ABSTRACT
Loop-mediated isothermal amplification (LAMP) is widely used in detection of pathogenic microorganisms including SARS-CoV-2. However, the performance of LAMP assay needs further exploration in the emerging SARS-CoV-2 variants test. Here, we design serials of primers and select an optimal set for LAMP-based on SARS-CoV-2 N gene for a robust and visual assay in SARS-CoV-2 diagnosis. The limit of detectable template reaches 10 copies of N gene per 25 µL reaction at isothermal 58â within 40 min. Importantly, the primers for LAMP assay locate at 12 to 213 nt of N gene, a highly conservative region, which serves as a compatible test in emerging SARS-CoV-2 variants. Comparison to a commercial qPCR assay, this LAMP assay exerts the high viability in diagnosis of 41 clinical samples. Our study optimizes an advantageous LAMP assay for colorimetric detection of SARS-CoV-2 and emerging variants, which is hopeful to be a promising test in COVID-19 surveillance.
ABSTRACT
As the world has been facing several deadly virus crises, including Zika virus disease, Ebola virus disease, severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), and Coronavirus disease 2019 (COVID-19), lateral flow assays (LFAs), which require minimal equipment for point-of-care of viral infectious diseases, are garnering much attention. Accordingly, there is an increasing demand to reduce the time and cost required for manufacturing LFAs. The current study introduces an equipment-free method of salt-mediated immobilization of nucleic acids (SAIoNs) for LFAs. Compared to general DNA immobilization methods such as streptavidin-biotin, UV-irradiation, and heat treatment, our method does not require special equipment (e.g., centrifuge, UV-crosslinker, heating device); therefore, it can be applied in a resource-limited environment with reduced production costs. The immobilization process was streamlined and completed within 30 min. Our method improved the color intensity signal approximately 14 times compared to the method without using SAIoNs and exhibited reproducibility with the long-term storage stability. The proposed method can be used to detect practical targets (e.g., SARS-CoV-2) and facilitates highly sensitive and selective detection of target nucleic acids with multiplexing capability and without any cross-reactivity. This novel immobilization strategy provides a basis for easily and inexpensively developing nucleic acid LFAs combined with various types of nucleic acid amplification.
ABSTRACT
Rapid diagnostics is pivotal to curb SARS-CoV-2 transmission, and saliva has emerged as a practical alternative to naso/oropharyngeal (NOP) specimens. We aimed to develop a direct RT-LAMP (reverse transcription loop-mediated isothermal amplification) workflow for viral detection in saliva, and to provide more information regarding its potential in curbing COVID-19 transmission. Clinical and contrived specimens were used to optimize formulations and sample processing protocols. Salivary viral load was determined in symptomatic patients to evaluate the clinical performance of the test and to characterize saliva based on age, gender and time from onset of symptoms. Our workflow achieved an overall sensitivity of 77.2% (n = 90), with 93.2% sensitivity, 97% specificity, and 0.895 Kappa for specimens containing >102 copies/µL (n = 77). Further analyses in saliva showed that viral load peaks in the first days of symptoms and decreases afterwards, and that viral load is ~10 times lower in females compared to males, and declines following symptom onset. NOP RT-PCR data did not yield relevant associations. This work suggests that saliva reflects the transmission dynamics better than NOP specimens, and reveals gender differences that may reflect higher transmission by males. This saliva RT-LAMP workflow can be applied to track viral spread and, to maximize detection, testing should be performed immediately after symptoms are presented, especially in females.
ABSTRACT
The outbreak of corona virus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to a global pandemic. The high infectivity of SARS-CoV-2 highlights the need for sensitive, rapid and on-site diagnostic assays of SARS-CoV-2 with high-throughput testing capability for large-scale population screening. The current detection methods in clinical application need to operate in centralized labs. Though some on-site detection methods have been developed, few tests could be performed for high-throughput analysis. We here developed a gold nanoparticle-based visual assay that combines with CRISPR/Cas12a-assisted RT-LAMP, which is called Cas12a-assisted RT-LAMP/AuNP (CLAP) assay for rapid and sensitive detection of SARS-CoV-2. In optimal condition, we could detect down to 4 copies/µL of SARS-CoV-2 RNA in 40 min. by naked eye. The sequence-specific recognition character of CRISPR/Cas12a enables CLAP a superior specificity. More importantly, the CLAP is easy for operation that can be extended to high-throughput test by using a common microplate reader. The CLAP assay holds a great potential to be applied in airports, railway stations, or low-resource settings for screening of suspected people. To the best of our knowledge, this is the first AuNP-based colorimetric assay coupled with Cas12 and RT-LAMP for on-site diagnosis of COVID-19. We expect CLAP assay will improve the current COVID-19 screening efforts, and make contribution for control and mitigation of the pandemic.
ABSTRACT
Coronavirus Disease 2019 (COVID-19) manifests as extreme acute respiratory conditions caused by a novel beta coronavirus named severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) which is reported to be the seventh coronavirus to infect humans. Like other SARS-CoVs it has a large positive-stranded RNA genome. But, specific furin site in the spike protein, mutation prone and phylogenetically mess open reading frame1ab (Orf1ab) separates SARS-CoV-2 from other RNA viruses. Since the outbreak (February-March 2020), researchers, scientists, and medical professionals are inspecting all possible facts and aspects including its replication, detection, and prevention strategies. This led to the prompt identification of its basic biology, genome characterization, structural and expression based functional information of proteins, and utilization of this information in optimizing strategies to prevent its spread. This review summarizes the recent updates on the basic molecular biology of SARS-CoV-2 and prevention strategies undertaken worldwide to tackle COVID-19. This recent information can be implemented for the development and designing of therapeutics against SARS-CoV-2.
ABSTRACT
Virus-induced infection such as SARS-CoV-2 is a serious threat to human health and the economic setback of the world. Continued advances in the development of technologies are required before the viruses undergo mutation. The low concentration of viruses in environmental samples makes the detection extremely challenging; simple, accurate and rapid detection methods are in urgent need. Of all the analytical techniques, electrochemical methods have the established capabilities to address the issues. Particularly, the integration of nanotechnology would allow miniature devices to be made available at the point-of-care. This review outlines the capabilities of electrochemical methods in conjunction with nanotechnology for the detection of SARS-CoV-2. Future directions and challenges of the electrochemical biosensors for pathogen detection are covered including wearable and conformal biosensors, detection of plant pathogens, multiplexed detection, and reusable biosensors for on-site monitoring, thereby providing low-cost and disposable biosensors.