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1.
J Biomol Tech ; 32(3): 206-213, 2021 09.
Article in English | MEDLINE | ID: covidwho-1625607

ABSTRACT

A highly efficient, selective, and sensitive method for analysis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in raw sewage was developed and tested to illustrate basic characteristics of the procedure. The method uses reverse transcriptase (RT) loop mediated isothermal amplification (LAMP) in a quantitative application, RT qLAMP. The applicability of this procedure to detection of SARS-CoV-2 in clinical samples has been documented in many reports since early 2020. Basic LAMP characteristics depending on the multiple primer design that produce highly selective and sensitive target amplification virtually free of interferences in complex sample media make it ideal for application to target recognition in raw sewage. Three previously described primer sets targeting ORF1a, E- and N-gene regions were selected and tested to define method performance characteristics and performance for SARS-CoV-2 detection in raw sewage samples from a municipal sewage system serving > 600 000, between July and October, 2020. The virus was detected in all samples from each of three independent interceptors near their treatment terminus. Virus quantities varied significantly between samples and between primer targets within samples. Sewage sampling dates corresponded to relatively low COVID-19 incidence rates reported by the local service area health department. The limited number of samples and aggregating downstream sampling locations did not permit resolving concentration differences. The most significant finding was the ability of the RT qLAMP method to detect SARS-CoV-2 in the raw sewage samples directly without preprocessing to isolate or concentrate the virus or to extract and concentrate viral RNA.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , Molecular Diagnostic Techniques , Nucleic Acid Amplification Techniques , RNA, Viral/genetics , RNA-Directed DNA Polymerase , Sensitivity and Specificity , Sewage
2.
J Biomol Tech ; 32(3): 134-136, 2021 09.
Article in English | MEDLINE | ID: covidwho-1625529

ABSTRACT

At this writing, over 100 million people have tested positive for Corona Virus Disease-19 (COVID-19), and the global death toll from this disease has reached nearly 3 million. Despite the many tests currently available, we have not yet achieved the testing capacity needed to limit the spread of the virus and mitigate suffering worldwide. We have developed the One Hour COVID Test to address this challenge. Our test leverages an easy-to-use, commercially available oral swab kit for sample collection paired with a novel RNA processing protocol and a simple colorimetric assay that requires minimal equipment. The test can be easily scaled via automation and takes 1 h from sample collection to result.


Subject(s)
COVID-19 , Colorimetry , COVID-19 Testing , Humans , Molecular Diagnostic Techniques , Nucleic Acid Amplification Techniques , Prothrombin Time , RNA, Viral/genetics , SARS-CoV-2 , Sensitivity and Specificity
3.
BMC Public Health ; 22(1): 82, 2022 01 13.
Article in English | MEDLINE | ID: covidwho-1622227

ABSTRACT

BACKGROUND: Antigen tests for SARS-CoV-2 offer advantages over nucleic acid amplification tests (NAATs, such as RT-PCR), including lower cost and rapid return of results, but show reduced sensitivity. Public health organizations recommend different strategies for utilizing NAATs and antigen tests. We sought to create a framework for the quantitative comparison of these recommended strategies based on their expected performance. METHODS: We utilized a decision analysis approach to simulate the expected outcomes of six testing algorithms analogous to strategies recommended by public health organizations. Each algorithm was simulated 50,000 times in a population of 100,000 persons seeking testing. Primary outcomes were number of missed cases, number of false-positive diagnoses, and total test volumes. Outcome medians and 95% uncertainty ranges (URs) were reported. RESULTS: Algorithms that use NAATs to confirm all negative antigen results minimized missed cases but required high NAAT capacity: 92,200 (95% UR: 91,200-93,200) tests (in addition to 100,000 antigen tests) at 10% prevalence. Selective use of NAATs to confirm antigen results when discordant with symptom status (e.g., symptomatic persons with negative antigen results) resulted in the most efficient use of NAATs, with 25 NAATs (95% UR: 13-57) needed to detect one additional case compared to exclusive use of antigen tests. CONCLUSIONS: No single SARS-CoV-2 testing algorithm is likely to be optimal across settings with different levels of prevalence and for all programmatic priorities. This analysis provides a framework for selecting setting-specific strategies to achieve acceptable balances and trade-offs between programmatic priorities and resource constraints.


Subject(s)
COVID-19 , SARS-CoV-2 , Algorithms , COVID-19 Testing , Decision Support Techniques , Humans , Nucleic Acid Amplification Techniques , Sensitivity and Specificity
4.
Front Immunol ; 12: 732756, 2021.
Article in English | MEDLINE | ID: covidwho-1597480

ABSTRACT

Coronavirus disease 2019 (COVID-19), which started out as an outbreak of pneumonia, has now turned into a pandemic due to its rapid transmission. Besides developing a vaccine, rapid, accurate, and cost-effective diagnosis is essential for monitoring and combating the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its related variants on time with precision and accuracy. Currently, the gold standard for detection of SARS-CoV-2 is Reverse Transcription Polymerase Chain Reaction (RT-PCR), but it lacks accuracy, is time-consuming and cumbersome, and fails to detect multi-variant forms of the virus. Herein, we have summarized conventional diagnostic methods such as Chest-CT (Computed Tomography), RT-PCR, Loop Mediated Isothermal Amplification (LAMP), Reverse Transcription-LAMP (RT-LAMP), as well new modern diagnostics such as CRISPR-Cas-based assays, Surface Enhanced Raman Spectroscopy (SERS), Lateral Flow Assays (LFA), Graphene-Field Effect Transistor (GraFET), electrochemical sensors, immunosensors, antisense oligonucleotides (ASOs)-based assays, and microarrays for SARS-CoV-2 detection. This review will also provide an insight into an ongoing research and the possibility of developing more economical tools to tackle the COVID-19 pandemic.


Subject(s)
COVID-19 Testing/methods , COVID-19/diagnosis , Clinical Laboratory Techniques/methods , Molecular Diagnostic Techniques/methods , SARS-CoV-2/genetics , COVID-19/epidemiology , COVID-19/virology , Humans , Immunoassay/methods , Nucleic Acid Amplification Techniques/methods , Oligonucleotide Probes/genetics , Pandemics , RNA, Viral/genetics , Reproducibility of Results , Reverse Transcriptase Polymerase Chain Reaction/methods , SARS-CoV-2/physiology , Sensitivity and Specificity
5.
Sci Rep ; 11(1): 24234, 2021 12 20.
Article in English | MEDLINE | ID: covidwho-1585791

ABSTRACT

The main strategy for response and control of COVID-19 demands the use of rapid, accurate diagnostic tests aimed at the first point of health care. During the emergency, an increase in asymptomatic and symptomatic cases results in a great demand for molecular tests, which is promoting the development and application of rapid diagnostic technologies. In this study, we describe the development and evaluation of RT-LAMP to detect SARS-CoV-2 based on three genes (ORF1ab, M and N genes) in monoplex and triplex format. RT-LAMP assays were compared with the gold standard method RT-qPCR. The triplex format (RdRp, M and N genes) allowed obtaining comparable results with de RT-qPCR (RdRp and E genes), presented a sensitivity of 98.9% and a specificity of 97.9%, opening the opportunity to apply this method to detect SARS-CoV-2 at primary health-care centers.


Subject(s)
Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/methods , RNA, Viral/metabolism , SARS-CoV-2/isolation & purification , COVID-19/diagnosis , COVID-19/virology , COVID-19 Nucleic Acid Testing/methods , Coronavirus RNA-Dependent RNA Polymerase/genetics , Humans , Limit of Detection , Nasopharynx/virology , Nucleocapsid Proteins/genetics , Point-of-Care Systems , RNA, Viral/genetics , SARS-CoV-2/genetics , SARS-CoV-2/metabolism , Viral Matrix Proteins/genetics
6.
Mikrochim Acta ; 189(1): 34, 2021 12 23.
Article in English | MEDLINE | ID: covidwho-1588792

ABSTRACT

DNA is recognized as a powerful biomarker for clinical diagnostics because its specific sequences are closely related to the cause and development of diseases. However, achieving rapid, low-cost, and sensitive detection of short-length target DNA still remains a considerable challenge. Herein, we successfully combine the catalytic hairpin assembly (CHA) technique with capillary action to develop a new and cost-effective method, a target DNA- and pH-responsive DNA hydrogel-based capillary assay, for the naked eye detection of 24 nt short single-stranded target DNA. Upon contact of target DNA, three individual hairpin DNAs hybridize with each other to sufficiently amplify Y-shaped DNA nanostructures (Y-DNA) until they are completely consumed via CHA cycling reactions. Each arm of the resultant Y-DNA contains sticky ends with i-motif DNA structure-forming sequences that can be self-assembled in an acidic environment (pH 5.0) to form target DNA- and pH-responsive DNA hydrogels by means of i-motif DNA-driven crosslinking. When inserting a capillary tube in the resultant solution, the liquid level inside clearly reduces due to the decrease in capillary force induced by the gels. In this way, the developed assay demonstrates sensitive and quantitative detection, with a detection limit of approximately 10 pM of 24 nt short complementary DNA (cDNA) targeting SARS-CoV-2 RNA genes at room temperature within 1 h. The assay is further shown to successfully detect target cDNA in serum, and it is also applied to detect several types of target sequences. Requiring no analytic equipment, precise temperature control, or enzymatic reactions, the developed DNA hydrogel-based capillary assay has potential as a promising naked eye detection platform for target DNA in resource-limited clinical settings.


Subject(s)
Chemistry Techniques, Analytical/methods , DNA, Catalytic/chemistry , DNA, Complementary/analysis , Hydrogels/chemistry , RNA, Viral/genetics , SARS-CoV-2/chemistry , Capillary Action , Chemistry Techniques, Analytical/instrumentation , DNA, Catalytic/genetics , DNA, Complementary/genetics , Hydrogen-Ion Concentration , Inverted Repeat Sequences , Limit of Detection , Nucleic Acid Amplification Techniques , Nucleic Acid Hybridization
7.
Biosens Bioelectron ; 200: 113900, 2022 Mar 15.
Article in English | MEDLINE | ID: covidwho-1588212

ABSTRACT

The coronavirus disease 2019 (COVID-19) pandemic has highlighted the major shortcoming of healthcare systems globally in their inability to diagnose the disease rapidly and accurately. At present, the molecular approaches for diagnosing COVID-19 primarily use reverse transcriptase polymerase chain reaction (RT-PCR) to create and amplify cDNA from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA. Although molecular tests are reported to be specific, false negatives are quite common. Furthermore, literally all these tests require a step involving RNA isolation which does not make them point-of-care (POC) in the true sense. Here, we report a lateral flow strip-based RNA extraction and amplification-free nucleic acid test (NAT) for rapid diagnosis of positive COVID-19 cases at POC. The assay uses highly specific 6-carboxyfluorescein (6-FAM) and biotin labeled antisense oligonucleotides (ASOs) as probes those are designed to target N-gene sequence of SARS-CoV-2. Additionally, we utilized cysteamine capped gold-nanoparticles (Cyst-AuNPs) to augment the signal further for enhanced sensitivity. Without any large-stationary equipment and highly trained staffers, the entire sample-to-answer approach in our case would take less than 30 min from a patient swab sample collection to final diagnostic result. Moreover, when evaluated with 60 clinical samples and verified with an FDA-approved TaqPath RT-PCR kit for COVID-19 diagnosis, the assay obtained almost 99.99% accuracy and specificity. We anticipate that the newly established low-cost amplification-free detection of SARS-CoV-2 RNA will aid in the development of a platform technology for rapid and POC diagnosis of COVID-19 and other pathogens.


Subject(s)
Biosensing Techniques , COVID-19 , Metal Nanoparticles , COVID-19 Testing , Gold , Humans , Nucleic Acid Amplification Techniques , Point-of-Care Systems , RNA, Viral/genetics , SARS-CoV-2 , Sensitivity and Specificity
8.
Biosens Bioelectron ; 200: 113925, 2022 Mar 15.
Article in English | MEDLINE | ID: covidwho-1588208

ABSTRACT

The current COVID-19 pandemic caused by SARS-CoV-2 is raging, seriously threatening people's lives. The establishment of rapid and accurate pathogen detection technology is not only critical in this epidemic, but also a reminder that we must always be prepared for possible future outbreaks. Therefore, we developed a Palm Germ-Radar (PaGeR) device for rapid and simple detection of COVID-19 from extracted patient sample RNA by RT-LAMP. The whole procedure of rapid COVID-19 detection is based on 4 simple steps: inactivation, extraction, amplification, and detection. SARS-CoV-2 down to 1 copy/µL could be detected selectively with naked-eye. Three detection methods (colorimetric, fluorometric and lateral dipstick readout) could be performed in PaGeR instrument. By employing the PaGeR, we successfully detected SARS-CoV-2 in clinical RNA samples isolated from swab specimens. The results showed that 15 out of 17 COVID-19 patients were diagnosed as positive while all 55 normal samples were diagnosed as negative. Therefore, the developed PaGeR instrument can realize the detection of COVID-19 with easily visualized results, providing a promising instrument for rapid detection in the community as well as at home.


Subject(s)
Biosensing Techniques , COVID-19 , Humans , Molecular Diagnostic Techniques , Nucleic Acid Amplification Techniques , Pandemics , RNA, Viral/genetics , Radar , Reverse Transcription , SARS-CoV-2 , Sensitivity and Specificity
9.
Rev Med Virol ; 31(6): e2215, 2021 11.
Article in English | MEDLINE | ID: covidwho-1573992

ABSTRACT

The novel coronavirus disease-2019 (Covid-19) public health emergency has caused enormous loss around the world. This pandemic is a concrete example of the existing gap between availability of advanced diagnostics and current need for cost-effective methodology. The advent of the loop-mediated isothermal amplification (LAMP) assay provided an innovative tool for establishing a rapid diagnostic technique based on the molecular amplification of pathogen RNA or DNA. In this review, we explore the applications, diagnostic effectiveness of LAMP test for molecular diagnosis and surveillance of severe acute respiratory syndrome coronavirus 2. Our results show that LAMP can be considered as an effective point-of-care test for the diagnosis of Covid-19 in endemic areas, especially for low- and middle-income countries.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19/diagnosis , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/methods , Point-of-Care Testing/organization & administration , SARS-CoV-2/genetics , Bibliometrics , COVID-19/epidemiology , COVID-19/pathology , COVID-19/virology , COVID-19 Nucleic Acid Testing/economics , COVID-19 Nucleic Acid Testing/instrumentation , Humans , Molecular Diagnostic Techniques/economics , Molecular Diagnostic Techniques/instrumentation , Nucleic Acid Amplification Techniques/economics , Nucleic Acid Amplification Techniques/instrumentation , Point-of-Care Testing/economics , RNA, Viral/genetics , SARS-CoV-2/pathogenicity , Sensitivity and Specificity
10.
Intern Med ; 60(21): 3497-3501, 2021 Nov 01.
Article in English | MEDLINE | ID: covidwho-1572223

ABSTRACT

A 40-year-old woman developed a fever, sore throat, and cough. Coronavirus disease 2019 (COVID-19) was suspected; chest CT showed pan-lobular ground-glass opacity in the bilateral lower lobes suggesting viral pneumonia. Although a reverse transcription loop-mediated isothermal amplification (RT-LAMP) test for COVID-19 using a nasopharyngeal swab was negative, she was hospitalized and isolated because COVID-19 could not be ruled out. After admission, multiplex polymerase chain reaction (PCR) with the FilmArray Respiratory Panel 2.1 from a nasopharyngeal swab was positive for human coronavirus (HCoV) OC43. Therefore, the diagnosis was pneumonia due to HCoV-OC43. Multiplex PCR is useful for differentiating pneumonia due to COVID-19 from that due to other viral pneumonias.


Subject(s)
Coronavirus Infections/diagnosis , Coronavirus OC43, Human , Pneumonia, Viral/diagnosis , Adult , COVID-19 , Coronavirus OC43, Human/genetics , Diagnosis, Differential , Female , Humans , Molecular Diagnostic Techniques , Multiplex Polymerase Chain Reaction , Nucleic Acid Amplification Techniques , Pneumonia, Viral/virology
11.
PLoS One ; 16(12): e0260732, 2021.
Article in English | MEDLINE | ID: covidwho-1571987

ABSTRACT

The Loopamp SARS-CoV-2 Detection Kit is used for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Loop-mediated isothermal amplification (LAMP) is based on a measurement principle that can be used with a relatively simple device. Detection using this kit requires viral RNA extraction from samples with the QIAGEN QIAamp Viral Mini Kit (QIAGEN extraction) or the Loopamp Viral RNA Extraction Kit (Eiken extraction), which are recommended by the manufacturer. However, the efficacy of LAMP-based SARS-CoV-2 detection using these extraction methods has not been compared. In this study, we aimed to compare the results of genome extraction and detection from nasopharyngeal swab samples using the QIAGEN and Eiken extraction kits. The present study involved patients who presented to the Rinku General Medical Center with suspected COVID-19 (25 positive and 26 negative cases). A comparison of the results obtained using each extraction method with those obtained via PCR showed that the positive, negative, and overall concordance rates between QIAGEN extraction and PCR were 96.0% (24/25 samples), 100% (26/26), and 98.0% (50/51; κ = 0.96, 95% CI = 0.69-1.00), respectively. Results with Eiken extraction were also favorable, with positive, negative, and overall concordance rates of 88.0% (22/25), 100% (26/26), and 94.1% (48/51; κ = 0.88, 95% CI = 0.61-1.00), respectively. Favorable results were obtained using both QIAGEN and Eiken extraction kits. Since Eiken extraction can be completed in a few minutes, it enables prompt and reliable testing for SARS-CoV-2 detection.


Subject(s)
COVID-19/diagnosis , Molecular Diagnostic Techniques/methods , Nasopharynx/virology , Nucleic Acid Amplification Techniques/methods , RNA, Viral/genetics , SARS-CoV-2/isolation & purification , Humans , Prospective Studies , Reagent Kits, Diagnostic , SARS-CoV-2/genetics , Sensitivity and Specificity
12.
Biosens Bioelectron ; 199: 113865, 2022 Mar 01.
Article in English | MEDLINE | ID: covidwho-1560782

ABSTRACT

Rapid and sensitive detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical for early diagnostics and timely medical treatment of coronavirus disease 2019 (COVID-19). However, current detection methods typically rely on expensive and bulky instrumentation. Here, we developed a simple, sensitive, instrument-free, CRISPR-based diagnostics of SARS-CoV-2 using a self-contained microfluidic system. The microfluidic chip integrates isothermal amplification, CRISPR cleavage, and lateral flow detection in a single, closed microfluidic platform, enabling contamination-free, visual detection. To simplify the operation and transportation of the device, we lyophilized the CRISPR reagents in the reaction chamber and pre-stored the liquid solutions in blisters. We employed a low-cost, portable hand warmer to incubate the microfluidic chip without the need for electricity. The self-contained microfluidic system can detect down to 100 copies of SARS-CoV-2 RNA. Further, we clinically validated our method by detecting 24 COVID-19 clinical nasopharyngeal swab samples, achieving excellent sensitivity (94.1%), specificity (100%), and accuracy (95.8%). This simple, sensitive, and affordable microfluidic system represents a promising tool for point-of-care diagnostics of COVID-19 and other infectious diseases.


Subject(s)
Biosensing Techniques , COVID-19 , CRISPR-Cas Systems , Humans , Microfluidics , Nucleic Acid Amplification Techniques , RNA, Viral/genetics , SARS-CoV-2 , Sensitivity and Specificity
13.
J Nanobiotechnology ; 19(1): 348, 2021 Oct 30.
Article in English | MEDLINE | ID: covidwho-1555350

ABSTRACT

Viral infections are the most common among diseases that globally require around 60 percent of medical care. However, in the heat of the pandemic, there was a lack of medical equipment and inpatient facilities to provide all patients with viral infections. The detection of viral infections is possible in three general ways such as (i) direct virus detection, which is performed immediately 1-3 days after the infection, (ii) determination of antibodies against some virus proteins mainly observed during/after virus incubation period, (iii) detection of virus-induced disease when specific tissue changes in the organism. This review surveys some global pandemics from 1889 to 2020, virus types, which induced these pandemics, and symptoms of some viral diseases. Non-analytical methods such as radiology and microscopy also are overviewed. This review overlooks molecular analysis methods such as nucleic acid amplification, antibody-antigen complex determination, CRISPR-Cas system-based viral genome determination methods. Methods widely used in the certificated diagnostic laboratory for SARS-CoV-2, Influenza A, B, C, HIV, and other viruses during a viral pandemic are outlined. A comprehensive overview of molecular analytical methods has shown that the assay's sensitivity, accuracy, and suitability for virus detection depends on the choice of the number of regions in the viral open reading frame (ORF) genome sequence and the validity of the selected analytical method.


Subject(s)
Clinical Laboratory Techniques , Virus Diseases/diagnosis , Viruses/isolation & purification , Biosensing Techniques , COVID-19/diagnosis , COVID-19/epidemiology , Humans , Nucleic Acid Amplification Techniques , Pandemics , SARS-CoV-2/genetics , SARS-CoV-2/immunology , SARS-CoV-2/isolation & purification , Viral Proteins/genetics , Viral Proteins/immunology , Virus Diseases/epidemiology , Viruses/classification , Viruses/genetics , Viruses/immunology
14.
Arch Virol ; 166(6): 1599-1605, 2021 Jun.
Article in English | MEDLINE | ID: covidwho-1549420

ABSTRACT

Pigeon paramyxovirus-1 (PPMV-1) is a strain of Newcastle disease virus (NDV) that has adapted to infect pigeons and poses a constant threat to the commercial poultry industry. Early detection via rapid and sensitive methods, along with timely preventative and mitigating actions, is important for reducing the spread of PPMV-1. Here, we report the development of a TaqMan loop-mediated isothermal amplification assay (TaqMan-LAMP) for rapid and specific detection of PPMV-1 based on the F gene. This system makes use of six novel primers and a TaqMan probe that targets nine distinct regions of the F gene that are highly conserved among PPMV-1 isolates. The results showed that the limit of detection was 10 copies µL-1 for PPMV-1 cDNA and 0.1 ng for PPMV-1 RNA. The reaction was completed within 25 min and was thus faster than conventional RT-PCR. Moreover, no cross-reactions with similar viruses or with peste des petits ruminants virus (PPRV) or NDV LaSota vaccine strains were observed under the same conditions. To evaluate the applicability of the assay, the TaqMan-LAMP assay and a commercial RT-PCR assay were compared using 108 clinical samples, and the concordance rate between two methods was found to be 96.3%. The newly developed PPMV-1 TaqMan-LAMP assay can therefore be used for simple, efficient, rapid, specific, and sensitive diagnosis of PPMV-1 infections.


Subject(s)
Molecular Diagnostic Techniques/veterinary , Newcastle disease virus/genetics , Newcastle disease virus/isolation & purification , Nucleic Acid Amplification Techniques/veterinary , Animals , Columbidae , Feces/virology , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/methods , RNA, Viral , Sensitivity and Specificity , Time Factors
15.
Analyst ; 146(19): 6026-6034, 2021 Sep 27.
Article in English | MEDLINE | ID: covidwho-1545657

ABSTRACT

The ongoing outbreaks of the coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have resulted in unprecedented challenges to global health. To effectively contain the COVID-19 transmission, rapid tests for detecting existing SARS-CoV-2 infections and assessing virus spread are critical. To address the huge need for ever-increasing tests, we developed a facile all-in-one nucleic acid testing assay by combining Si-OH activated glass bead (aGB)-based viral RNA fast extraction and in situ colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) detection in a single tube. aGBs demonstrate a strong ability to capture viral RNA in a guanidinium-based lysis buffer, and the purified aGBs/RNA composite, without RNA elution step, could be directly used to perform RT-LAMP assay. The assay was well characterized by using a novel SARS-CoV-2-like coronavirus GX/P2V, and showed a limit of detection (LOD) of 15 copies per µL in simulated clinical samples within 50 min. We further demonstrated our assay by testing simulated SARS-CoV-2 pseudovirus samples, showing an LOD of 32 copies per µL and high specificity without cross-reactivity with the most closely related GX/P2V or host DNA/RNA. The all-in-one approach developed in this study has the potential as a simple, scalable, and time-saving alternative for point-of-care testing of SARS-CoV-2 in low-income regions, as well as a promising tool for at-home testing.


Subject(s)
COVID-19 , SARS-CoV-2 , Colorimetry , Humans , Molecular Diagnostic Techniques , Nucleic Acid Amplification Techniques , Point-of-Care Testing , RNA, Viral/genetics , Sensitivity and Specificity
16.
Biomed Pharmacother ; 144: 112353, 2021 Dec.
Article in English | MEDLINE | ID: covidwho-1544808

ABSTRACT

Almost 80% of people confronting COVID-19 recover from COVID-19 disease without any particular treatments. They experience heterogeneous symptoms; a wide range of respiratory symptoms, cough, dyspnea, fever, and viral pneumonia. However, some others need urgent intervention and special treatment to get rid of this widespread disease. So far, there isn't any unique drug for the potential treatment of COVID 19. However, some available therapeutic drugs used for other diseases seem beneficial for the COVID-19 treatment. On the other hand, there is a robust global concern for developing an efficient COVID-19 vaccine to control the COVID-19 pandemic sustainably. According to the WHO report, since 8 October 2021, 320 vaccines have been in progress. 194 vaccines are in the pre-clinical development stage that 126 of them are in clinical progression. Here, in this paper, we have comprehensively reviewed the most recent and updated information about coronavirus and its mutations, all the potential therapeutic approaches for treating COVID-19, developed diagnostic systems for COVID- 19 and the available COVID-19 vaccines and their mechanism of action.


Subject(s)
COVID-19 Vaccines/therapeutic use , COVID-19/drug therapy , COVID-19/prevention & control , Biosensing Techniques/methods , COVID-19/diagnosis , COVID-19 Nucleic Acid Testing/methods , Dexamethasone/therapeutic use , Glucocorticoids/therapeutic use , Humans , Molecular Diagnostic Techniques/methods , Mutation , Nucleic Acid Amplification Techniques/methods , Point-of-Care Testing , SARS-CoV-2/genetics , World Health Organization
17.
Biosens Bioelectron ; 197: 113771, 2022 Feb 01.
Article in English | MEDLINE | ID: covidwho-1536447

ABSTRACT

Due to the similar clinical symptoms of influenza (Flu) and coronavirus disease 2019 (COVID-19), there is a looming infection threat of concurrent Flu viruses and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this work, we introduce a customized isothermal amplification integrated lateral flow strip (LFS) that is capable performing duplex reverse transcription-recombinase polymerase amplification (RT-RPA) and colorimetric LFS in a sequential manner. With customized amplification primer sets targeted to SARS-CoV-2 (opening reading frame 1a/b and nucleoprotein genes) and Flu viruses (Flu A and Flu B), the platform allows the rapid and simultaneous visual screening of SARS-CoV-2 and Flu viruses (Flu A and Flu B) without cross reactivity, false positives, and false negatives. Moreover, it maximally eases the detection, reduces the detection time (1 h), and improves the assay performance to detect as low as 10 copies of the viral RNA. Its clinical application is powerfully demonstrated with 100% accuracy for evaluating 15 SARS-CoV-2-positive clinical samples, 10 Flu viruses-positive clinical samples, and 5 negative clinical samples, which were pre-confirmed by standard qRT-PCR. We envision this portable device can meet the increasing need of online monitoring the serious infectious diseases that substantially affects health care systems worldwide.


Subject(s)
Biosensing Techniques , COVID-19 , Influenza, Human , Humans , Influenza, Human/diagnosis , Nucleic Acid Amplification Techniques , SARS-CoV-2 , Sensitivity and Specificity
18.
Sci Rep ; 11(1): 22493, 2021 11 18.
Article in English | MEDLINE | ID: covidwho-1526101

ABSTRACT

The COVID-19 pandemic caused by SARS-CoV-2 has infected millions worldwide, therefore there is an urgent need to increase our diagnostic capacity to identify infected cases. Although RT-qPCR remains the gold standard for SARS-CoV-2 detection, this method requires specialised equipment in a diagnostic laboratory and has a long turn-around time to process the samples. To address this, several groups have recently reported the development of loop-mediated isothermal amplification (LAMP) as a simple, low cost and rapid method for SARS-CoV-2 detection. Herein we present a comparative analysis of three LAMP-based assays that target different regions of the SARS-CoV-2: ORF1ab RdRP, ORF1ab nsp3 and Gene N. We perform a detailed assessment of their sensitivity, kinetics and false positive rates for SARS-CoV-2 diagnostics in LAMP or RT-LAMP reactions, using colorimetric or fluorescent detection. Our results independently validate that all three assays can detect SARS-CoV-2 in 30 min, with robust accuracy at detecting as little as 1000 RNA copies and the results can be visualised simply by color changes. Incorporation of RT-LAMP with fluorescent detection further increases the detection sensitivity to as little as 100 RNA copies. We also note the shortcomings of some LAMP-based assays, including variable results with shorter reaction time or lower load of SARS-CoV-2, and false positive results in some experimental conditions and clinical saliva samples. Overall for RT-LAMP detection, the ORF1ab RdRP and ORF1ab nsp3 assays have faster kinetics for detection but varying degrees of false positives detection, whereas the Gene N assay exhibits no false positives in 30 min reaction time, which highlights the importance of optimal primer design to minimise false-positives in RT-LAMP. This study provides validation of the performance of LAMP-based assays as a rapid, highly sensitive detection method for SARS-CoV-2, which have important implications in development of point-of-care diagnostics for SARS-CoV-2.


Subject(s)
COVID-19 Nucleic Acid Testing , COVID-19 , Molecular Diagnostic Techniques , Nucleic Acid Amplification Techniques , SARS-CoV-2/genetics , Saliva/metabolism , Adult , COVID-19/diagnosis , COVID-19/genetics , COVID-19/metabolism , Female , Humans , Male , Saliva/virology
19.
Analyst ; 146(22): 6917-6923, 2021 Nov 08.
Article in English | MEDLINE | ID: covidwho-1526109

ABSTRACT

Nucleic acid amplification is a widely used diagnostic tool, although it requires a relatively time-consuming and complicated extraction step. To address this issue outside the laboratory, we investigated a sample preparation system and determined that a silica membrane and silica-coated beads are powerful tools for the extraction from raw samples: nucleic acids are kept in the silica membrane, retained during a single wash step, and released at the elution step. The eluent is appropriate for the quantitative real-time polymerase chain reaction (qPCR) and loop-mediated amplification (LAMP) assay in terms of purity and quantity. We also built an innovative equipment-free nucleic acid extraction squeeze system which requires less than 20 min. The sample with improved purity augments the specificity and sensitivity. This system is simple, user-friendly, low-cost, and equipment-free, thus making nucleic acid extraction more accessible and affordable for researchers and untrained users. Furthermore, when combined with the reverse-transcription quantitative real-time polymerase chain reaction method, the method will accelerate the detection of diseases. The same goes when combined with the LAMP assay, especially in developing countries.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , Molecular Diagnostic Techniques , Nucleic Acid Amplification Techniques , Pathology, Molecular , RNA, Viral/genetics , Sensitivity and Specificity
20.
BMC Infect Dis ; 21(1): 1162, 2021 Nov 17.
Article in English | MEDLINE | ID: covidwho-1523285

ABSTRACT

BACKGROUND: Current assays for detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) rely on time consuming, costly and laboratory based methods for virus isolation, purification and removing inhibitors. To address this limitation, we propose a simple method for testing RNA from nasopharyngeal swab samples that bypasses the RNA purification step. METHODS: In the current project, we have described two extraction-free reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays for the detection of SARS-CoV-2 by using E gene and RdRp gene as the targets. RESULTS: Here, results showed that reverse transcription loop-mediated isothermal amplification assays with 88.4% sensitive (95% CI: 74.9-96.1%) and 67.4% sensitive (95% CI: 51.5-80.9%) for E gene and RdRp gene, respectively. CONCLUSION: Without the need of RNA purification, our developed RT-LAMP assays for direct detection of SARS-CoV-2 from nasopharyngeal swab samples could be turned into alternatives to qRT-PCR for rapid screening.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , Molecular Diagnostic Techniques , Nucleic Acid Amplification Techniques , RNA, Viral/genetics , Reverse Transcription , Sensitivity and Specificity
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