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1.
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 , /genetics , Genome, Viral , Mutation
2.
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)
/methods , Molecular Diagnostic Techniques/standards , Nucleic Acid Amplification Techniques/standards , RNA, Viral/genetics , /genetics , Artifacts , /standards , DNA Primers/genetics , DNA Primers/metabolism , DNA Probes/genetics , DNA Probes/metabolism , Humans , Reagent Kits, Diagnostic , Sensitivity and Specificity
3.
ACS Sens ; 6(3): 1086-1093, 2021 03 26.
Article in English | MEDLINE | ID: covidwho-1120724

ABSTRACT

The outbreak of COVID-19 caused a worldwide public health crisis. Large-scale population screening is an effective means to control the spread of COVID-19. Reverse transcription-polymerase chain reaction (RT-qPCR) and serology assays are the most available techniques for SARS-CoV-2 detection; however, they suffer from either less sensitivity and accuracy or low instrument accessibility for screening. To balance the sensitivity, specificity, and test availability, here, we developed enhanced colorimetry, which is termed as a magnetic pull-down-assisted colorimetric method based on the CRISPR/Cas12a system (M-CDC), for SARS-CoV-2 detection. By this method, SARS-CoV-2 RNA from synthetic sequences and cultured viruses can be detected by the naked eye based on gold nanoparticle (AuNP) probes, with a detection limit of 50 RNA copies per reaction. With CRISPR/Cas12a-assisted detection, SARS-CoV-2 can be specifically distinguished from other closely related viruses. M-CDC was further used to analyze 41 clinical samples, whose performance was 95.12%, consistent with that of an approved Clinical RT-qPCR Diagnosis kit. The developed M-CDC method is not dependent on sophisticated instruments, which makes it potentially valuable to be applied for SARS-CoV-2 screening under poor conditions.


Subject(s)
/methods , RNA, Viral/analysis , /genetics , Bacterial Proteins , CRISPR-Associated Proteins , CRISPR-Cas Systems , Cell Line, Tumor , Colorimetry , DNA/chemistry , DNA Probes , Endodeoxyribonucleases , Gold/chemistry , Humans , Metal Nanoparticles/chemistry
4.
Mol Cell Probes ; 56: 101707, 2021 04.
Article in English | MEDLINE | ID: covidwho-1087137

ABSTRACT

BACKGROUND: DNA mismatches can affect the efficiency of PCR techniques if the intended target has mismatches in primer or probe regions. The accepted rule is that mismatches are detrimental as they reduce the hybridization temperatures, yet a more quantitative assessment is rarely performed. METHODS: We calculate the hybridization temperatures of primer/probe sets after aligning to SARS-CoV-2, SARS-CoV-1 and non-SARS genomes, considering all possible combinations of single, double and triple consecutive mismatches. We consider the mismatched hybridization temperature within a range of 5 ∘C to the fully matched reference temperature. RESULTS: We obtained the alignments of 19 PCR primers sets that were recently reported for the detection of SARS-CoV-2 and to 21665 SARS-CoV-2 genomes as well as 323 genomes of other viruses of the coronavirus family of which 10 are SARS-CoV-1. We find that many incompletely aligned primers become fully aligned to most of the SARS-CoV-2 when mismatches are considered. However, we also found that many cross-align to SARS-CoV-1 and non-SARS genomes. CONCLUSIONS: Some primer/probe sets only align substantially to most SARS-CoV-2 genomes if mismatches are taken into account. Unfortunately, by the same mechanism, almost 75% of these sets also align to some SARS-CoV-1 and non-SARS viruses. It is therefore recommended to consider mismatch hybridization for the design of primers whenever possible, especially to avoid undesired cross-reactivity.


Subject(s)
Base Pair Mismatch , DNA Primers/metabolism , DNA Probes/metabolism , DNA, Viral/metabolism , Nucleic Acid Hybridization , Polymerase Chain Reaction/methods , /genetics , Genome, Viral , Sequence Alignment , Temperature
5.
Biosens Bioelectron ; 177: 113005, 2021 Apr 01.
Article in English | MEDLINE | ID: covidwho-1033431

ABSTRACT

The coronavirus disease 2019 (COVID-19) pandemic has been a major public health challenge in 2020. Early diagnosis of COVID-19 is the most effective method to control disease spread and prevent further mortality. As such, a high-precision and rapid yet economic assay method is urgently required. Herein, we propose an innovative method to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) using isothermal amplification of nucleic acids on a mesh containing multiple microfluidic pores. Hybridization of pathogen DNA and immobilized probes forms a DNA hydrogel by rolling circle amplification and, consequently, blocks the pores to prevent fluid movement, as observed. Following optimization of several factors, including pore size, mesh location, and precision microfluidics, the limit of detection (LOD) for SARS-CoV-2 was determined to be 0.7 aM at 15-min incubation. These results indicate rapid, easy, and effective detection with a moderate-sized LOD of the target pathogen by remote point-of-care testing and without the requirement of any sophisticated device.


Subject(s)
/methods , Hydrogels/chemistry , Immobilized Nucleic Acids/chemistry , Point-of-Care Testing , /isolation & purification , Biosensing Techniques/economics , Biosensing Techniques/instrumentation , Biosensing Techniques/methods , /economics , DNA Probes/chemistry , DNA Probes/genetics , Equipment Design , Humans , Immobilized Nucleic Acids/genetics , Lab-On-A-Chip Devices , Limit of Detection , Molecular Diagnostic Techniques/economics , Molecular Diagnostic Techniques/instrumentation , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/economics , Nucleic Acid Amplification Techniques/instrumentation , Nucleic Acid Amplification Techniques/methods , /genetics
6.
Mikrochim Acta ; 187(11): 624, 2020 10 23.
Article in English | MEDLINE | ID: covidwho-888208

ABSTRACT

A label-free electrochemical strategy is proposed combining equivalent substitution effect with AuNPs-assisted signal amplification. According to the differences of S1 protein in various infectious bronchitis virus (IBV) strains, a target DNA sequence that can specifically recognize H120 RNA forming a DNA-RNA hybridized double-strand structure has been designed. Then, the residual single-stranded target DNA is hydrolyzed by S1 nuclease. Therefore, the content of target DNA becomes equal to the content of virus RNA. After equivalent coronavirus, the target DNA is separated from DNA-RNA hybridized double strand by heating, which can partly hybridize with probe 2 modified on the electrode surface and probe 1 on AuNPs' surface. Thus, AuNPs are pulled to the surface of the electrode and the abundant DNA on AuNPs' surface could adsorb a large amount of hexaammineruthenium (III) chloride (RuHex) molecules, which produce a remarkably amplified electrochemical response. The voltammetric signal of RuHex with a peak near - 0.28 V vs. Ag/AgCl is used as the signal output. The proposed method shows a detection range of 1.56e-9 to 1.56e-6 µM with the detection limit of 2.96e-10 µM for IBV H120 strain selective quantification detection, exhibiting good accuracy, stability, and simplicity, which shows a great potential for IBV detection in vaccine research and avian infectious bronchitis diagnosis. Graphical abstract.


Subject(s)
Biosensing Techniques/methods , Coronavirus Infections/virology , Coronavirus/isolation & purification , Electrochemical Techniques/methods , Infectious bronchitis virus/isolation & purification , Spike Glycoprotein, Coronavirus/chemistry , Animals , Biosensing Techniques/standards , Capsid Proteins/genetics , Chickens , Coronavirus/genetics , DNA Probes , Gold , In Situ Hybridization , Infectious bronchitis virus/genetics , Limit of Detection , Metal Nanoparticles/chemistry , RNA, Viral/genetics , RNA, Viral/isolation & purification , Species Specificity
7.
Emerg Microbes Infect ; 9(1): 1175-1179, 2020 Dec.
Article in English | MEDLINE | ID: covidwho-361278

ABSTRACT

Different primers/probes sets have been developed all over the world for the nucleic acid detection of SARS-CoV-2 by quantitative real time polymerase chain reaction (qRT-PCR) as a standard method. In our recent study, we explored the feasibility of droplet digital PCR (ddPCR) for clinical SARS-CoV-2 nucleic acid detection compared with qRT-PCR using the same primer/probe sets issued by Chinese Center for Disease Control and Prevention (CDC) targeting viral ORF1ab or N gene, which showed that ddPCR could largely minimize the false negatives reports resulted by qRT-PCR [Suo T, Liu X, Feng J, et al. ddPCR: a more sensitive and accurate tool for SARS-CoV-2 detection in low viral load specimens. medRxiv [Internet]. 2020;2020.02.29.20029439. Available from: https://medrxiv.org/content/early/2020/03/06/2020.02.29.20029439.abstract]. Here, we further stringently compared the performance of qRT-PCR and ddPCR for 8 primer/probe sets with the same clinical samples and conditions. Results showed that none of 8 primer/probe sets used in qRT-PCR could significantly distinguish true negatives and positives with low viral load (10-4 dilution). Moreover, false positive reports of qRT-PCR with UCDC-N1, N2 and CCDC-N primers/probes sets were observed. In contrast, ddPCR showed significantly better performance in general for low viral load samples compared to qRT-PCR. Remarkably, the background readouts of ddPCR are relatively lower, which could efficiently reduce the production of false positive reports.


Subject(s)
Betacoronavirus/genetics , Coronavirus Infections/diagnosis , Coronavirus Infections/virology , Multiplex Polymerase Chain Reaction , Pneumonia, Viral/diagnosis , Pneumonia, Viral/virology , Real-Time Polymerase Chain Reaction , DNA Primers , DNA Probes , Humans , Multiplex Polymerase Chain Reaction/methods , Pandemics , Real-Time Polymerase Chain Reaction/methods , Real-Time Polymerase Chain Reaction/standards , Sensitivity and Specificity , Viral Load
8.
Anal Chem ; 92(14): 9699-9705, 2020 07 21.
Article in English | MEDLINE | ID: covidwho-342681

ABSTRACT

A novel coronavirus (SARS-CoV-2) was recently identified in patients with acute respiratory disease and spread quickly worldwide. A specific and rapid diagnostic method is important for early identification. The reverse-transcription recombinase-aided amplification (RT-RAA) assay is a rapid detection method for several pathogens. Assays were performed within 5-15 min as a one-step single tube reaction at 39 °C. In this study, we established two RT-RAA assays for the S and orf1ab gene of SARS-CoV-2 using clinical specimens for validation. The analytical sensitivity of the RT-RAA assay was 10 copies for the S and one copy for the orf1ab gene per reaction. Cross-reactions were not observed with any of the other respiratory pathogens. A 100% agreement between the RT-RAA and real-time PCR assays was accomplished after testing 120 respiratory specimens. These results demonstrate that the proposed RT-RAA assay will be beneficial as it is a faster, more sensitive, and more specific tool for the detection of SARS-CoV-2.


Subject(s)
Betacoronavirus/chemistry , Betacoronavirus/genetics , Coronavirus Infections/diagnosis , Nucleic Acid Amplification Techniques/methods , Pneumonia, Viral/diagnosis , Polymerase Chain Reaction/methods , Bacteria/chemistry , Bacteria/genetics , Cross Reactions , DNA Probes , Genes, Viral , Humans , Pandemics , Plasmids , Polyproteins , Respiratory Tract Infections/microbiology , Respiratory Tract Infections/virology , Sensitivity and Specificity , Viral Proteins/genetics , Viruses/chemistry , Viruses/genetics
9.
Emerg Infect Dis ; 26(8): 1944-1946, 2020 08.
Article in English | MEDLINE | ID: covidwho-326416
10.
Mol Cell Probes ; 53: 101599, 2020 10.
Article in English | MEDLINE | ID: covidwho-306367

ABSTRACT

•Most of the COVID-19 cases in Nepal are in the Southern districts of Nepal bordering India with travel histories to India.•Very few positive cases of COVID-19 are detected in Nepal which could either be due to early national lockdown.•Low PCR positivity rates could also be due to inefficiency of the PCR methods.•Whole genomes of 93 clinical samples from COVID-19 patients were analyzed to find the primer and probe binding sites.•Mutations in probe binding sites were found which could impact PCR efficiency resulting in false negative results.


Subject(s)
Betacoronavirus/genetics , Coronavirus Infections/diagnosis , Coronavirus Infections/epidemiology , Genome, Viral , Pandemics , Pneumonia, Viral/diagnosis , Pneumonia, Viral/epidemiology , RNA, Viral/genetics , Reverse Transcriptase Polymerase Chain Reaction/standards , Betacoronavirus/classification , Betacoronavirus/pathogenicity , Clinical Laboratory Techniques/methods , Coronavirus Infections/transmission , Coronavirus Infections/virology , DNA Probes/standards , False Negative Reactions , Humans , India/epidemiology , Mutation , Nepal/epidemiology , Pneumonia, Viral/transmission , Pneumonia, Viral/virology , Reagent Kits, Diagnostic/standards , Severity of Illness Index , Transients and Migrants
11.
Clin Chem ; 66(8): 1047-1054, 2020 08 01.
Article in English | MEDLINE | ID: covidwho-209847

ABSTRACT

BACKGROUND: The current outbreak of SARS-CoV-2 has spread to almost every country with more than 5 million confirmed cases and over 300,000 deaths as of May 26, 2020. Rapid first-line testing protocols are needed for outbreak control and surveillance. METHODS: We used computational and manual designs to generate a suitable set of reverse transcription recombinase polymerase amplification (RT-RPA) primer and exonuclease probe, internally quenched (exo-IQ), sequences targeting the SARS-CoV-2 N gene. RT-RPA sensitivity was determined by amplification of in vitro transcribed RNA standards. Assay selectivity was demonstrated with a selectivity panel of 32 nucleic acid samples derived from common respiratory viruses. To validate the assay against full-length SARS-CoV-2 RNA, total viral RNA derived from cell culture supernatant and 19 nasopharyngeal swab samples (8 positive and 11 negative for SARS-CoV-2) were screened. All results were compared to established RT-qPCR assays. RESULTS: The 95% detection probability of the RT-RPA assay was determined to be 7.74 (95% CI: 2.87-27.39) RNA copies per reaction. The assay showed no cross-reactivity to any other screened coronaviruses or respiratory viruses of clinical significance. The developed RT-RPA assay produced 100% diagnostic sensitivity and specificity when compared to RT-qPCR (n = 20). CONCLUSIONS: With a run time of 15 to 20 minutes and first results being available in under 7 minutes for high RNA concentrations, the reported assay constitutes one of the fastest nucleic acid based detection methods for SARS-CoV-2 to date and may provide a simple-to-use alternative to RT-qPCR for first-line screening at the point of need.


Subject(s)
Betacoronavirus/genetics , Coronavirus Infections/diagnosis , Nucleic Acid Amplification Techniques/methods , Pneumonia, Viral/diagnosis , RNA, Viral/metabolism , Betacoronavirus/isolation & purification , Coronavirus Infections/virology , DNA Probes/chemistry , DNA Probes/metabolism , Exonucleases/metabolism , Humans , Pandemics , Pneumonia, Viral/virology , Point-of-Care Testing , Real-Time Polymerase Chain Reaction , Sensitivity and Specificity
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