbreak, smartphone-based aptasensors for a portable and point-of-care test (POCT) of COVID-19 detection will be of great importance in the future.


Subject(s)
Aptamers, Nucleotide/chemistry , Biosensing Techniques/methods , Viral Proteins/analysis , Viruses/isolation & purification , Biosensing Techniques/instrumentation , Colorimetry/methods , Electrochemical Techniques/methods , Fluorescence Resonance Energy Transfer , Humans , Smartphone , Spectrum Analysis, Raman , Viral Proteins/chemistry , Viruses/chemistry
8.
Biotechniques ; 70(4): 218-225, 2021 04.
Article in English | MEDLINE | ID: covidwho-1170031

ABSTRACT

Evaluation of the performance of a new set of primers defined from the ORF1ab sequence, and its combination with a previously published set of primers from the N sequence, to detect SARS-CoV-2 RNA by the loop-mediated isothermal amplification technique is presented. The ORF1ab primer set enables visual detection of SARS-CoV-2 RNA in 16 min. In addition, a simultaneous reaction with both ORF1ab and N primers allows for higher sensitivity of detection, particularly when low numbers of copies are present (250 viral RNA copies). Further, the protocol is able to detect viral RNA in saliva samples. The procedure reported could be easily implemented in the generation of a new and sensitive rapid point-of care device for SARS-CoV-2 RNA visual detection.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19/diagnosis , Colorimetry/methods , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/methods , SARS-CoV-2/genetics , COVID-19/virology , DNA Primers , Humans , Polyproteins/genetics , Viral Proteins/genetics
9.
Biosens Bioelectron ; 182: 113173, 2021 Jun 15.
Article in English | MEDLINE | ID: covidwho-1152282

ABSTRACT

Respiratory syncytial virus (RSV) infection is the most common clinical infectious disease threatening the safety of human life. Herein, we provided a sensitive and specific method for detection and differentiation of RSV subgroups A (RSVA) and B (RSVB) with colorimetric toehold switch sensors in a paper-based cell-free system. In this method, we applied the toehold switch, an RNA-based riboswitch, to regulate the translation level of ß-galactosidase (lacZ) gene. In the presence of target trigger RNA, the toehold switch sensor was activated and the expressed LacZ hydrolyzed chromogenic substrates to produce a colorimetric result that can be observed directly with the naked eye in a cell-free system. In addition, nucleic acid sequence-based amplification (NASBA) was used to improve the sensitivity by amplifying target trigger RNAs. Under optimal conditions, our method produced a visible result for the detection of RSVA and RSVB with the detection limit of 52 aM and 91 aM, respectively. The cross-reaction of this method was validated with other closely related respiratory viruses, including human coronavirus HKU1 (HCoV-HKU1), and Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Furthermore, we used the paper-based carrier material that allows stable storage of our detection elements and rapid detection outside laboratory. In conclusion, this method can sensitively and specifically differentiate RSVA and RSVB and generate a visible colorimetric result without specialized operators and sophisticated equipment. Based on these advantages above, this method serves as a simple and portable detector in resource-poor areas and point-of-care testing (POCT) scenarios.


Subject(s)
Biosensing Techniques , Cell-Free System , Colorimetry/methods , Respiratory Syncytial Virus, Human/isolation & purification , Betacoronavirus/isolation & purification , Humans , RNA, Viral , SARS-CoV-2/isolation & purification
10.
Nat Commun ; 12(1): 1467, 2021 03 05.
Article in English | MEDLINE | ID: covidwho-1118805

ABSTRACT

Efforts to contain the spread of SARS-CoV-2 have spurred the need for reliable, rapid, and cost-effective diagnostic methods which can be applied to large numbers of people. However, current standard protocols for the detection of viral nucleic acids while sensitive, require a high level of automation and sophisticated laboratory equipment to achieve throughputs that allow whole communities to be tested on a regular basis. Here we present Cap-iLAMP (capture and improved loop-mediated isothermal amplification) which combines a hybridization capture-based RNA extraction of gargle lavage samples with an improved colorimetric RT-LAMP assay and smartphone-based color scoring. Cap-iLAMP is compatible with point-of-care testing and enables the detection of SARS-CoV-2 positive samples in less than one hour. In contrast to direct addition of the sample to improved LAMP (iLAMP), Cap-iLAMP prevents false positives and allows single positive samples to be detected in pools of 25 negative samples, reducing the reagent cost per test to ~1 Euro per individual.


Subject(s)
COVID-19 Testing/methods , COVID-19/diagnosis , COVID-19/virology , Colorimetry/methods , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/methods , Nucleic Acid Hybridization/methods , Point-of-Care Testing , SARS-CoV-2/isolation & purification , Coronavirus Nucleocapsid Proteins/genetics , Humans , Phosphoproteins/genetics , RNA, Viral/genetics , SARS-CoV-2/genetics , Sensitivity and Specificity
11.
Anal Chem ; 93(8): 4126-4133, 2021 03 02.
Article in English | MEDLINE | ID: covidwho-1078274

ABSTRACT

The outbreak of the pandemic caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) calls for an urgent unmet need for developing a facial and cost-effective detection method. The requirement of well-trained personnel and sophisticated instrument of current primary mean (reverse transcription polymerase chain reaction, RT-PCR) may hinder the practical application worldwide. In this regard, a reverse transcription recombinase polymerase amplification (RT-RPA) coupled with CRISPR-Cas12a colorimetric assay is proposed for the SARS-CoV-2 detection. The methodology we have described herein utilizes DNA-modified gold nanoparticles (AuNPs) as a universal colorimetric readout and can specifically target ORF1ab and N regions of the SARS-CoV-2 genome. After the virus genome is amplified through RT-RPA, the resulting abundant dsDNA will bind and activate Cas12a. Under trans-cleavage degradation, the capped DNA substrate will be hydrolyzed gradually from AuNPs, demonstrating a change in the surface plasmon resonance (SPR), which can be facially monitored by UV-vis absorbance spectroscopy and naked eye observation. The high amplification efficiency from RT-RPA and Cas12a trans-cleavage process bring the sensitivity of our method to 1 copy of viral genome sequence per test. Notably, under the dual variations inspecting from the isothermal amplification and Cas12a activation process, the false positive events from other beta coronavirus members can be effectively avoided and thus significantly improve the specificity. Furthermore, the reliability of this colorimetric assay is validated by standard clinical samples from the hospital laboratory department. Through integration of the inherently high sensitivity and specificity from an RPA-coupled Cas12a system with the intrinsic simplicity of AuNP-based colorimetric assay, our method increases the practical testing availability of SARS-CoV-2.


Subject(s)
CRISPR-Cas Systems , Colorimetry/methods , DNA/chemistry , Nucleic Acid Amplification Techniques/methods , RNA, Viral/analysis , SARS-CoV-2/isolation & purification , Bacterial Proteins , Base Sequence , COVID-19/diagnosis , CRISPR-Associated Proteins , Coronavirus Nucleocapsid Proteins/genetics , DNA/genetics , Endodeoxyribonucleases , Gold/chemistry , Humans , Metal Nanoparticles/chemistry , Phosphoproteins/genetics , Polyproteins/genetics , RNA, Viral/genetics , Reverse Transcription , SARS-CoV-2/chemistry , Surface Plasmon Resonance , Viral Proteins/genetics
12.
Anal Methods ; 13(2): 169-178, 2021 01 21.
Article in English | MEDLINE | ID: covidwho-1039652

ABSTRACT

We demonstrate a loop-mediated isothermal amplification (LAMP) method to detect and amplify SARS-CoV-2 genetic sequences using a set of in-house designed initiators that target regions encoding the N protein. We were able to detect and amplify SARS-CoV-2 nucleic acids in the range of 62 to 2 × 105 DNA copies by this straightforward method. Using synthetic SARS-CoV-2 samples and RNA extracts from patients, we demonstrate that colorimetric LAMP is a quantitative method comparable in diagnostic performance to RT-qPCR (i.e., sensitivity of 92.85% and specificity of 81.25% in a set of 44 RNA extracts from patients analyzed in a hospital setting).


Subject(s)
COVID-19 Nucleic Acid Testing/methods , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/methods , RNA/analysis , SARS-CoV-2/chemistry , Viral Load/methods , COVID-19/diagnosis , Colorimetry/methods , Coronavirus Nucleocapsid Proteins , DNA/analysis , DNA/chemistry , Fluorescent Dyes/chemistry , Humans , Intercalating Agents/chemistry , Phenolsulfonphthalein/chemistry , Phosphoproteins , RNA/chemistry
13.
Pan Afr Med J ; 37: 78, 2020.
Article in English | MEDLINE | ID: covidwho-926884

ABSTRACT

Introduction: the most recently discovered severe acute respiratory syndrome Coronavirus 2 (SARS-COV-2) that causes COVID-19, subjected the entire world in turmoil health-wise and economically. With higher burden of malaria in Nigeria and other sub-Saharan African countries coupled with fragile healthcare system and delivery, these may pose a threat in the diagnosis and management of COVID-19 patients co-infected with malaria. Free radicals have been implicated in the progression and pathogenesis of malaria and COVID-19 through Fenton's reaction and cytokine storm respectively. Methods: the current research comprises of seventy-four (74) participants; 20 apparently healthy controls and 54 COVID-19 patients (34 among which were co-infected with malaria). Serum levels of 8-iso PGF2α and Alphatocopherol were determined among the study participants using ELISA technique and colorimetric assay, respectively. Results: results revealed statistically significant elevation of 8-iso PGF2α in COVID-19 patients co-infected with malaria compared to COVID-19 patients only, and this may be due to increase production of free radicals. Furthermore, a significant decrease of Alphatocopherol was observed in COVID-19 co-infected with malaria compared to COVID-19 patients due to increase utilization of antioxidants in counterbalancing the negative effect of free radicals generated. Conclusion: conclusively, SARS-COV-2 patients co-infected with malaria might be predisposed to oxidative stress and low Alphatocopherol. The increase in oxidative stress is proportional to malaria parasite density and inversely related to Alphatocopherol levels. This implies that oxidative stress is notably higher and such patients may have a severer form of the COVID-19. Increased 8-iso-PGF2α in co-infection and decreased alphatocopherol levels can reflect the severity and adverse outcomes compared to COVID-19 naïve because of their tremendous involvement in the pathogenesis and progression of diseases.


Subject(s)
COVID-19/blood , Coinfection/blood , Dinoprost/analogs & derivatives , Malaria/blood , SARS-CoV-2 , alpha-Tocopherol/blood , Biomarkers/blood , COVID-19 Testing/methods , Case-Control Studies , Coinfection/diagnosis , Colorimetry/methods , Cross-Sectional Studies , Dinoprost/blood , Female , Humans , Malaria/diagnosis , Malaria/parasitology , Male , Nigeria , Oxidative Stress , Pandemics , Reverse Transcriptase Polymerase Chain Reaction
14.
Analyst ; 146(2): 471-477, 2021 Jan 21.
Article in English | MEDLINE | ID: covidwho-915796

ABSTRACT

COVID-19, caused by the infection of SARS-CoV-2, has emerged as a rapidly spreading infection. The disease has now reached the level of a global pandemic and as a result a more rapid and simple detection method is imperative to curb the spread of the virus. We aimed to develop a visual diagnostic platform for SARS-CoV-2 based on colorimetric RT-LAMP with levels of sensitivity and specificity comparable to that of commercial qRT-PCR assays. In this work, the primers were designed to target a conserved region of the RNA-dependent RNA polymerase gene (RdRp). The assay was characterized for its sensitivity and specificity, and validated with clinical specimens collected in Thailand. The developed colorimetric RT-LAMP assay could amplify the target gene and enabled visual interpretation in 60 min at 65 °C. No cross-reactivity with six other common human respiratory viruses (influenza A virus subtypes H1 and H3, influenza B virus, respiratory syncytial virus types A and B, and human metapneumovirus) and five other human coronaviruses (MERS-CoV, HKU-1, OC43, 229E and NL63) was observed. The limit of detection was 25 copies per reaction when evaluated with contrived specimens. However, the detection rate at this concentration fell to 95.8% when the incubation time was reduced from 60 to 30 min. The diagnostic performance of the developed RT-LAMP assay was evaluated in 2120 clinical specimens and compared with the commercial qRT-PCR. The results revealed high sensitivity and specificity of 95.74% and 99.95%, respectively. The overall accuracy of the RT-LAMP assay was determined to be 99.86%. In summary, our results indicate that the developed colorimetric RT-LAMP provides a simple, sensitive and reliable approach for the detection of SARS-CoV-2 in clinical samples, implying its beneficial use as a diagnostic platform for COVID-19 screening.


Subject(s)
COVID-19 Testing/methods , COVID-19/diagnosis , Colorimetry/methods , Mass Screening/methods , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/methods , RNA, Viral/genetics , SARS-CoV-2/genetics , COVID-19/genetics , COVID-19/virology , Humans , RNA, Viral/analysis , Reverse Transcription , SARS-CoV-2/isolation & purification
15.
Biosens Bioelectron ; 172: 112765, 2021 Jan 15.
Article in English | MEDLINE | ID: covidwho-893624

ABSTRACT

To accurately diagnose COVID-19 infection and its time-dependent progression, the rapid, sensitive, and noninvasive determination of immunoglobulins A specific to SARS-CoV-2 (IgA) in saliva and serum is needed to complement tests that detect immunoglobulins G and M. We have developed a dual optical/chemiluminescence format of a lateral flow immunoassay (LFIA) immunosensor for IgA in serum and saliva. A recombinant nucleocapsid antigen specifically captures SARS-CoV-2 antibodies in patient specimens. A labelled anti-human IgA reveals the bound IgA fraction. A dual colorimetric and chemiluminescence detection enables the affordable and ultrasensitive determination of IgA to SARS-CoV-2. Specifically, a simple smartphone-camera-based device measures the colour signal provided by nanogold-labelled anti-human IgA. For the ultrasensitive chemiluminescence transduction, we used a contact imaging portable device based on cooled CCD, and measured the light signal resulting from the reaction of the HRP-labelled anti-human IgA with a H2O2/luminol/enhancers substrate. A total of 25 serum and 9 saliva samples from infected and/or recovered individuals were analysed by the colorimetric LFIA, which was sensitive and reproducible enough for the semi-quantification of IgA in subjects with a strong serological response and in the early stage of COVID-19 infection. Switching to CL detection, the same immunosensor exhibited higher detection capability, revealing the presence of salivary IgA in infected individuals. For the patients included in the study (n = 4), the level of salivary IgA correlated with the time elapsed from diagnosis and with the severity of the disease. This IgA-LFIA immunosensor could be useful for noninvasively monitoring early immune responses to COVID-19 and for investigating the diagnostic/prognostic utility of salivary IgA in the context of large-scale screening to assess the efficacy of SARS-CoV-2 vaccines.


Subject(s)
Antibodies, Viral/analysis , Biosensing Techniques/instrumentation , COVID-19 Serological Testing/instrumentation , COVID-19/diagnosis , SARS-CoV-2/immunology , Antibody Specificity , Biosensing Techniques/methods , COVID-19/immunology , COVID-19/virology , COVID-19 Serological Testing/methods , Colorimetry/instrumentation , Colorimetry/methods , Equipment Design , Humans , Immunoglobulin A/blood , Immunoglobulin A, Secretory/analysis , Luminescent Measurements/instrumentation , Luminescent Measurements/methods , Saliva/immunology
16.
Clin Chem ; 67(2): 415-424, 2021 01 30.
Article in English | MEDLINE | ID: covidwho-887266

ABSTRACT

BACKGROUND: Rapid, reliable, and widespread testing is required to curtail the ongoing COVID-19 pandemic. Current gold-standard nucleic acid tests are hampered by supply shortages in critical reagents including nasal swabs, RNA extraction kits, personal protective equipment, instrumentation, and labor. METHODS: To overcome these challenges, we developed a rapid colorimetric assay using reverse-transcription loop-mediated isothermal amplification (RT-LAMP) optimized on human saliva samples without an RNA purification step. We describe the optimization of saliva pretreatment protocols to enable analytically sensitive viral detection by RT-LAMP. We optimized the RT-LAMP reaction conditions and implemented high-throughput unbiased methods for assay interpretation. We tested whether saliva pretreatment could also enable viral detection by conventional reverse-transcription quantitative polymerase chain reaction (RT-qPCR). Finally, we validated these assays on clinical samples. RESULTS: The optimized saliva pretreatment protocol enabled analytically sensitive extraction-free detection of SARS-CoV-2 from saliva by colorimetric RT-LAMP or RT-qPCR. In simulated samples, the optimized RT-LAMP assay had a limit of detection of 59 (95% confidence interval: 44-104) particle copies per reaction. We highlighted the flexibility of LAMP assay implementation using 3 readouts: naked-eye colorimetry, spectrophotometry, and real-time fluorescence. In a set of 30 clinical saliva samples, colorimetric RT-LAMP and RT-qPCR assays performed directly on pretreated saliva samples without RNA extraction had accuracies greater than 90%. CONCLUSIONS: Rapid and extraction-free detection of SARS-CoV-2 from saliva by colorimetric RT-LAMP is a simple, sensitive, and cost-effective approach with broad potential to expand diagnostic testing for the virus causing COVID-19.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19/diagnosis , Nucleic Acid Amplification Techniques/methods , RNA, Viral/analysis , SARS-CoV-2/isolation & purification , Saliva/virology , COVID-19/epidemiology , Colorimetry/methods , Endopeptidase K/chemistry , Humans , Limit of Detection , Pandemics , Point-of-Care Testing , SARS-CoV-2/chemistry
17.
ACS Sens ; 5(10): 3043-3048, 2020 10 23.
Article in English | MEDLINE | ID: covidwho-801107

ABSTRACT

Mass testing is fundamental to face the pandemic caused by the coronavirus SARS-CoV-2 discovered at the end of 2019. To this aim, it is necessary to establish reliable, fast, and cheap tools to detect viral particles in biological material so to identify the people capable of spreading the infection. We demonstrate that a colorimetric biosensor based on gold nanoparticle (AuNP) interaction induced by SARS-CoV-2 lends itself as an outstanding tool for detecting viral particles in nasal and throat swabs. The extinction spectrum of a colloidal solution of multiple viral-target gold nanoparticles-AuNPs functionalized with antibodies targeting three surface proteins of SARS-CoV-2 (spike, envelope, and membrane)-is red-shifted in few minutes when mixed with a solution containing the viral particle. The optical density of the mixed solution measured at 560 nm was compared to the threshold cycle (Ct) of a real-time PCR (gold standard for detecting the presence of viruses) finding that the colorimetric method is able to detect very low viral load with a detection limit approaching that of the real-time PCR. Since the method is sensitive to the infecting viral particle rather than to its RNA, the achievements reported here open a new perspective not only in the context of the current and possible future pandemics, but also in microbiology, as the biosensor proves itself to be a powerful though simple tool for measuring the viral particle concentration.


Subject(s)
Betacoronavirus/chemistry , Colorimetry/methods , Coronavirus Infections/diagnosis , Coronavirus Infections/virology , Nasal Mucosa/virology , Pharynx/virology , Pneumonia, Viral/diagnosis , Pneumonia, Viral/virology , Biosensing Techniques , COVID-19 , Gold , Humans , Membrane Proteins/chemistry , Metal Nanoparticles , Pandemics , Photochemistry , Polymerase Chain Reaction , SARS-CoV-2 , Specimen Handling , Spike Glycoprotein, Coronavirus/chemistry , Threshold Limit Values , Viral Envelope Proteins/chemistry
18.
Analyst ; 145(23): 7680-7686, 2020 Nov 23.
Article in English | MEDLINE | ID: covidwho-798256

ABSTRACT

This work reports the development of a rapid, simple and inexpensive colorimetric paper-based assay for the detection of the severe acute respiratory symptom coronavirus 2 (SARS-CoV-2) humanized antibody. The paper device was prepared with lamination for easy sample handling and coated with the recombinant SARS-CoV-2 nucleocapsid antigen. This assay employed a colorimetric reaction, which is followed by horseradish peroxidase (HRP) conjugated detecting antibody in the presence of the 3,3',5,5'-tetramethylbenzidine (TMB) substrate. The colorimetric readout was evaluated and quantified for specificity and sensitivity. The characterization of this assay includes determining the linear regression curve, the limit of detection (LOD), the repeatability, and testing complex biological samples. We found that the LOD of the assay was 9.00 ng µL-1 (0.112 IU mL-1). The relative standard deviation was approximately 10% for a sample number of n = 3. We believe that our proof-of-concept assay has the potential to be developed for clinical screening of the SARS-CoV-2 humanized antibody as a tool to confirm infected active cases or to confirm SARS-CoV-2 immune cases during the process of vaccine development.


Subject(s)
Antibodies, Monoclonal, Humanized/blood , Antibodies, Viral/blood , COVID-19 Testing/methods , Colorimetry/methods , Enzyme-Linked Immunosorbent Assay/methods , Paper , SARS-CoV-2/immunology , Antibodies, Monoclonal, Humanized/immunology , Antibodies, Viral/immunology , Armoracia/enzymology , Benzidines/chemistry , COVID-19/diagnosis , COVID-19 Testing/instrumentation , Colorimetry/instrumentation , Coronavirus Nucleocapsid Proteins/immunology , Enzyme-Linked Immunosorbent Assay/instrumentation , Horseradish Peroxidase/chemistry , Humans , Limit of Detection , Phosphoproteins/immunology , Proof of Concept Study , SARS-CoV-2/chemistry
19.
Anal Bioanal Chem ; 412(28): 7861-7869, 2020 Nov.
Article in English | MEDLINE | ID: covidwho-738672

ABSTRACT

Sanitizing solutions against bacterial and viral pathogens are of utmost importance in general and, in particular, in these times of pandemic due to Sars-Cov2. They frequently consist of chlorine-based solutions, or in the direct input of a certain amount of chlorine in water supply systems and swimming pools. Colorimetry is one of the techniques used to measure the crucial persistence of chlorine in water, including household chlorine test kits commonly based on colorimetric indicators. Here, we show a simple and cheap colorimetric method based on 3,3',5,5'-tetramethylbenzidine (TMB), commonly used as chromogenic reagent for enzyme-linked immunosorbent assays. TMB is converted by chlorine to a colored molecule through a pH-dependent multi-step oxidation process where the chromaticity of TMB is directly proportional to chlorine content. This molecule offers several advantages over other commonly used reagents in terms of safety, sensitivity, and, peculiarly, hue modulation, giving rise to the detection of chlorine in water with a multi-color change of the indicator solution (transparent/blue/green/yellow). Moreover, through the appropriate setting of reaction conditions, such coloration is finely tunable to cover the range of chlorine concentration recommended by international health agencies for treatment of drinking water and swimming pools and to test homemade solutions prepared by dilution of household bleach during health emergency events such as during the current pandemic. Graphical abstract.


Subject(s)
Benzidines/chemistry , Chlorine/analysis , Chromogenic Compounds/chemistry , Colorimetry/methods , Disinfectants/analysis , Water/chemistry , Guidelines as Topic , Limit of Detection , Oxidation-Reduction , Spectrophotometry, Ultraviolet
20.
Virus Res ; 288: 198129, 2020 10 15.
Article in English | MEDLINE | ID: covidwho-719033

ABSTRACT

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.


Subject(s)
Betacoronavirus/genetics , CRISPR-Cas Systems , Clinical Laboratory Techniques/methods , Colorimetry/methods , Coronavirus Infections/diagnosis , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/methods , Pneumonia, Viral/diagnosis , COVID-19 , COVID-19 Testing , Clinical Laboratory Techniques/instrumentation , Colorimetry/instrumentation , Coronavirus Infections/virology , Endodeoxyribonucleases/chemistry , Humans , Molecular Diagnostic Techniques/instrumentation , Nucleic Acid Amplification Techniques/instrumentation , Pandemics , Pneumonia, Viral/virology , Point-of-Care Systems , Rheology , SARS-CoV-2 , Sensitivity and Specificity
SELECTION OF CITATIONS
SEARCH DETAIL
...