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The Cq ratio-based RT-LAMP method. (A) The real-time quantitative fluorescent RT-LAMP result of the conserved and R203M primers over a range of templates from 10 3 −10 8 copies/mL. The amplification curves: wild-type plasmid using the conserved primers (solid lines), wild-type plasmid using the mutant primers (dotted lines), and R203M mutant plasmid using mutant primers (dashed lines). (B) The Cq ratio of the mutant plasmid and wild-type plasmid (left). The Cq ratio of the Delta variant and non-Delta variant (right). Box plots indicate the minimum and maximum value (whiskers), median (middle line), and 25th and 75th percentiles (box). The cutoff of the Cq ratio is set at 1.80. The statistical analysis was performed using the Wilcoxon/Kruskal-Wallis test, ***P < 0.001.
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The highly infectious Delta variant strain of SARS-CoV-2 remains globally dominant and undermines COVID-19 vaccines. Rapid detection of the Delta variant is crucial for the identification and quarantine of infected individuals. In this study, our aim was to design and validate a genotyping RT-LAMP method to detect Delta variants specifically. R203M...
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... developed the Cq ratio-based RT-LAMP method using R203M mutant-type and wild-type plasmids in triplicate experiments with different concentrations. As shown in from Figure 3A, the time gap between the R203M type and the wild type always maintained a sufficient difference in the RT-LAMP reaction with different concentrations of the template (from 10 3 copies/mL to 10 8 copies/mL). The mutant Cq value was divided by the conservative Cq value to obtain the final Cq ratio. ...
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... amplification templates of these two Cq values were the same type of plasmid template with the same concentration. The results and comparison for the Cq ratio of the two plasmid templates containing different alleles are shown in Figure 3B (left). A Cq ratio threshold of 1.80 was established (the threshold is the middle value of the ratio of the two groups). ...
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... Cq ratio threshold of 1.80 was established (the threshold is the middle value of the ratio of the two groups). Then, we performed the same serial dilution and amplification tests using RNA extracted from Delta variant samples and non-Delta variant samples, and the Cq ratios are shown in Figure 3B (right). The RNA of non-Delta variants contains different types sequenced as 20A, 20I (Alpha, V1) and 20H (Beta, V2), including common gene sequence types near the R203M mutation (Supplemental Table 3). ...
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The waves of COVID-19 infections driven by its variants continue to nullify the success we achieved through efficacious vaccines, social restrictions, testing and quarantine policies. This paper models the two major variants-driven waves by two sets of susceptible-infected-quarantined-recovered-vaccinated-deceased coupled dynamics that are modulate...
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... In the present study, we first examined the effectiveness of colorimetric RT-LAMP in detecting the globally widespread Delta variant of SARS-CoV-2, which is identified by the L452R SNP mutation in the RBD of the spike protein. Our research endorses previous studies which suggests that RT-LAMP could potentially serve as a rapid and cost-efficient diagnostic method for detecting SARS-CoV-2 29-33 and identifying the Delta variant 34,35 . ...
The rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has triggered global difficulties for both individuals and economies, with new variants continuing to emerge. The Delta variant of SARS-CoV-2 remains most prevalent worldwide, and it affects the efficacy of coronavirus disease 2019 (COVID-19) vaccination. Expedited testing to detect the Delta variant of SARS-CoV-2 and monitor viral transmission is necessary. This study aimed to develop and evaluate a colorimetric reverse-transcription loop-mediated isothermal amplification (RT-LAMP) technique targeting the L452R mutation in the S gene for the specific detection of the Delta variant. In the test, positivity was indicated as a color change from purple to yellow. The assay’s 95% limit of detection was 57 copies per reaction for the L452R (U1355G)-specific standard plasmid. Using 126 clinical samples, our assay displayed 100% specificity, 97.06% sensitivity, and 98.41% accuracy in identifying the Delta variant of SARS-CoV-2 compared to real-time RT-PCR. To our knowledge, this is the first colorimetric RT-LAMP assay that can differentiate the Delta variant from its generic SARS-CoV-2, enabling it as an approach for studying COVID-19 demography and facilitating proper effective control measure establishment to fight against the reemerging variants of SARS-CoV-2 in the future.
... The RT-LAMP assay for SARS-CoV-2 was completely concordant with standard RT-qPCR. The test differentiates between delta variants and non-delta variants, and the area under the curve was 1.00 [48] ( Fig. 1). The microdevice integrated RT-LAMP to purify RNA via microbead and real-time monitor RT-LAMP with a miniaturized optical detector (J. ...
The identification of influenza viruses and SARS-CoV-2 has garnered increasing attention due of their longstanding global menace to human life and health. The point-of-care test is a potential approach for identifying influenza viruses and SARS-CoV-2 in clinical settings, leading to timely discovery, documentation, and treatment. The primary difficulties encountered with conventional detection techniques for influenza viruses and SARS-CoV-2 are the limited or inadequate ability to identify the presence of the viruses, the lack of speed, precision, accuracy, sensitivity, and specificity, often resulting in a failure to promptly notify disease control authorities. Recently, point-of-care test methods, along with nucleic acid amplification, optics, electrochemistry, lateral/vertical flow, and minimization, have been demonstrated the characteristics of reliability, sensitivity, specificity, stability, and portability. A point-of-care test offers promising findings in the early detection of influenza viruses and SARS-CoV-2 in both scientific research and practical use. In this review, we will go over the principles, advantages, limitations, and real-world applications of point-of-care diagnostics. The significance of constraints of detection, throughput, sensitivity, and specificity in the analysis of clinical samples in settings with restricted resources is underscored. This discussion concludes with their prospects and challenges.
... The overall mutation analysis detected the presence of the D614G variant in both respiratory samples; this is the main protein S variant observed worldwide, with increased infectivity, efficiency, [21][22][23] and high mortality. [24] A N protein variant detected in respiratory samples is R203M, this change in N protein already occurred 2,247,623 times (21.78% of all samples with N sequence) in 203 countries. ...
... In addition, this variant may have increased adaptability and enhanced infectious capacity. [23] There is evidence that supports a selection process in SARS-CoV-2 viral populations, mediated by selective pressure, giving rises to multiple variants present in the same host or among different hosts (intra-host and inter-host variability). [7] This process is also dependent on the tissue in which the viral infection is located. ...
Background: Genomic sequencing data from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have provided important
information on the emergence of variants of interest. Some research has focused on determining intra-host variability and evolution, and
others have focused on viral genetic variability in different tissues, as well as implications for virus infectivity and pathogenicity. However,
the inter-host variability of SARS-CoV-2 in respiratory compartments and its implications has not yet been explained. To determine a possible
pattern of tissue‑specific inter‑host variability at different levels of the respiratory system associating this information with clinically important
characteristics. Methods: In this work, we analyzed the inter-host variability of SARS-CoV-2 using the Shannon entropy method with genomic
data from NCBI database. Fifty-seven SARS-CoV-2 viral genomic sequences were included in the study. Oronasopharyngeal (n = 30) and
tracheal aspirate (n = 27) sample sequences were analyzed by bioinformatics methods to determine the viral variability. Results: Different
patterns of variability were observed in SARS-CoV-2 in the respiratory tract, which may indicate tissue compartmentalization phenomena.
Genetic changes found in the N y S gene could affect the detection and spread of the virus. The overall analysis of the viral genomes identifies
the variants of worldwide distribution. The viral diversity found in the different parts of the respiratory tract was high and uniformly distributed
throughout the genome. Conclusions: Tracking inter-host variations in SARS-CoV-2 could help to understand the effects of viral evolution,
provide information for diagnosis, drug, and vaccine design.
... Compared with other nucleic acid amplification methods, LAMP technology is among the most developed isothermal amplification techniques and exhibits several advantages, including simple operation and low cost [22]. Currently, the major methods for the detection of LAMP products are agarose gel electrophoresis, real-time PCR or turbidimetric assays [23][24][25]. However, most of these methods require specialised personnel, long assay times (>1 h) and expensive instrumentation. ...
Acinetobacter baumannii (A. baumannii) is among the main pathogens that cause nosocomial infections. The ability to rapidly and accurately detect A. baumannii and its drug resistance is essential for blocking secondary infections and guiding treatments. In this study, we reported a nucleic acid fluorescent lateral flow assay (NFLFA) to identify A. baumannii and carbapenem-resistant A. baumannii (CRAB) in a rapid and quantitative manner by integrating loop-mediated isothermal amplification (LAMP) and silica–based multilayered quantum dot nanobead tag (Si@MQB). First, a rapid LAMP system was established and optimised to support the effective amplification of two bacterial genes in 35 min. Then, the antibody-modified Si@MQB was introduced to capture the two kinds of amplified DNA sequences and simultaneously detect them on two test lines of a LFA strip, which greatly improved the detection sensitivity and stability of the commonly used AuNP-based nucleic acid LFA. With these strategies, the established LAMP-NFLFA achieved detection limits of 199 CFU/mL and 287 CFU/mL for the RecA (house-keeping gene) and blaOXA-23 (drug resistance gene) genes, respectively, within 43 min. Furthermore, the assay exhibited good repeatability and specificity for detecting target pathogens in real complex specimens and environments; thus, the proposed assay undoubtedly provides a promising and low-cost tool for the on-site monitoring of nosocomial infections.
... 65 Sequencing for diagnosis involves obtaining the whole genome information of SARS-CoV-2 through metagenomic sequencing, [66][67][68] including mainstream NGS Illumina and nanopore sequencing. 69,70 Its advantage is its high specificity and ability to simultaneously identify other respiratory viruses, compensating for the high false positives of RT-PCR. 16 Additionally, new molecular detection technologies are being developed or are expected to be used for SARS-CoV-2 nucleic acid detection. ...
Since the development of Sanger sequencing in 1977, sequencing technology has played a pivotal role in molecular biology research by enabling the interpretation of biological genetic codes. Today, nanopore sequencing is one of the leading third-generation sequencing technologies. With its long reads, portability, and low cost, nanopore sequencing is widely used in various scientific fields including epidemic prevention and control, disease diagnosis, and animal and plant breeding. Despite initial concerns about high error rates, continuous innovation in sequencing platforms and algorithm analysis technology has effectively addressed its accuracy. During the coronavirus disease (COVID-19) pandemic, nanopore sequencing played a critical role in detecting the severe acute respiratory syndrome coronavirus-2 virus genome and containing the pandemic. However, a lack of understanding of this technology may limit its popularization and application. Nanopore sequencing is poised to become the mainstream choice for preventing and controlling COVID-19 and future epidemics while creating value in other fields such as oncology and botany. This work introduces the contributions of nanopore sequencing during the COVID-19 pandemic to promote public understanding and its use in emerging outbreaks worldwide. We discuss its application in microbial detection, cancer genomes, and plant genomes and summarize strategies to improve its accuracy.
... In particular, RPA has key features, such as a simple primer design, no initial heating step, low and constant operational temperature, multiplex capability, and enhanced tolerance to inhibitors [21,22]. Two rapid molecular studies using a multi-step procedure for the detection of Delta-specific mutation R203M and three other VOC-specific mutations showed high accuracy in clinical samples taking about 60 min [23,24]. Similarly, a single-copy sensitive assay for the Delta mutation L452R using a two-step procedure showed a reaction time of 75 min [25]. ...
Rapid molecular testing for severe acute respiratory coronavirus 2 (SARS-CoV-2) variants may contribute to the development of public health measures, particularly in resource-limited areas. Reverse transcription recombinase polymerase amplification using a lateral flow assay (RT-RPA-LF) allows rapid RNA detection without thermal cyclers. In this study, we developed two assays to detect SARS-CoV-2 nucleocapsid (N) gene and Omicron BA.1 spike (S) gene-specific deletion–insertion mutations (del211/ins214). Both tests had a detection limit of 10 copies/µL in vitro and the detection time was approximately 35 min from incubation to detection. The sensitivities of SARS-CoV-2 (N) RT-RPA-LF by viral load categories were 100% for clinical samples with high (>9015.7 copies/µL, cycle quantification (Cq): < 25) and moderate (385.5–9015.7 copies/µL, Cq: 25–29.9) viral load, 83.3% for low (16.5–385.5 copies/µL, Cq: 30–34.9), and 14.3% for very low (<16.5 copies/µL, Cq: 35–40). The sensitivities of the Omicron BA.1 (S) RT-RPA-LF were 94.9%, 78%, 23.8%, and 0%, respectively, and the specificity against non-BA.1 SARS-CoV-2-positive samples was 96%. The assays seemed more sensitive than rapid antigen detection in moderate viral load samples. Although implementation in resource-limited settings requires additional improvements, deletion–insertion mutations were successfully detected by the RT-RPA-LF technique.
... Facing new viruses' variants, generous time and professional technicians are required to construct probe molecules of new virus. Other genetic methods, such as gene sequencing, reverse transcription loop mediated isothermal amplification (RT-LAMP) or CRISPR (clustered regular interspaced short palindromic repeats) diagnosis, also have some inherent shortcomings [95][96][97][98][99]. In the past decades, SERS has proved itself to be a highly selective tool in the field of virus diagnosis. ...
... By analyzing the corresponding Raman vibrational spectra, the molecular structure of the substance to be measured can be analyzed. In particular, it can be used for Genome sequencing High accuracy and reliability, gold standard for identifying variants Low sensitivity, specialized laboratories and technical skills, be time-consuming and expensive [95] RT-qPCR High sensitivity, reliability primer/probe mismatches, expensive equipment [94,112] RT-LAMP High specificity, portability, be rapid and costly low tolerance to highly variable target sequences, limitations of a single reaction [96,97] CRISPR High specificity and sensitivity, experimental simplicity, versatility ...
Surface-Enhanced Raman Scattering (SERS) technology, as a powerful tool to identify molecular species by collecting molecular spectral signals at the single-molecule level, has achieved substantial progresses in the fields of environmental science, medical diagnosis, food safety, and biological analysis. As deepening research is delved into SERS sensing, more and more high-performance or multifunctional SERS substrate materials emerge, which are expected to push Raman sensing into more application fields. Especially in the field of biological analysis, intrinsic and extrinsic SERS sensing schemes have been widely used and explored due to their fast, sensitive and reliable advantages. Herein, recent developments of SERS substrates and their applications in biomolecular detection (SARS-CoV-2 virus, tumor etc.), biological imaging and pesticide detection are summarized. The SERS concepts (including its basic theory and sensing mechanism) and the important strategies (extending from nanomaterials with tunable shapes and nanostructures to surface bio-functionalization by modifying affinity groups or specific biomolecules) for improving SERS biosensing performance are comprehensively discussed. For data analysis and identification, the applications of machine learning methods and software acquisition sources in SERS biosensing and diagnosing are discussed in detail. In conclusion, the challenges and perspectives of SERS biosensing in the future are presented.
... To date, multiple variants of SARS-CoV-2 have emerged, and the rapid and sensitive detection of the variants is crucial. 32,33 We investigated the detection performance of SADQD-based ICA for SARS-CoV-2 variants by detecting the recombinant proteins from several major mutant strains. Recombinant proteins of Alpha, Beta, Delta, and Omicron variants were obtained from Beijing Sino Biological Inc. ...
Timely, accurate, and rapid diagnosis of SARS-CoV-2 is a key factor in controlling the spread of the epidemic and guiding treatments. Herein, a flexible and ultrasensitive immunochromatographic assay (ICA) was proposed based on a colorimetric/fluorescent dual-signal enhancement strategy. We first fabricated a highly stable dual-signal nanocomposite (SADQD) by continuously coating one layer of 20 nm AuNPs and two layers of quantum dots onto a 200 nm SiO2 nanosphere to provide strong colorimetric signals and enhanced fluorescence signals. Two kinds of SADQD with red and green fluorescence were conjugated with spike (S) antibody and nucleocapsid (N) antibody, respectively, and used as dual-fluorescence/colorimetric tags for the simultaneous detection of S and N proteins on one test line of ICA strip, which can not only greatly reduce the background interference and improve the detection accuracy but also achieve a higher colorimetric sensitivity. The detection limits of the method for target antigens via colorimetric and fluorescence modes were as low as 50 and 2.2 pg/mL, respectively, which were 5 and 113 times more sensitive than those from the standard AuNP-ICA strips, respectively. This biosensor will provide a more accurate and convenient way to diagnose COVID-19 in different application scenarios.
... In addition, the technique identifies the presence/absence of the virus without specifically recognizing variants. To our knowledge, to date, only one group has developed an RT-LAMP assay for specific discrimination of the SARS-CoV-2 Delta variant detecting the R203M mutation [36]. Lastly, CRISPR is a novel, rapid, low-cost, and highly sensitive assay. ...
Since the emergence of the COVID-19 pandemic in December 2019, the SARS-CoV-2 virus continues to evolve into many variants emerging around the world. To enable regular surveillance and timely adjustments in public health interventions, it is of the utmost importance to accurately monitor and track the distribution of variants as rapidly as possible. Genome sequencing is the gold standard for monitoring the evolution of the virus, but it is not cost-effective, rapid and easily accessible. We have developed a microarray-based assay that can distinguish known viral variants present in clinical samples by simultaneously detecting mutations in the Spike protein gene. In this method, the viral nucleic acid, extracted from nasopharyngeal swabs, after RT-PCR, hybridizes in solution with specific dual-domain oligonucleotide reporters. The domains complementary to the Spike protein gene sequence encompassing the mutation form hybrids in solution that are directed by the second domain (“barcode” domain) at specific locations on coated silicon chips. The method utilizes characteristic fluorescence signatures to unequivocally differentiate, in a single assay, different known SARS-CoV-2 variants. In the nasopharyngeal swabs of patients, this multiplex system was able to genotype the variants which have caused waves of infections worldwide, reported by the WHO as being of concern (VOCs), namely Alpha, Beta, Gamma, Delta and Omicron variants.
... The target binds to the receptor, and fluorescent and quantum dot markers are used to detect the optical reaction so that the signal value can be detected on the test line or control line. LFA has the characteristics of portability, low cost and efficiency, which have allowed it to become an ideal choice for point-of-care testing (POCT), and has been widely used in the rapid detection of various targets, such as bacteria, viruses, parasites, mycotoxins, and with the continuous innovation and development of LFA technology, a photothermal test strip assay that combines test strips with a portable photothermal card reader was established for the sensitive, rapid and quantitative detection of residues of food hazards Charlermroj et al., 2021;Wang Y. et al., 2022b;Yang J. et al., 2022). An LFA based on LAMP to identify tissue of cattle origin has been developed with high specificity and sensitivity (Jawla et al., 2021). ...
The number of words: 4645, the number of figures: 4, the number of tables: 1The outbreak of COVID-19 in December 2019 caused a global pandemic of acute respiratory disease, and with the increasing virulence of mutant strains and the number of confirmed cases, this has resulted in a tremendous threat to global public health. Therefore, an accurate diagnosis of COVID-19 is urgently needed for rapid control of SARS-CoV-2 transmission. As a new molecular biology technology, loop-mediated isothermal amplification (LAMP) has the advantages of convenient operation, speed, low cost and high sensitivity and specificity. In the past two years, rampant COVID-19 and the continuous variation in the virus strains have demanded higher requirements for the rapid detection of pathogens. Compared with conventional RT–PCR and real-time RT–PCR methods, genotyping RT-LAMP method and LAMP plus peptide nucleic acid (PNA) probe detection methods have been developed to correctly identified SARS-CoV-2 variants, which is also why LAMP technology has attracted much attention. LAMP detection technology combined with lateral flow assay, microfluidic technology and other sensing technologies can effectively enhance signals by nucleic acid amplification and help to give the resulting output in a faster, more convenient and user-friendly way. At present, LAMP plays an important role in the detection of SARS-CoV-2.
