Recent developments and trends of automatic nucleic acid detection systems.

Nucleic acid detection, widely used in clinical diagnosis, biological analysis, and environmental monitoring, is of great significance for disease diagnosis and basic research. With the outbreak of COVID-19, the demand for fast and high-throughput nucleic acid detection from large numbers of samples has increased sharply. Automated nucleic acid detection systems can meet these needs, and also play important roles in disease screening and infectious disease prevention and control. In this review, we introduce and compare the current mainstream nucleic acid automatic detection instruments and equipment, then discuss the future demands of nucleic acid detection.

Fully Automated CRISPR-LAMP Platform for SARS-CoV-2 Delta and Omicron Variants.

Integrated clustered regularly interspaced short palindromic repeat (CRISPR)-loop-mediated amplification (LAMP) technology is of great importance in CRISPR-based diagnostic systems, which urgently needs to be developed to improve diagnostic accuracy. A labor-free, contamination-free, and fully automated droplet manipulation platform for the CRISPR-LAMP technology has not been developed before. Herein, we propose a fully automated CRISPR-LAMP platform, which can precisely manipulate the CRISPR-LAMP droplet and perform combined reactions with high sensitivity and specificity. SARS-CoV-2 Spike T478K, D614G, P681R, and P681H mutations, typical point mutations of B.1.617.2 (Delta) and Omicron variants, are monitored with this platform with a detection limit of 102 copies/µL. Allele discrimination between the mutants and wild type is significant with the designed one/two-mismatch CRISPR RNA (crRNA) at a limit of 102 copies/µL. Chemically synthesized and modified crRNAs greatly increase the CRISPR-LAMP signal, which advance the wide application. Combined with the previously developed RdRp CRISPR-LAMP assay, clinical results showed that Spike T478K and P681H can discriminate the mutant type form the wild type with 70% (49.66-85.50%, 95% confidence interval) and 78% (57.27-90.62%, 95% confidence interval) sensitivity, respectively, and 100% specificity (51.68-100%, 95% confidence interval), and the RdRp target can detect SARS-CoV-2 strains with 85% sensitivity (65.39-95.14%, 95% confidence interval) and 100% specificity (51.68-100%, 95% confidence interval). We believe that this automatic digital microfluid (DMF) system can advance the integrated CRISPR-LAMP technology with higher stability, sensitivity, and practicability, also for other CRISPR-associated diagnostic platforms.

Exposure Risk to Medical Staff in a Nasopharyngeal Swab Sampling Cabin under Four Different Ventilation Strategies

Medical staff working in a nasopharyngeal swab sampling cabin are exposed to a higher exposure risk of COVID-19. In this study, computational fluid dynamics (CFD) are used to evaluate the exposure risk to medical staff in a nasopharyngeal swab sampling cabin of Chinese customs under four different ventilation strategies, i.e., multiple supply fans ventilation (MSFV), multiple exhaust fans ventilation (MEFV), single exhaust fan and outer windows closed ventilation (SEFV), and single exhaust fan and outer windows opened ventilation (SEFV-W). The impact of physical partitions on exposure risk is also discussed. The results show that MSFV performed best in reducing exposure risk. No significant difference was found between MEFV and SEFV. SEFV-W performed better than SEFV with a ventilation rate of 10–50 L/(s∙Person), while it performed worse with a ventilation rate of 50–90 L/(s∙Person). The exposure risk to medical staff did not decrease linearly with the increase in the ventilation flow rate under the four ventilation strategies. For MSFV, the installation of partitions is conducive to the reduction in the exposure risk. This study is expected to provide some guidance for ventilation designs in sampling cabins.

Recent developments and trends of automatic nucleic acid detection systems

Nucleic acid detection, widely used in clinical diagnosis, biological analysis, and environmental monitoring, is of great significance for disease diagnosis and basic research. With the outbreak of COVID-19, the demand for fast and high-throughput nucleic acid detection from large numbers of samples has increased sharply. Automated nucleic acid detection systems can meet these needs, and also play important roles in disease screening and infectious disease prevention and control. In this review, we introduce and compare the current mainstream nucleic acid automatic detection instruments and equipment, then discuss the future demands of nucleic acid detection.

Universally Stable and Precise CRISPR-LAMP Detection Platform for Precise Multiple Respiratory Tract Virus Diagnosis Including Mutant SARS-CoV-2 Spike N501Y.

Nowadays, rapid and accurate diagnosis of respiratory tract viruses is an urgent need to prevent another epidemic outbreak. To overcome this problem, we have developed a clustered, regularly interspaced short palindromic repeats (CRISPR) loop mediated amplification (LAMP) technology to detect influenza A virus, influenza B virus, respiratory syncytial A virus, respiratory syncytial B virus, and severe acute respiratory syndrome coronavirus 2, including variants of concern (B.1.1.7), which utilized CRISPR-associated protein 12a (Cas12a) to advance LAMP technology with the sensitivity increased 10 times. To reduce aerosol contamination in CRISPR-LAMP technology, an uracil-DNA-glycosylase-reverse transcription-LAMP system was also developed which can effectively remove dUTP-incorporated LAMP amplicons. In vitro Cas12a cleavage reaction with 28 crRNAs showed that there were no position constraints for Cas12a/CRISPR RNA (crRNA) recognition and cleavage in LAMP amplicons, and even the looped position of LAMP amplicons could be effectively recognized and cleaved. Wild-type or spike N501Y can be detected with a limit of detection of 10 copies/µL (wild-type) even at a 1% ratio level on the background (spike N501Y). Combining UDG-RT-LAMP technology, CRISPR-LAMP design, and mutation detection design, we developed a CRISPR-LAMP detection platform that can precisely diagnose pathogens with better stability and significantly improved point mutation detection efficiency.

On-site analysis of COVID-19 on the surfaces in wards.

SARS-Cov-2 has erupted across the globe, and confirmed cases of COVID-19 pose a high infection risk. Infected patients typically receive their treatment in specific isolation wards, where they are confined for at least 14 days. The virus may contaminate any surface of the room, especially frequently touched surfaces. Therefore, surface contamination in wards should be monitored for disease control and hygiene purposes. Herein, surface contamination in the ward was detected on-site using an RNA extraction-free rapid method. The whole detection process, from surface sample collection to readout of the detection results, was finished within 45 min. The nucleic acid extraction-free method requires minimal labor. More importantly, the tests were performed on-site and the results were obtained almost in real-time. The test confirmed that 31 patients contaminated seven individual sites. Among the sampled surfaces, the electrocardiogram fingertip presented a 72.7% positive rate, indicating that this surface is an important hygiene site. Meanwhile, the bedrails showed the highest correlation with other surfaces, so should be detected daily. Another surface with high contamination risk was the door handle in the bathroom. To our knowledge, we present the first on-site analysis of COVID-19 surface contamination in wards. The results and applied technique provide a potential further reference for disease control and hygiene suggestions.

Development of a Sensitive Immunochromatographic Method Using Lanthanide Fluorescent Microsphere for Rapid Serodiagnosis of COVID-19.

The SARS-CoV-2 infection that caused the COVID-19 pandemic quickly spread worldwide within two months. Rapid diagnosis of the disease and isolation of patients are effective ways to prevent and control the spread of COVID-19. Therefore, a sensitive immunofluorescent assay method was developed for rapid detection of special IgM and IgG of COVID-19 in human serum within 10 min. The recombinant nucleocapsid protein of 2019 novel coronavirus was used as capture antigen. Lanthanide, Eu(III) fluorescent microsphere, was used to qualitatively/semiquantitatively determine the solid phase immunochromatographic assay. A total of 28 clinical positive and 77 negative serum or plasma samples were included in the test. Based on the analysis of serum or plasma from COVID-19 patients and healthy people, the sensitivity and specificity of the immunochromatographic assay were calculated as 98.72% and 100% (IgG), and 98.68% and 93.10% (IgM), respectively. The results demonstrated that rapid immunoassay has high sensitivity and specificity and was useful for rapid serodiagnosis of COVID-19.