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1.
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
2.
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
3.
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
4.
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
5.
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
6.
Sci Rep ; 11(1): 21385, 2021 11 01.
Article in English | MEDLINE | ID: covidwho-1493218

ABSTRACT

Shortages of reverse transcriptase (RT)-polymerase chain reaction (PCR) reagents and related equipment during the COVID-19 pandemic have demonstrated the need for alternative, high-throughput methods for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)-mass screening in clinical diagnostic laboratories. A robust, SARS-CoV-2 RT-loop-mediated isothermal amplification (RT-LAMP) assay with high-throughput and short turnaround times in a clinical laboratory setting was established and compared to two conventional RT-PCR protocols using 323 samples of individuals with suspected SARS-CoV-2 infection. Limit of detection (LoD) and reproducibility of the isolation-free SARS-CoV-2 RT-LAMP test were determined. An almost perfect agreement (Cohen's kappa > 0.8) between the novel test and two classical RT-PCR protocols with no systematic difference (McNemar's test, P > 0.05) was observed. Sensitivity and specificity were in the range of 89.5 to 100% and 96.2 to 100% dependent on the reaction condition and the RT-PCR method used as reference. The isolation-free RT-LAMP assay showed high reproducibility (Tt intra-run coefficient of variation [CV] = 0.4%, Tt inter-run CV = 2.1%) with a LoD of 95 SARS-CoV-2 genome copies per reaction. The established SARS-CoV-2 RT-LAMP assay is a flexible and efficient alternative to conventional RT-PCR protocols, suitable for SARS-CoV-2 mass screening using existing laboratory infrastructure in clinical diagnostic laboratories.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19/diagnosis , COVID-19/epidemiology , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/methods , Pandemics , Reverse Transcriptase Polymerase Chain Reaction/methods , SARS-CoV-2/genetics , COVID-19/virology , Genome, Viral , Humans , Infection Control/methods , Limit of Detection , Mass Screening/methods , RNA, Viral/genetics , RNA, Viral/isolation & purification , RNA-Directed DNA Polymerase/genetics , Reproducibility of Results , Reverse Transcription/genetics , Sensitivity and Specificity
7.
Microbiol Spectr ; 9(2): e0084621, 2021 10 31.
Article in English | MEDLINE | ID: covidwho-1476400

ABSTRACT

Isothermal amplification-based tests have been introduced as rapid, low-cost, and simple alternatives to real-time reverse transcriptase PCR (RT-PCR) tests for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection. The clinical performance of two isothermal amplification-based tests (Atila Biosystems iAMP coronavirus disease of 2019 [COVID-19] detection test and OptiGene COVID-19 direct plus RT-loop-mediated isothermal amplification [LAMP] test) was compared with that of clinical RT-PCR assays using different sampling strategies. A total of 1,378 participants were tested across 4 study sites. Compared with standard of care RT-PCR testing, the overall sensitivity and specificity of the Atila iAMP test for detection of SARS-CoV-2 were 76.2% and 94.9%, respectively, and increased to 88.8% and 89.5%, respectively, after exclusion of an outlier study site. Sensitivity varied based on the anatomic site from which the sample was collected. Sensitivity for nasopharyngeal sampling was 65.4% (range across study sites, 52.8% to 79.8%), for midturbinate was 88.2%, for saliva was 55.1% (range across study sites, 42.9% to 77.8%), and for anterior nares was 66.7% (range across study sites, 63.6% to 76.5%). The specificity for these anatomic collection sites ranged from 96.7% to 100%. Sensitivity improved in symptomatic patients (overall, 82.7%) and those with a higher viral load (overall, 92.4% for cycle threshold [CT] of ≤25). Sensitivity and specificity of the OptiGene direct plus RT-LAMP test, which was conducted at a single study site, were 25.5% and 100%, respectively. The Atila iAMP COVID test with midturbinate sampling is a rapid, low-cost assay for detecting SARS-CoV-2, especially in symptomatic patients and those with a high viral load, and could be used to reduce the risk of SARS-CoV-2 transmission in clinical settings. Variation of performance between study sites highlights the need for site-specific clinical validation of these assays before clinical adoption. IMPORTANCE Numerous SARS-CoV-2 detection assays have been developed and introduced into the market under emergency use authorizations (EUAs). EUAs are granted primarily based on small studies of analytic sensitivity and specificity with limited clinical validations. A thorough clinical performance evaluation of SARS-CoV-2 assays is important to understand the strengths, limitations, and specific applications of these assays. In this first large-scale multicentric study, we evaluated the clinical performance and operational characteristics of two isothermal amplification-based SARS-CoV-2 tests, namely, (i) iAMP COVID-19 detection test (Atila BioSystems, USA) and (ii) COVID-19 direct plus RT-LAMP test (OptiGene Ltd., UK), compared with those of clinical RT-PCR tests using different sampling strategies (i.e., nasopharyngeal, self-sampled anterior nares, self-sampled midturbinate, and saliva). An important specific use for these isothermal amplification-based, rapid, low-cost, and easy-to-perform SARS-CoV-2 assays is to allow for a safer return to preventive clinical encounters, such as cancer screening, particularly in low- and middle-income countries that have low SARS-CoV-2 vaccination rates.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19/diagnosis , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/methods , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification , Humans , Limit of Detection , Mass Screening , Nasopharynx/virology , Point-of-Care Systems , RNA, Viral/analysis , Reverse Transcriptase Polymerase Chain Reaction/methods , Specimen Handling , Viral Load
8.
Biosensors (Basel) ; 11(10)2021 Oct 13.
Article in English | MEDLINE | ID: covidwho-1470794

ABSTRACT

Loop-mediated isothermal amplification (LAMP) has been recently studied as an alternative method for cost-effective diagnostics in the context of the current COVID-19 pandemic. Recent reports document that LAMP-based diagnostic methods have a comparable sensitivity and specificity to that of RT-qPCR. We report the use of a portable Arduino-based LAMP-based amplification system assisted by pH microelectrodes for the accurate and reliable diagnosis of SARS-CoV-2 during the first 3 min of the amplification reaction. We show that this simple system enables a straightforward discrimination between samples containing or not containing artificial SARS-CoV-2 genetic material in the range of 10 to 10,000 copies per 50 µL of reaction mix. We also spiked saliva samples with SARS-CoV-2 synthetic material and corroborated that the LAMP reaction can be successfully monitored in real time using microelectrodes in saliva samples as well. These results may have profound implications for the design of real-time and portable quantitative systems for the reliable detection of viral pathogens including SARS-CoV-2.


Subject(s)
COVID-19/diagnosis , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/methods , SARS-CoV-2/genetics , COVID-19/virology , Coronavirus Nucleocapsid Proteins/genetics , Humans , Microelectrodes , Molecular Diagnostic Techniques/instrumentation , Nucleic Acid Amplification Techniques/instrumentation , Phosphoproteins/genetics , Point-of-Care Systems , RNA, Viral/analysis , RNA, Viral/metabolism , Reaction Time , SARS-CoV-2/isolation & purification , Saliva/virology
9.
Sci Rep ; 11(1): 20471, 2021 10 14.
Article in English | MEDLINE | ID: covidwho-1469980

ABSTRACT

Dual-labeled PNA probe used RT-LAMP molecular rapid assay targeting SARS-CoV-2 ORF1ab and N genes was developed, and the analytical, clinical performances for detection of SARS-CoV-2 RNA extracted from clinical nasopharyngeal swab specimens were evaluated in this study. Data showed that this assay is highly specific for SARS-CoV-2, and the absolute detection limit is 1 genomic copy per microliter of viral RNA which can be considered to be comparable to gold-standard molecular diagnostic method real-time reverse transcriptase PCR. Both clinical sensitivity and specificity against a commercial real-time RT-PCR assay were determined as identical. In conclusion, the PNA RT-LAMP assay showed high analytical and clinical accuracy which are identical to real-time RT-PCR which has been routinely used for the detection of SARS-CoV-2.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19/diagnosis , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/methods , RNA, Viral/analysis , SARS-CoV-2/isolation & purification , Coronavirus Nucleocapsid Proteins/genetics , Genes, Viral , Humans , Limit of Detection , Phosphoproteins/genetics , Polyproteins/genetics , RNA, Viral/genetics , SARS-CoV-2/genetics , Sensitivity and Specificity , Viral Proteins/genetics
10.
Microbiol Spectr ; 9(2): e0020521, 2021 10 31.
Article in English | MEDLINE | ID: covidwho-1467671

ABSTRACT

The extraordinary global demand for reagents and diagnostic instruments needed for timely detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has rapidly affected their availability. In order to meet diagnostic needs, it has been necessary to develop new diagnostic procedures. To date, molecular diagnostic tools have represented the gold standard for diagnosis of SARS-CoV-2 infection, and thus an alternative and real-time PCR system was required. To this aim, a molecular rapid test which works with direct real-time RT-PCR may be a relevant aid. In the present work, the accuracy, sensitivity, and specificity of the bKIT Virus Finder COVID-19 rapid molecular test by Hyris Ltd. was evaluated. Moreover, the influence of a different swab storage medium composition was examined relative to that of a routinely used comparator assay. The Hyris Ltd. assay showed an overall agreement of 100% with the comparator based on a panel consisting of 74 retrospective positive nasopharyngeal swabs (NPSs), collected either in universal transport medium (UTM) or using ESwab. No false-positive result was achieved on samples that previously tested negative. Cross-reactivity screening on microorganisms that commonly colonize the human upper respiratory tract was not detected, excluding the risk of false-positive results. Simultaneously, drugs frequently administered to cure respiratory diseases did not interfere with the analytical performance of the assay. Our results showed that the Hyris Ltd. bKIT Virus Finder COVID-19 is a reliable assay for rapid qualitative detection of SARS-CoV-2, providing the advantage of less complex and unambiguous interpretation of results. Indeed, skilled technicians are not required, and thus the Hyris system is suitable as a rapid and easy system for SARS-CoV-2 diagnosis. IMPORTANCE In order to overcome the increased demand for diagnostic tools for the timely detection of SARS-CoV-2 infection, we tested the bKIT Virus Finder COVID-19 molecular rapid test by Hyris Ltd. The new system was confirmed as a reliable assay for rapid SARS-CoV-2 detection, since sensitivity and specificity parameters were fully satisfied. Moreover, the bKIT Virus Finder COVID-19 provides the advantage of easy results interpretation, since skilled technicians are not required, and thus the Hyris system is a valuable SARS-CoV-2 rapid diagnosis system.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19/diagnosis , Molecular Diagnostic Techniques/methods , Humans , Limit of Detection , Nasopharynx/virology , Real-Time Polymerase Chain Reaction/methods , SARS-CoV-2/genetics , Specimen Handling
11.
Pan Afr Med J ; 40: 2, 2021.
Article in English | MEDLINE | ID: covidwho-1449273

ABSTRACT

In response to the current COVID-19 pandemic, numerous commercial assays have been developed for the detection of SARS-CoV-2 for use in the clinical diagnostic laboratories. To date, there is limited comparison of testing methods performed in different hospital laboratory sites. The aims of the study were to evaluate the analytical performance of Cepheid Xpert Xpress SARS-CoV-2 when compared to RT-PCR. This is a cross-sectional study. A total of 155 nasopharyngeal swabs were taken in duplicate from patients presenting with suspected COVID-19 to 8 hospitals in Oman. One swab was tested by the hospital laboratory and the duplicate swab was sent to the national Central Public Health Laboratory (CPHL) for testing. We compared the analytical performance of the commercially available point of care Cepheid Xpert Xpress SARS-CoV-2 assay which was used in the 8 different hospitals with assays including Liferiver, Sansure, TIB MOL BIOL, Kingfisher and COBAS 6800 by Roche which were performed at the CPHL. Testing of the duplicate swabs revealed excellent agreement of results with the viral loads of Ct values ranging from 16-43 for the E gene, 18-44 for the N gene and 17-44 for the ORF gene using the Liferiver assay. The overall sample sensitivity and specificity of the Cepheid Xpert Xpress SARS-CoV-2 assay were both 100% and there was 100% agreement across specimens. We conclude that the rapid GeneXpert and RT-PCR kits assessed in this study may be used for routine diagnostic testing of COVID-19 patients by experienced clinical microbiology diagnostic laboratories. Our results highlight the importance of rapid molecular testing at different sites within a country in a public health emergency.


Subject(s)
COVID-19 Testing/methods , COVID-19/diagnosis , Clinical Laboratory Techniques , SARS-CoV-2/isolation & purification , Cross-Sectional Studies , Humans , Laboratories, Hospital , Molecular Diagnostic Techniques/methods , Oman , Point-of-Care Testing , RNA, Viral/genetics , Sensitivity and Specificity , Specimen Handling , Viral Load
12.
Biosensors (Basel) ; 11(10)2021 Oct 02.
Article in English | MEDLINE | ID: covidwho-1444102

ABSTRACT

The COVID-19 pandemic has changed people's lives and has brought society to a sudden standstill, with lockdowns and social distancing as the preferred preventative measures. To lift these measurements and reduce society's burden, developing an easy-to-use, rapid, and portable system to detect SARS-CoV-2 is mandatory. To this end, we developed a portable and semi-automated device for SARS-CoV-2 detection based on reverse transcription loop-mediated isothermal amplification followed by a CRISPR/Cas12a reaction. The device contains a heater element mounted on a printed circuit board, a cooler fan, a proportional integral derivative controller to control the temperature, and designated areas for 0.2 mL Eppendorf® PCR tubes. Our system has a limit of detection of 35 copies of the virus per microliter, which is significant and has the capability of being used in crisis centers, mobile laboratories, remote locations, or airports to diagnose individuals infected with SARS-CoV-2. We believe the current methodology that we have implemented in this article is beneficial for the early screening of infectious diseases, in which fast screening with high accuracy is necessary.


Subject(s)
COVID-19/diagnosis , CRISPR-Cas Systems/genetics , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/methods , SARS-CoV-2/genetics , COVID-19/virology , COVID-19 Testing/instrumentation , COVID-19 Testing/methods , Humans , Limit of Detection , Molecular Diagnostic Techniques/instrumentation , Nucleic Acid Amplification Techniques/instrumentation , Point-of-Care Systems , RNA, Viral/analysis , RNA, Viral/metabolism , SARS-CoV-2/isolation & purification
13.
PLoS One ; 16(9): e0257350, 2021.
Article in English | MEDLINE | ID: covidwho-1435609

ABSTRACT

SARS-CoV-2 has spread worldwide and has become a global health problem. As a result, the demand for inputs for diagnostic tests rose dramatically, as did the cost. Countries with inadequate infrastructure experience difficulties in expanding their qPCR testing capacity. Therefore, the development of sensitive and specific alternative methods is essential. This study aimed to develop, standardize, optimize, and validate conventional RT-PCR targeting the N gene of SARS-CoV-2 in naso-oropharyngeal swab samples compared to qPCR. Using bioinformatics tools, specific primers were determined, with a product expected to be 519 bp. The reaction conditions were optimized using a commercial positive control, and the detection limit was determined to be 100 fragments. To validate conventional RT-PCR, we determined a representative sampling of 346 samples from patients with suspected infection whose diagnosis was made in parallel with qPCR. A sensitivity of 92.1% and specificity of 100% were verified, with an accuracy of 95.66% and correlation coefficient of 0.913. Under current Brazilian conditions, this method generates approximately 60% savings compared to qPCR costs. Conventional RT-PCR, validated herein, showed sufficient results for the detection of SARS-CoV-2 and can be used as an alternative for epidemiological studies and interspecies correlations.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19/diagnosis , Nose/virology , Nucleocapsid Proteins/genetics , Oropharynx/virology , Real-Time Polymerase Chain Reaction/methods , SARS-CoV-2/genetics , Adolescent , Brazil , COVID-19/virology , DNA Primers/genetics , Female , Humans , Male , Molecular Diagnostic Techniques/methods , RNA, Viral/genetics , Reference Standards , Sensitivity and Specificity , Specimen Handling/methods
15.
PLoS One ; 16(9): e0257563, 2021.
Article in English | MEDLINE | ID: covidwho-1416905

ABSTRACT

The COVID-19 pandemic caused by the SARS-CoV-2 is a serious health threat causing worldwide morbidity and mortality. Real-time reverse transcription PCR (RT-qPCR) is currently the standard for SARS-CoV-2 detection. Although various nucleic acid-based assays have been developed to aid the detection of SARS-CoV-2 from COVID-19 patient samples, the objective of this study was to develop a diagnostic test that can be completed in 30 minutes without having to isolate RNA from the samples. Here, we present an RNA amplification detection method performed using reverse transcription loop-mediated isothermal amplification (RT-LAMP) reactions to achieve specific, rapid (30 min), and sensitive (<100 copies) fluorescent detection in real-time of SARS-CoV-2 directly from patient nasopharyngeal swab (NP) samples. When compared to RT-qPCR, positive NP swab samples assayed by fluorescent RT-LAMP had 98% (n = 41/42) concordance and negative NP swab samples assayed by fluorescent RT-LAMP had 87% (n = 59/68) concordance for the same samples. Importantly, the fluorescent RT-LAMP results were obtained without purification of RNA from the NP swab samples in contrast to RT-qPCR. We also show that the fluorescent RT-LAMP assay can specifically detect live virus directly from cultures of both SARS-CoV-2 wild type (WA1/2020), and a SARS-CoV-2 B.1.1.7 (alpha) variant strain with equal sensitivity to RT-qPCR. RT-LAMP has several advantages over RT-qPCR including isothermal amplification, speed (<30 min), reduced costs, and similar sensitivity and specificity.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19/diagnosis , Diagnostic Tests, Routine/methods , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/methods , Real-Time Polymerase Chain Reaction/methods , SARS-CoV-2/isolation & purification , Humans , RNA, Viral/isolation & purification , Sensitivity and Specificity
16.
Theranostics ; 11(18): 9133-9161, 2021.
Article in English | MEDLINE | ID: covidwho-1410987

ABSTRACT

During an epidemic or pandemic, the primary task is to rapidly develop precise diagnostic approaches and effective therapeutics. Oligonucleotide aptamer-based pathogen detection assays and control therapeutics are promising, as aptamers that specifically recognize and block pathogens can be quickly developed and produced through simple chemical synthesis. This work reviews common aptamer-based diagnostic techniques for communicable diseases and summarizes currently available aptamers that target various pathogens, including the SARS-CoV-2 virus. Moreover, this review discusses how oligonucleotide aptamers might be leveraged to control pathogen propagation and improve host immune system responses. This review offers a comprehensive data source to the further develop aptamer-based diagnostics and therapeutics specific for infectious diseases.


Subject(s)
Aptamers, Nucleotide , Bacteria/genetics , Communicable Diseases/diagnosis , Molecular Diagnostic Techniques/methods , Viruses/genetics , Aptamers, Nucleotide/pharmacology , Biosensing Techniques , COVID-19 Testing/methods , Communicable Disease Control , Communicable Diseases/microbiology , Communicable Diseases/virology , Enzyme-Linked Immunosorbent Assay/methods , Host-Pathogen Interactions/immunology , Humans , SELEX Aptamer Technique , Virus Internalization
18.
J Clin Virol ; 127: 104383, 2020 06.
Article in English | MEDLINE | ID: covidwho-1385847

ABSTRACT

BACKGROUND: Numerous nucleic acid amplification assays have recently received emergency use authorization (EUA) for the diagnosis of SARS-CoV-2 infection, and there is a need to assess their test performance relative to one another. OBJECTIVES: The aim of this study was to compare the test performance of the Hologic Panther Fusion SARS-CoV-2 assay targeting two regions of open reading frame 1ab (ORF1ab) to a high complexity molecular-based, laboratory-developed EUA from Stanford Health Care (SHC) targeting the SARS-CoV-2 envelope (E) gene. STUDY DESIGN: We performed a diagnostic comparison study by testing nasopharyngeal samples on the two assays. Assay agreement was assessed by overall percent agreement and Cohen's kappa coefficient. RESULTS: A total of 184 nasopharyngeal samples were tested using the two assays, of which 180 showed valid results and were included for the comparative analysis. Overall percent agreement between the assays was 98.3 % (95 % confidence interval (CI) 95.2-99.7) and kappa coefficient was 0.97 (95 % CI 0.93-1.0). One sample was detected on the SHC laboratory developed test (LDT) and not on the Panther Fusion, and had a Ct of 35.9. Conversely, 2 samples were detected on the Panther Fusion and not on the LDT, and had Ct values of 37.2 and 36.6. CONCLUSION: The Panther Fusion SARS-CoV-2 assay and the SHC LDT perform similarly on clinical nasopharyngeal swab specimens. Other considerations, including reagent availability, turnaround time, labor requirements, cost and instrument throughput should guide the decision of which assay to perform.


Subject(s)
Coronavirus Infections/diagnosis , Molecular Diagnostic Techniques/methods , Molecular Diagnostic Techniques/standards , Pneumonia, Viral/diagnosis , Reagent Kits, Diagnostic/standards , Viral Envelope Proteins/isolation & purification , Betacoronavirus/genetics , COVID-19 , Coronavirus Envelope Proteins , Humans , Nasopharynx/virology , Pandemics , Reproducibility of Results , SARS-CoV-2 , Viral Envelope Proteins/genetics
19.
Jpn J Infect Dis ; 74(4): 325-332, 2021 Jul 21.
Article in English | MEDLINE | ID: covidwho-1380108

ABSTRACT

Studies describing reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay-based infection control strategies (LAMP-based ICSs) for coronavirus disease 2019 (COVID-19) are limited. We reviewed the medical records of cases in which RT-LAMP was performed. Standard ICSs and LAMP-based ICSs were implemented during the study period. The strategies were intended to impose longer periods of infection control precautions (ICPs) for specific patients, such as those with a history of exposure to COVID-19 patients and/or bilateral ground glass opacities (bGGO) on chest computed tomography (CT). Of 212 patients, which included 13 confirmed COVID-19 patients in the diagnostic cohort, exposure to COVID-19 patients (P <0.0001) and chest CT bGGO (P = 0.0022) were identified as significant predictors of COVID-19. In the 173 hospitalized patients in which the results of the first RT-LAMP were negative, the duration of ICPs was significantly longer in patients with exposure to COVID-19 and/or a high clinical index of suspicion and patients with bGGO than in the remaining patients (P = 0.00046 and P = 0.0067, respectively). Additionally, no confirmed COVID-19 cases indicating nosocomial spread occurred during the study period. Establishing a comprehensive system that combines rational LAMP-based ICSs with standard ICSs might be useful for preventing nosocomial spread.


Subject(s)
COVID-19 Testing/methods , COVID-19/diagnosis , COVID-19/virology , Infection Control/methods , Reverse Transcription/genetics , SARS-CoV-2/genetics , Adult , Clinical Laboratory Techniques/methods , Female , Hospitals , Humans , Male , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/methods , RNA, Viral/genetics , Sensitivity and Specificity , Tokyo , Young Adult
20.
Virol J ; 18(1): 178, 2021 08 30.
Article in English | MEDLINE | ID: covidwho-1379795

ABSTRACT

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


Subject(s)
COVID-19 Testing/methods , COVID-19/virology , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/methods , SARS-CoV-2/genetics , COVID-19/diagnosis , Humans , Reverse Transcription , SARS-CoV-2/isolation & purification , SARS-CoV-2/physiology , Sensitivity and Specificity
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