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1.
PLoS One ; 16(12): e0260850, 2021.
Article in English | MEDLINE | ID: covidwho-1613341

ABSTRACT

Novel strains of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) harboring nucleotide changes (mutations) in the spike gene have emerged and are spreading rapidly. These mutations are associated with SARS-CoV-2 transmissibility, virulence, or resistance to some neutralizing antibodies. Thus, the accurate detection of spike mutants is crucial for controlling SARS-CoV-2 transmission and identifying neutralizing antibody-resistance caused by amino acid changes in the receptor-binding domain. Here, we developed five SARS-CoV-2 spike gene primer pairs (5-SSG primer assay; 69S, 144S, 417S, 484S, and 570S) and verified their ability to detect nine key spike mutations (ΔH69/V70, T95I, G142D, ΔY144, K417T/N, L452R, E484K/Q, N501Y, and H655Y) using a Sanger sequencing-based assay. The 5-SSG primer assay showed 100% specificity and a conservative limit of detection with a median tissue culture infective dose (TCID50) values of 1.4 × 102 TCID50/mL. The accuracy of the 5-SSG primer assay was confirmed by next generation sequencing. The results of these two approaches showed 100% consistency. Taken together, the ability of the 5-SSG primer assay to accurately detect key SARS-CoV-2 spike mutants is reliable. Thus, it is a useful tool for detecting SARS-CoV-2 spike gene mutants in a clinical setting, thereby helping to improve the management of patients with COVID-19.


Subject(s)
Mutation , SARS-CoV-2/genetics , Sequence Analysis, RNA/methods , Spike Glycoprotein, Coronavirus/genetics , DNA Primers/genetics , High-Throughput Nucleotide Sequencing , Humans , Limit of Detection , Protein Domains , Spike Glycoprotein, Coronavirus/chemistry
2.
PLoS One ; 16(11): e0258263, 2021.
Article in English | MEDLINE | ID: covidwho-1511816

ABSTRACT

Clinical and surveillance testing for the SARS-CoV-2 virus relies overwhelmingly on RT-qPCR-based diagnostics, yet several popular assays require 2-3 separate reactions or rely on detection of a single viral target, which adds significant time, cost, and risk of false-negative results. Furthermore, multiplexed RT-qPCR tests that detect at least two SARS-CoV-2 genes in a single reaction are typically not affordable for large scale clinical surveillance or adaptable to multiple PCR machines and plate layouts. We developed a RT-qPCR assay using the Luna Probe Universal One-Step RT-qPCR master mix with publicly available primers and probes to detect SARS-CoV-2 N gene, E gene, and human RNase P (LuNER) to address these shortcomings and meet the testing demands of a university campus and the local community. This cost-effective test is compatible with BioRad or Applied Biosystems qPCR machines, in 96 and 384-well formats, with or without sample pooling, and has a detection sensitivity suitable for both clinical reporting and wastewater surveillance efforts.


Subject(s)
COVID-19/virology , Ribonuclease P/genetics , SARS-CoV-2/genetics , Waste Water/virology , DNA Primers/genetics , Humans , RNA, Viral/genetics , Real-Time Polymerase Chain Reaction/methods , Sensitivity and Specificity , Specimen Handling/methods , Wastewater-Based Epidemiological Monitoring
3.
Jpn J Infect Dis ; 74(5): 465-472, 2021 Sep 22.
Article in English | MEDLINE | ID: covidwho-1436361

ABSTRACT

Soon after the 2019 outbreak of coronavirus disease 2019 in Wuhan, China, a protocol for real-time RT-PCR assay detection of severe acute respiratory syndrome coronavirus (SARS-CoV-2) was established by the National Institute of Infectious Diseases (NIID) in Japan. The protocol used Charité's nucleocapsid (Sarbeco-N) and NIID nucleocapsid (NIID-N2) assays. During the following months, SARS-CoV-2 spread and caused a global pandemic, and various SARS-CoV-2 sequences were registered in public databases, such as the Global Initiative on Sharing All Influenza Data (GISAID). In this study, we evaluated the S2 assay (NIID-S2) that was newly developed to replace the Sarbeco-N assay and the performance of the NIID-N2 and NIID-S2 assays, referring to mismatches in the primer/probe targeted region. We found that the analytical sensitivity and specificity of the NIID-S2 set were comparable to those of the NIID-N2 assay, and the detection rate for clinical specimens was identical to that of the NIID-N2 assay. Furthermore, among the available sequences (approximately 192,000), the NIID-N2 and NIID-S2 sets had 2.6% and 1.2% mismatched sequences, respectively, although most of these mismatches did not affect the amplification efficiency, except the 3' end of the NIID-N2 forward primer. These findings indicate that the previously developed NIID-N2 assay is suitable for the detection of SARS-CoV-2 with support from the newly developed NIID-S2 set.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19/diagnosis , SARS-CoV-2/isolation & purification , Coronavirus Nucleocapsid Proteins/genetics , DNA Primers/genetics , Humans , Japan , Phosphoproteins/genetics , RNA, Viral/analysis , RNA, Viral/genetics , SARS-CoV-2/genetics , Sensitivity and Specificity , Spike Glycoprotein, Coronavirus/genetics
4.
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
5.
Genomics ; 113(5): 3174-3184, 2021 09.
Article in English | MEDLINE | ID: covidwho-1320193

ABSTRACT

As mutations in SARS-CoV-2 virus accumulate rapidly, novel primers that amplify this virus sensitively and specifically are in demand. We have developed a webserver named CoVrimer by which users can search for and align existing or newly designed conserved/degenerate primer pair sequences against the viral genome and assess the mutation load of both primers and amplicons. CoVrimer uses mutation data obtained from an online platform established by NGDC-CNCB (12 May 2021) to identify genomic regions, either conserved or with low levels of mutations, from which potential primer pairs are designed and provided to the user for filtering based on generalized and SARS-CoV-2 specific parameters. Alignments of primers and probes can be visualized with respect to the reference genome, indicating variant details and the level of conservation. Consequently, CoVrimer is likely to help researchers with the challenges posed by viral evolution and is freely available at http://konulabapps.bilkent.edu.tr:3838/CoVrimer/.


Subject(s)
DNA Primers/chemistry , SARS-CoV-2/genetics , Sequence Analysis, DNA/methods , Software , Conserved Sequence , DNA Primers/genetics , Genome, Viral , Mutation
6.
J Virol Methods ; 295: 114215, 2021 09.
Article in English | MEDLINE | ID: covidwho-1275556

ABSTRACT

BACKGROUND: This study aimed to evaluate the impact of four different reverse transcription quantitative PCR (RT-qPCR) master mixes on the performance of SARS-CoV-2 diagnostic PCRs using three primer/probe assays targeting the N gene (A, B and C). The dynamic range and lowest detected quantity was determined using a SARS-CoV-2 partial N gene RNA transcript dilution series (100,000-1 copy/µl) and verified using 72 nose and throat swabs, 29 of which tested positive for SARS-CoV-2 RNA. RESULTS: Assay C consistently detected the lowest quantity of partial N gene RNA transcript with all mastermixes. The Takara One Step PrimeScript™ III RT-PCR Kit mastermix enabled all primer pairs to detect the entire dynamic range evaluated, with the Qiagen Quantifast and Thermofisher TaqPath 1-Step kits also performing well. Sequences from all three primer/probe sets tested in this study (assay A, B and C) have 100 % homology to ≥97 % of the of SARS-CoV-2 sequences available up to 31st December 2020 (n = 291,483 sequences). CONCLUSIONS: This work demonstrates that specific assays (in this case assay C) can perform well in terms of dynamic range and lowest detected quantity regardless of the mastermix used. However we also show that, by choosing the most appropriate mastermix, poorer performing primer pairs are also able to detect all of the template dilutions investigated. This work increases the potential options when choosing assays for SARS-CoV-2 diagnosis and provides solutions to enable them to work with optimal analytical sensitivity.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , Coronavirus Nucleocapsid Proteins/genetics , SARS-CoV-2/isolation & purification , COVID-19/diagnosis , COVID-19 Nucleic Acid Testing/instrumentation , DNA Primers/genetics , Humans , Nose/virology , Pharynx/virology , Phosphoproteins/genetics , RNA, Viral/genetics , Reagent Kits, Diagnostic , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , SARS-CoV-2/genetics , Sensitivity and Specificity , Sequence Homology, Nucleic Acid
7.
Mol Cell Probes ; 58: 101748, 2021 08.
Article in English | MEDLINE | ID: covidwho-1272616

ABSTRACT

Covid-19 disease caused by SARS-CoV-2 is still being transmitted in developed and developing countries irrespective of healthcare setups. India with 1.3 billion people in the world is severely affected by Covid-19 with 11.3 million cases and 157 000 deaths so far. We have assessed the mismatches in WHO recommended rRT-PCR assays primer and probe binding regions against SARS-CoV-2 Indian genome sequences through in-silico bioinformatics analysis approach. Primers and probe sequences belonging to CN-CDC-ORF1ab from China and HKU-ORF1b from Hong Kong targeting ORF1ab gene while NIH-TH-N from Thailand, HKU-N from Hong Kong and US-CDCN-2 from USA targeting N genes displayed accurate matches (>98.3%) with the 2019 novel corona virus sequences from India. On the other hand, none of the genomic sequences displayed exact match with the primer/probe sequences belonging to Charité-ORF1b from Germany targeting ORF1ab gene. We think it will be worthwhile to release this information to the clinical and medical communities working in Indian Covid-19 frontline taskforce to tackle the recently emerging Covid-19 outbreaks as of March-2021.


Subject(s)
COVID-19/diagnosis , Computer Simulation , Genome, Viral/genetics , Mutation , RNA, Viral/genetics , SARS-CoV-2/genetics , COVID-19/epidemiology , COVID-19/virology , DNA Primers/genetics , DNA Probes/genetics , Disease Outbreaks , Humans , India/epidemiology , Open Reading Frames/genetics , Real-Time Polymerase Chain Reaction/methods , Reproducibility of Results , SARS-CoV-2/physiology , Sensitivity and Specificity
8.
Biomed Res Int ; 2021: 6653950, 2021.
Article in English | MEDLINE | ID: covidwho-1263958

ABSTRACT

The study is aimed at establishing the optimal parameters for RNA purification of pooled specimens, in SARS-CoV-2 assay. This research work evaluates the difference of extracted RNA purity of pooled samples with and without treatment with isopropyl alcohol and its effect on real-time RT-PCR. As per the protocol of the Indian Council of Medical Research (ICMR), 5 sample pools were analysed using qRT-PCR. A total of 100 pooled samples were selected for the study by mixing 50 µL of one COVID-19 positive nasopharyngeal/oropharyngeal (NP/OP) specimen and 50 µL each of 4 known negative specimens. Pool RNA was extracted using the column-based method, and 1 set of pooled extracted RNA was tested as such, while RNA of the second set was treated additionally with chilled isopropyl alcohol (modified protocol). Further, the purity of extracted RNA in both the groups was checked using Microvolume Spectrophotometers (Nanodrop) followed by RT-PCR targeting E-gene and RNaseP target. The results showed that the purity index of extracted RNA of untreated pooled specimens was inferior to isopropyl alcohol-treated templates, which was observed to be 85% sensitivity and 100% specificity. The average Cq (E gene) in the unpurified and purified pool RNA group was 34.66 and 31.48, respectively. The nanodrop data suggested that purified RNA concentration was significantly increased with an average value of 24.73 ± 1.49 ng/uL, which might be the reason for high sensitivity and specificity. Thus, this group testing of SARS-CoV-2 cases using pools of 5 individual samples would be the best alternative for saving molecular reagents, personnel time, and can increase the overall testing capacity. However, purity of RNA is one of the important determinants to procure unfailing results, thus, this additional purification step must be included in the protocol after RNA has been extracted using commercially available kit before performing qRT-PCR.


Subject(s)
COVID-19/diagnosis , Coronavirus Envelope Proteins/genetics , RNA, Viral/isolation & purification , Real-Time Polymerase Chain Reaction/methods , SARS-CoV-2/genetics , 2-Propanol/chemistry , Biomarkers/analysis , COVID-19/virology , DNA Primers/chemical synthesis , DNA Primers/genetics , Humans , Nasopharynx/virology , Oropharynx/virology , RNA, Viral/genetics , Real-Time Polymerase Chain Reaction/economics , Real-Time Polymerase Chain Reaction/standards , Reproducibility of Results , Sensitivity and Specificity
9.
Mol Cell Probes ; 58: 101744, 2021 08.
Article in English | MEDLINE | ID: covidwho-1253367

ABSTRACT

To increase the repertoire of PCR based laboratory developed tests (LDTs) for the detection of SARS-CoV-2, we describe a new multiplex assay (SORP), targeting the SARS-CoV-2's, Spike and ORF8 genes. The widely used human RNaseP internal control was modified to specifically co-amplify the RNaseP mRNA. The SORP triplex assay was tested on a cohort (n = 372; POS = 144/NEG = 228) of nasopharyngeal flocked swab (NPFS) specimens, previously tested for the presence of SARS-CoV-2 using a PCR assay targeting E and RdRp genes. The overall sensitivity and specificity of the SORP assay was: 99.31% (95% CI: 96.22-99.98%), 100.0% (95% CI: 98.4-100%) respectively. The SORP assay could also detect a panel of variants of concern (VOC) from the B1.1.7 (UK) and B1.351 (SA) lineage. In summary, access to a repertoire of new SARS-CoV-2 LDT's would assist diagnostic laboratories in developing strategies to overcome some of the testing issues encountered during high-throughput SARS-CoV-2 testing.


Subject(s)
COVID-19 Testing/methods , COVID-19/diagnosis , Clinical Laboratory Techniques/methods , Multiplex Polymerase Chain Reaction/methods , Reverse Transcriptase Polymerase Chain Reaction/methods , SARS-CoV-2/genetics , COVID-19/virology , DNA Primers/genetics , DNA Probes/genetics , Humans , Molecular Diagnostic Techniques/methods , Reproducibility of Results , Ribonuclease P/genetics , SARS-CoV-2/physiology , Sensitivity and Specificity , Spike Glycoprotein, Coronavirus/genetics , Viral Proteins/genetics
10.
J Virol Methods ; 295: 114197, 2021 09.
Article in English | MEDLINE | ID: covidwho-1240483

ABSTRACT

OBJECTIVES: The SARS-CoV-2 pandemic has created an unprecedented need for rapid large-scale diagnostic testing to prompt clinical and public health interventions. Currently, several quantitative reverse-transcription polymerase chain reaction (RT-qPCR) assays recommended by the World Health Organization are being used by clinical and public health laboratories and typically target regions of the RNA-dependent RNA polymerase (RdRp), envelope (E) and nucleocapsid (N) coding region. However, it is currently unclear if results from different tests are comparable. This study aimed to clarify the clinical performances of the primer/probe sets designed by US CDC and Charité/Berlin to help clinical laboratories in assay selection for SARS-CoV-2 routine detection. METHODS: We compared the clinical performances of the recommended primer/probe sets using one hundred nasopharyngeal swab specimens from patients who were clinically diagnosed with COVID-19. An additional 30 "pre-intervention screening" samples from patients who were not suspected of COVID-19 were also included in this study. We also performed sequence alignment between 31064 European SARS-CoV-2 and variants of concern genomes and the recommended primer/probe sets. RESULTS: The present study demonstrates substantial differences in SARS-CoV-2 RNA detection sensitivity among the primer/probe sets recommended by the World Health Organization especially for low-level viral loads. The alignment of thousands of SARS-CoV-2 sequences reveals that the genetic diversity remains relatively low at the primer/probe binding sites. However, multiple nucleotide mismatches might contribute to false negatives. CONCLUSION: An understanding of the limitations depending on the targeted genes and primer/probe sets may influence the selection of molecular detection assays by clinical laboratories.


Subject(s)
DNA Primers/genetics , Genome, Viral/genetics , SARS-CoV-2/isolation & purification , COVID-19/diagnosis , COVID-19/virology , COVID-19 Nucleic Acid Testing , Coronavirus/genetics , Humans , RNA, Viral/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , SARS-CoV-2/genetics , Sensitivity and Specificity , Sequence Alignment , Viral Load , Viral Proteins/genetics
11.
J Virol Methods ; 294: 114171, 2021 08.
Article in English | MEDLINE | ID: covidwho-1226315

ABSTRACT

Respiratory syncytial virus (RSV) is a common cause of acute respiratory disease worldwide, especially in young children. The World Health Organization (WHO) has initiated an RSV Surveillance Pilot program that aims to perform worldwide RSV surveillance, requiring the development of reliable and rapid molecular methods to detect and identify RSV. A duplex real-time RT-PCR assay developed for simultaneous detection of both A and B subtypes of RSV was included as part of this program. This duplex assay targeted a conserved region of the RSV polymerase gene and was validated for analytical sensitivity, specificity, reproducibility and clinical performance with a wide range of respiratory specimens. The assay was highly specific for RSV and did not react with non-RSV respiratory pathogens, including the SARS-CoV-2 virus.


Subject(s)
Molecular Diagnostic Techniques/methods , RNA, Viral/isolation & purification , Respiratory Syncytial Virus, Human/isolation & purification , Reverse Transcriptase Polymerase Chain Reaction/methods , DNA Primers/genetics , Humans , Limit of Detection , Nasopharynx/virology , RNA-Dependent RNA Polymerase/genetics , Reproducibility of Results , Ribonuclease P/genetics , SARS-CoV-2/genetics , Sensitivity and Specificity
12.
Sci Rep ; 11(1): 8988, 2021 04 26.
Article in English | MEDLINE | ID: covidwho-1203449

ABSTRACT

Rapid tests for active SARS-CoV-2 infections rely on reverse transcription polymerase chain reaction (RT-PCR). RT-PCR uses reverse transcription of RNA into complementary DNA (cDNA) and amplification of specific DNA (primer and probe) targets using polymerase chain reaction (PCR). The technology makes rapid and specific identification of the virus possible based on sequence homology of nucleic acid sequence and is much faster than tissue culture or animal cell models. However the technique can lose sensitivity over time as the virus evolves and the target sequences diverge from the selective primer sequences. Different primer sequences have been adopted in different geographic regions. As we rely on these existing RT-PCR primers to track and manage the spread of the Coronavirus, it is imperative to understand how SARS-CoV-2 mutations, over time and geographically, diverge from existing primers used today. In this study, we analyze the performance of the SARS-CoV-2 primers in use today by measuring the number of mismatches between primer sequence and genome targets over time and spatially. We find that there is a growing number of mismatches, an increase by 2% per month, as well as a high specificity of virus based on geographic location.


Subject(s)
DNA Primers/genetics , DNA Probes/genetics , Reverse Transcriptase Polymerase Chain Reaction/methods , SARS-CoV-2/genetics , Genome, Viral , Mutation
13.
Vopr Virusol ; 66(1): 17-28, 2021 03 07.
Article in Russian | MEDLINE | ID: covidwho-1121949

ABSTRACT

This review presents the basic principles of application of the loop-mediated isothermal amplification (LAMP) reaction for the rapid diagnosis of coronavirus infection caused by SARS-CoV-2. The basic technical details of the method, and the most popular approaches of specific and non-specific detection of amplification products are briefly described. We also discuss the first published works on the use of the method for the detection of the nucleic acid of the SARS-CoV-2 virus, including those being developed in the Russian Federation. For commercially available and published LAMP-based assays, the main analytical characteristics of the tests are listed, which are often comparable to those based on the method of reverse transcription polymerase chain reaction (RT-PCR), and in some cases are even superior. The advantages and limitations of this promising methodology in comparison to other methods of molecular diagnostics, primarily RT-PCR, are discussed, as well as the prospects for the development of technology for the detection of other infectious agents.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19/diagnosis , Molecular Diagnostic Techniques/standards , Nucleic Acid Amplification Techniques/standards , RNA, Viral/genetics , SARS-CoV-2/genetics , Artifacts , COVID-19/virology , COVID-19 Nucleic Acid Testing/standards , DNA Primers/genetics , DNA Primers/metabolism , DNA Probes/genetics , DNA Probes/metabolism , Humans , Reagent Kits, Diagnostic , Sensitivity and Specificity
14.
PLoS One ; 16(3): e0248042, 2021.
Article in English | MEDLINE | ID: covidwho-1115310

ABSTRACT

A newly identified coronavirus, designated as severe acute respiratory syndrome coronavirus 2 (SARS CoV-2), has spread rapidly from its epicenter in China to more than 150 countries across six continents. In this study, we have designed three reverse-transcription loop-mediated isothermal amplification (RT-LAMP) primer sets to detect the RNA-dependent RNA polymerase (RdRP), Envelope (E) and Nucleocapsid protein (N) genes of SARS CoV-2. For one tube reaction, the detection limits for five combination SARS CoV-2 LAMP primer sets (RdRP/E, RdRP/N, E/N, RdRP/E/N and RdRP/N/Internal control (actin beta)) were evaluated with a clinical nasopharyngeal swab sample. Among the five combination, the RdRP/E and RdRP/N/IC multiplex LAMP assays showed low detection limits. The sensitivity and specificity of the RT-LAMP assay were evaluated and compared to that of the widely used Allplex™ 2019-nCoV Assay (Seegene, Inc., Seoul, South Korea) and PowerChek™ 2019-nCoV Real-time PCR kit (Kogenebiotech, Seoul, South Korea) for 130 clinical samples from 91 SARS CoV-2 patients and 162 NP specimens from individuals with (72) and without (90) viral respiratory infections. The multiplex RdRP (FAM)/N (CY5)/IC (Hex) RT-LAMP assay showed comparable sensitivities (RdRP: 93.85%, N: 94.62% and RdRP/N: 96.92%) to that of the Allplex™ 2019-nCoV Assay (100%) and superior to those of PowerChek™ 2019-nCoV Real-time PCR kit (RdRP: 92.31%, E: 93.85% and RdRP/E: 95.38%).


Subject(s)
COVID-19/diagnosis , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/methods , SARS-CoV-2/genetics , COVID-19/genetics , COVID-19 Testing/methods , Clinical Laboratory Techniques/methods , Coronavirus Infections/virology , DNA Primers/genetics , Humans , Nucleocapsid Proteins/genetics , RNA, Viral/genetics , Real-Time Polymerase Chain Reaction/methods , Reverse Transcription/genetics , SARS-CoV-2/pathogenicity , Sensitivity and Specificity
15.
Front Cell Infect Microbiol ; 11: 613304, 2021.
Article in English | MEDLINE | ID: covidwho-1088903

ABSTRACT

Background: The emerging Coronavirus Disease-2019 (COVID-19) has challenged the public health globally. With the increasing requirement of detection for SARS-CoV-2 outside of the laboratory setting, a rapid and precise Point of Care Test (POCT) is urgently needed. Methods: Targeting the nucleocapsid (N) gene of SARS-CoV-2, specific primers, and probes for reverse transcription recombinase-aided amplification coupled with lateral flow dipstick (RT-RAA/LFD) platform were designed. For specificity evaluation, it was tested with human coronaviruses, human influenza A virus, influenza B viruses, respiratory syncytial virus, and hepatitis B virus, respectively. For sensitivity assay, it was estimated by templates of recombinant plasmid and pseudovirus of SARS-CoV-2 RNA. For clinical assessment, 100 clinical samples (13 positive and 87 negatives for SARS-CoV-2) were tested via quantitative reverse transcription PCR (RT-qPCR) and RT-RAA/LFD, respectively. Results: The limit of detection was 1 copies/µl in RT-RAA/LFD assay, which could be conducted within 30 min at 39°C, without any cross-reaction with other human coronaviruses and clinical respiratory pathogens. Compared with RT-qPCR, the established POCT assay offered 100% specificity and 100% sensitivity in the detection of clinical samples. Conclusion: This work provides a convenient POCT tool for rapid screening, diagnosis, and monitoring of suspected patients in SARS-CoV-2 endemic areas.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19/diagnosis , Real-Time Polymerase Chain Reaction/methods , SARS-CoV-2/genetics , COVID-19/virology , COVID-19 Nucleic Acid Testing/instrumentation , Coronavirus Nucleocapsid Proteins/genetics , DNA Primers/genetics , Humans , Phosphoproteins/genetics , Point-of-Care Testing , RNA, Viral/genetics , Real-Time Polymerase Chain Reaction/instrumentation , Recombinases/metabolism , Reverse Transcription , SARS-CoV-2/isolation & purification , Sensitivity and Specificity
16.
Sci Rep ; 11(1): 947, 2021 01 13.
Article in English | MEDLINE | ID: covidwho-1065932

ABSTRACT

In this paper, deep learning is coupled with explainable artificial intelligence techniques for the discovery of representative genomic sequences in SARS-CoV-2. A convolutional neural network classifier is first trained on 553 sequences from the National Genomics Data Center repository, separating the genome of different virus strains from the Coronavirus family with 98.73% accuracy. The network's behavior is then analyzed, to discover sequences used by the model to identify SARS-CoV-2, ultimately uncovering sequences exclusive to it. The discovered sequences are validated on samples from the National Center for Biotechnology Information and Global Initiative on Sharing All Influenza Data repositories, and are proven to be able to separate SARS-CoV-2 from different virus strains with near-perfect accuracy. Next, one of the sequences is selected to generate a primer set, and tested against other state-of-the-art primer sets, obtaining competitive results. Finally, the primer is synthesized and tested on patient samples (n = 6 previously tested positive), delivering a sensitivity similar to routine diagnostic methods, and 100% specificity. The proposed methodology has a substantial added value over existing methods, as it is able to both automatically identify promising primer sets for a virus from a limited amount of data, and deliver effective results in a minimal amount of time. Considering the possibility of future pandemics, these characteristics are invaluable to promptly create specific detection methods for diagnostics.


Subject(s)
DNA Primers/genetics , Deep Learning , Limit of Detection , Polymerase Chain Reaction/methods , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification
17.
Biotechniques ; 70(3): 167-174, 2021 03.
Article in English | MEDLINE | ID: covidwho-1063262

ABSTRACT

The ongoing pandemic has demonstrated the utility of widespread surveillance and diagnostic detection of the novel SARS-CoV-2. Reverse-transcription loop-mediated isothermal amplification (RT-LAMP) has enabled broader testing, but current LAMP tests only detect single targets and require separate reactions for controls. With flu season in the Northern Hemisphere, the ability to screen for multiple targets will be increasingly important, and the ability to include internal controls in RT-LAMP allows for improved efficiency. Here we describe multiplexed RT-LAMP with four targets (SARS-CoV-2, influenza A, influenza B, human RNA) in a single reaction using real-time and end point fluorescence detection. Such increased functionality of RT-LAMP will enable even broader adoption of this molecular testing approach and aid in the fight against this public health threat.


Subject(s)
Influenza A virus/genetics , Influenza B virus/genetics , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/methods , RNA, Viral/genetics , SARS-CoV-2/genetics , COVID-19 Nucleic Acid Testing/methods , DNA Primers/genetics , Fluorescence , RNA, Viral/analysis , Reverse Transcription , Sensitivity and Specificity
19.
PLoS One ; 15(12): e0244882, 2020.
Article in English | MEDLINE | ID: covidwho-1004475

ABSTRACT

SARS-CoV-2 testing is crucial to controlling the spread of this virus, yet shortages of nucleic acid extraction supplies and other key reagents have hindered the response to COVID-19 in the US. Several groups have described loop-mediated isothermal amplification (LAMP) assays for SARS-CoV-2, including testing directly from nasopharyngeal swabs and eliminating the need for reagents in short supply. Frequent surveillance of individuals attending work or school is currently unavailable to most people but will likely be necessary to reduce the ~50% of transmission that occurs when individuals are nonsymptomatic. Here we describe a fluorescence-based RT-LAMP test using direct nasopharyngeal swab samples and show consistent detection in clinically confirmed primary samples with a limit of detection (LOD) of ~625 copies/µl, approximately 100-fold lower sensitivity than qRT-PCR. While less sensitive than extraction-based molecular methods, RT-LAMP without RNA extraction is fast and inexpensive. Here we also demonstrate that adding a lysis buffer directly into the RT-LAMP reaction improves the sensitivity of some samples by approximately 10-fold. Furthermore, purified RNA in this assay achieves a similar LOD to qRT-PCR. These results indicate that high-throughput RT-LAMP testing could augment qRT-PCR in SARS-CoV-2 surveillance programs, especially while the availability of qRT-PCR testing and RNA extraction reagents is constrained.


Subject(s)
COVID-19 Nucleic Acid Testing , COVID-19 , Molecular Diagnostic Techniques , Nucleic Acid Amplification Techniques , RNA, Viral/genetics , SARS-CoV-2/genetics , COVID-19/diagnosis , COVID-19/genetics , DNA Primers/chemistry , DNA Primers/genetics , Humans , Limit of Detection , Nasopharynx/virology
20.
Expert Rev Mol Diagn ; 21(1): 119-129, 2021 01.
Article in English | MEDLINE | ID: covidwho-1003446

ABSTRACT

Introduction: With the ongoing SARS-CoV-2 pandemic, different articles have been published highlighting the superiority of droplet digital PCR (ddPCR) over the gold-standard reverse transcription PCR (RT-PCR) in SARS-CoV-2 detection. However, few studies have been reported on developing multiplex ddPCR assays for SARS-CoV-2 detection and their performance. This study shows steps on how to develop different ddPCR SAR-CoV-2 assays including higher order multiplex assays for SARS-CoV-2 detection and antiviral screening.Methods: Using multiple primer/probe sets, we developed, optimized, and analyzed the performance of simplex (1 target), duplex (2 targets), triplex probe mix (3 targets), and quadruplex (4 targets) SARS-CoV-2 ddPCR assays based on a two-color ddPCR detection system.Results: Results showed that the quadruplex assay had similar limits of detection and accuracy to the lower multiplex assays. Analyzing 94 clinical samples demonstrated that the ddPCR triplex probe mix assay had better sensitivity than the RT-qPCR assay. Additionally, the ddPCR multiplex assay showed that remdesivir could inhibit the growth of SARS-CoV-2 in vitro while another testing drug could not.Conclusion: Our research shows that developing multiplex ddPCR assays is possible by combing probe mix and amplitude-based multiplexing, which will help in developing multiplexed ddPCR assays for different SARS-CoV-2 applications.


Subject(s)
COVID-19 Nucleic Acid Testing , COVID-19/diagnosis , Multiplex Polymerase Chain Reaction/methods , SARS-CoV-2/isolation & purification , Antiviral Agents/pharmacology , DNA Primers/genetics , False Positive Reactions , Humans , Limit of Detection , Pandemics , RNA, Viral/isolation & purification , Real-Time Polymerase Chain Reaction/methods , Reproducibility of Results , Sensitivity and Specificity , Temperature , Viral Load/methods
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