A Simple, Pipette-free, and Power-free Device for Virus Nucleic Acid Detection

A portable, inexpensive, and easy-to-manufacture microfluidic device is developed for the detection of SARS-CoV-2 dsDNA fragments. In this device, four reaction chambers separated by carbon fiber rods are pre-loaded with isothermal amplification and CRISPR-Cas12a reagents. The reaction is carried out by simply pulling the rods, without the need for manual pipetting. To facilitate power-free pathogen detection, the entire detection is designed to be heated with a disposable hand warmer. After the CRISPR reaction, the fluorescence signal generated by positive samples is identified by naked eye, using an inexpensive flashlight. This simple and sensitive device will serve as a new model for the next-generation viral diagnostics in either hospital or resource-limited settings. © 2023 SPIE.

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.

FEN1-aided recombinase polymerase amplification (FARPA) for one-pot and multiplex detection of nucleic acids with an ultra-high specificity and sensitivity.

Recombinase polymerase amplification (RPA) running at 37-42 °C is fast, efficient and less-implemented; however, the existing technologies of nucleic acid testing based on RPA have some limitations in specificity of single-base recognition and multiplexing capability. Herein, we report a highly specific and multiplex RPA-based nucleic acid detection platform by combining flap endonuclease 1 (FEN1)-catalysed invasive reactions with RPA, termed as FEN1-aided RPA (FARPA). The optimal conditions enable RPA and FEN1-based fluorescence detection to occur automatically and sequentially within a 25-min turnaround time and FARPA exhibits sensitivity to 5 target molecules. Due to the ability of invasive reactions in discriminating single-base variation, this one-pot FARPA is much more specific than the Exo probe-based or CRISPR-based RPA methods. Using a universal primer pair derived from tags in reverse transcription primers, multiplex FARPA was successfully demonstrated by the 3-plex assay for the detection of SARS-CoV-2 pathogen (the ORF1ab, the N gene, and the human RNase P gene as the internal control), the 2-plex assay for the discrimination of SARS-CoV-2 wild-type from variants (Alpha, Beta, Epsilon, Delta, or Omicrons), and the 4-plex assay for the screening of arboviruses (zika virus, tick-borne encephalitis virus, yellow fever virus, and chikungunya virus). We have validated multiplex FARPA with 103 nasopharyngeal swabs for SARS-CoV-2 detection. The results showed a 100% agreement with RT-qPCR assays. Moreover, a hand-held FARPA analyser was constructed for the visualized FARPA due to the switch-like endpoint read-out. This FARPA is very suitable for pathogen screening and discrimination of viral variants, greatly facilitating point-of-care diagnostics.

Recent advances on CRISPR/Cas system-enabled portable detection devices for on-site agri-food safety assay

Agri-food safety has been considered as one of the most important public concerns worldwide. From farm to table, food crops and foods are extremely vulnerable to the contamination by a variety of pollutants from their growth and processing. Moreover, the SARS-CoV-2 detected in the food supply chain during COVID-19 pandemic has posed a greater challenge for rapid and on-site detection of agri-food contaminants in complex and volatile environments. Therefore, the development of rapid, accurate, and on-site detection technologies and portable detection devices is of great importance to ensure the agri-food security. This review comprehensively summarized the recent advances on the construction of CRISPR/Cas systems-based biosensing technologies and their portable detection devices, as well as their promising applications in the field of agri-food safety. First of all, the classification and working principles of CRISPR/Cas systems were introduced. Then, the latest advances on the CRISPR/Cas system-based on-site detection technologies and portable detection devices were also systematically summarized. Most importantly, the state-of-the-art applications of CRISPR/Cas systems-based on-site detection technologies and portable detection devices in the fields of agri-food safety were comprehensively summarized. Impressively, the future opportunities and challenges in this emerging and promising field were proposed. Emerging CRISPR/Cas system-based on-site detection technologies have showed a great potential in the detection of agri-food safety. Impressively, the integration of CRISPR/Cas systems-based biosensing technologies with portable detection devices (e.g., nanopore-based detection devices, lateral flow assay, smartphone-based detection devices, and microfluidic devices) is very promising for the on-site detection of agri-food contaminants. Additionally, CRISPR/Cas system-based biosensing technologies can be further integrated with much more innovative technologies for the development of novel detection platforms to realize the more reliable on-site detection of agri-food safety.

Automatic Diagnosis of COVID-19 Based on Machine Learning

The lives and health of individuals are significantly threatened by the extremely infectious and dangerous Corona Virus Disease 2019 (COVID-19). For the containment of the epidemic, quick and precise COVID-19 detection and diagnosis are essential. Currently, artificial diagnosis based on medical imaging and nucleic acid detection are the major approaches used for COVID-19 detection and diagnosis. However, nucleic acid detection takes a long time and requires a dedicated test box, while manual diagnosis based on medical images relies too much on professional knowledge, and analysis takes a long time, and it is difficult to find hidden lesions. Thanks to the rapid development of pattern recognition algorithms, building a COVID-19 diagnostic model based on machine learning and clinical symptoms has become a feasible rapid detection solution. In this paper, support vector machines and random forest algorithms are used to build a COVID-19 diagnostic model, respectively. Based on the quantitative comparison of the performance of the two methods, the future development trends in this field are discussed. © 2023 IEEE.

An extraction-free one-step CRISPR-assisted detection platform and a potential Streptococcus pneumoniae at-home self-testing kit.

A rapid, accurate, easy-to-use nucleic acid detection technology is essential for disease diagnosis and control. Herein, we improved CRISPR-top (cluster regularly interspaced short palindromic repeats-mediated testing in one-pot) to develop Extraction-free one-step CRISPR-assistant detection (ExCad), a simple, rapid, accurate gene detection tool for unextracted colonies and samples. We established a pretreatment protocol to rapidly liquify sputum samples and release nucleic acids within 10 min. The ExCad results can be visualised by a real-time fluorescence reader or the naked eye under blue light. We developed an ExCad-Sp assay to detect Streptococcus pneumoniae from unextracted strains and specimens, and optimised the assay conditions. Assay feasibility was evaluated using sputum samples from 32 patients, and it achieved 92.9 % (13/14) sensitivity, 100 % (18/18) specificity, 100 % (13/13) positive predictive value, and 94.7 % (18/19) negative predictive value compared with bacteria culture. The ExCad-Sp assay has potential for developing an at-home self-testing kit for S. pneumoniae.

Recent advances in cascade isothermal amplification techniques for ultra-sensitive nucleic acid detection.

Nucleic acid amplification techniques have always been one of the hot spots of research, especially in the outbreak of COVID-19. From the initial polymerase chain reaction (PCR) to the current popular isothermal amplification, each new amplification techniques provides new ideas and methods for nucleic acid detection. However, limited by thermostable DNA polymerase and expensive thermal cycler, PCR is difficult to achieve point of care testing (POCT). Although isothermal amplification techniques overcome the defects of temperature control, single isothermal amplification is also limited by false positives, nucleic acid sequence compatibility, and signal amplification capability to some extent. Fortunately, efforts to integrating different enzymes or amplification techniques that enable to achieve intercatalyst communication and cascaded biotransformations may overcome the corner of single isothermal amplification. In this review, we systematically summarized the design fundamentals, signal generation, evolution, and application of cascade amplification. More importantly, the challenges and trends of cascade amplification were discussed in depth.

Light-Start CRISPR-Cas12a Reaction with Caged crRNA Enables Rapid and Sensitive Nucleic Acid Detection.

The clustered regularly interspaced short palindromic repeats (CRISPR) system is a promising platform for nucleic acid detection. Regulating the CRISPR reaction would be extremely useful to improve the detection efficiency and speed of CRISPR diagnostic applications. Here, we have developed a light-start CRISPR-Cas12a reaction by employing caged CRISPR RNA (crRNA). When combined with recombinase polymerase amplification, a robust photocontrolled one-pot assay is achieved. The photocontrolled one-pot assay is simpler and is 50-fold more sensitive than the conventional assay. This improved detection efficiency also facilitates the development of a faster CRISPR diagnostic method. The detection of clinical samples demonstrated that 10-20 min is sufficient for effective detection, which is much faster than the current gold-standard technique PCR. We expect this advance in CRISPR diagnostics to promote its widespread detection applications in biomedicine, agriculture, and food safety.

Blockchain-Based Security Check Framework in the State of the COVID-19 Epidemic

At present, one of the effective ways to deal with the widespread of COVID-19 is to control the source of infection. As the gate of population flow in various places, the security inspection department plays a vital role in screening positive patients in the population;To solve the problems of credibility and lack of human resources, this paper establishes a security check framework based on the blockchain and combines machine learning with the blockchain: the blockchain records the abnormal results of COVID-19 nucleic acid detection and the abnormal conditions detected by the security inspection system (such as no mask, high temperature);Use machine learning to realize mask recognition and other functions. The architecture, data flow, and key elements are presented and discussed. The study findings could solve the security problem under the epidemic and provide relevant enlightenment for the effective combination and application of machine learning and blockchain. © 2022 IEEE.

A real-time monitoring platform of colorimetric LAMP for developing rapid visual detection kits of SARS-CoV-2

Visual detection of nucleic acids is important to diagnose the serious acute infectious diseases such as coronavirus disease 2019 (COVID-19). During this pandemic, reliable visual detection kits have been in high demand for screening and prevention of the virus. While developing these visual detection kits, a real-time monitoring platform is usually applied to study the amplification and detection processes of nucleic acids and optimize the detecting conditions. Herein, we developed a real-time monitoring platform of colorimetric loop-mediated isothermal amplification (LAMP) to investigate the amplification and detection processes of nucleic acids. Using this platform, we could obtain the real-time amplification curves, and optimize the reaction temperature, color change, and detection time. Based on the optimized conditions, a visual detection kit for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was successfully developed with a sensitivity of 102 copies µL−1 in 12 min. This real-time monitoring platform has advantages of simple construction, steady performance, high sensitivity, and outstanding anti-pollution capability, and could replace the traditional colorimetric methods by photographing and reading values. This platform would accelerate the development of visual detection kits for colorimetric LAMP, help to explore the amplification and transcription of nucleic acids, and provide support for the prevention of emerging biological threats. © 2023

FELICX: A robust nucleic acid detection method using flap endonuclease and CRISPR-Cas12.

Nucleic acid detection is crucial for monitoring diseases for which rapid, sensitive, and easy-to-deploy diagnostic tools are needed. CRISPR-based technologies can potentially fulfill this need for nucleic acid detection. However, their widespread use has been restricted by the requirement of a protospacer adjacent motif in the target and extensive guide RNA optimization. In this study, we developed FELICX, a technique that can overcome these limitations and provide a useful alternative to existing technologies. FELICX comprises flap endonuclease, Taq ligase and CRISPR-Cas for diagnostics (X) and can be used for detecting nucleic acids and single-nucleotide polymorphisms. This method can be deployed as a point-of-care test, as only two temperatures are needed without thermocycling for its functionality, with the result generated on lateral flow strips. As a proof-of-concept, we showed that up to 0.6 copies/µL of DNA and RNA could be detected by FELICX in 60 min and 90 min, respectively, using simulated samples. Additionally, FELICX could be used to probe any base pair, unlike other CRISPR-based technologies. Finally, we demonstrated the versatility of FELICX by employing it for virus detection in infected human cells, the identification of antibiotic-resistant bacteria, and cancer diagnostics using simulated samples. Based on its unique advantages, we envision the use of FELICX as a next-generation CRISPR-based technology in nucleic acid diagnostics.

CRISPR-cas technology: A key approach for SARS-CoV-2 detection.

The CRISPR (Clustered Regularly Spaced Short Palindromic Repeats) system was first discovered in prokaryotes as a unique immune mechanism to clear foreign nucleic acids. It has been rapidly and extensively used in basic and applied research owing to its strong ability of gene editing, regulation and detection in eukaryotes. Hererin in this article, we reviewed the biology, mechanisms and relevance of CRISPR-Cas technology and its applications in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) diagnosis. CRISPR-Cas nucleic acid detection tools include CRISPR-Cas9, CRISPR-Cas12, CRISPR-Cas13, CRISPR-Cas14, CRISPR nucleic acid amplification detection technology, and CRISPR colorimetric readout detection system. The above CRISPR technologies have been applied to the nucleic acid detection, including SARS-CoV-2 detection. Common nucleic acid detection based on CRISPR derivation technology include SHERLOCK, DETECTR, and STOPCovid. CRISPR-Cas biosensing technology has been widely applied to point-of-care testing (POCT) by targeting recognition of both DNA molecules and RNA Molecules.

Anatomical and Vision-Guided Path Generation Method for Nasopharyngeal Swabs Sampling

Due to the global COVID-19 pandemic, there is a strong demand for pharyngeal swab sampling and nucleic acid testing. Research has shown that the positive rate of nasopharyngeal swabs is higher than that of oropharyngeal swabs. However, because of the high complexity and visual obscuring of the interior nasal cavity, it is impossible to obtain the sampling path information directly from the conventional imaging principle. Through the combination of anatomical geometry and spatial visual features, in this paper, we present a new approach to generate nasopharyngeal swabs sampling path. Firstly, this paper adopts an RGB-D camera to identify and locate the subject's facial landmarks. Secondly, the mid-sagittal plane of the subject's head is fitted according to these landmarks. At last, the path of the nasopharyngeal swab movement in the nasal cavity is determined by anatomical geometry features of the nose. In order to verify the validity of the method, the location accuracy of the facial landmarks and the fitting accuracy of mid-sagittal plane of the head are verified. Experiments demonstrate that this method provides a feasible solution with high efficiency, safety and accuracy. Besides, it can solve the problem that the nasopharyngeal robot cannot generate path based on traditional imaging principles. It also provides a key method for automatic and intelligent sampling of nasopharyngeal swabs, and it is of great clinical value to reduce the risk of cross-infection. © 2023, The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd.

Experimental study on the heating/cooling and temperature uniformity performance of the microchannel temperature control device for nucleic acid PCR amplification reaction of COVID-19

The nucleic acid detection is an effective way for the prevention and control of COVID-19. PCR amplification is an important process in the nucleic acid detection. At present, PCR amplification has the problem of low heating/cooling rates, and poor temperature uniformity. This paper proposes a microchannel temperature control device for the nucleic acid detection. Five groups of parallel serpentine channels are used to increase the cooling rate of the PCR amplification. A gradual thermal conductivity design is applied to the reaction module to increase the temperature uniformity. The experimental results show that the best temperature uniformity is obtained when the materials of the inner and outer layers of the reaction module are copper and aluminum alloys, respectively. The limit and average heating/cooling rate are 7.2, 6.12, 5.52 and 5.28 °C/s, respectively, when the input power of the thermoelectric cooler is 11.07 W/cm2, the temperature and flow rate of the cooling water are 15℃ and 700 ml/min, and the thermal conductivity of the thermal grease is 6 W/(m·K). Compared with the commercial fan-fin cooling method, the limit and average heating/cooling rates are increased by 38.02%, 80.82%, 86.49% and 208.77%, respectively, with the help of microchannel cooling method. © 2023 Elsevier Ltd

New Coronavirus 2 (SARS-CoV-2) Detection Method from Human Nucleic Acid Sequences Using Capsule Networks

The new coronavirus SARS-CoV-2 is an infectious virus with a long incubation period, which was first detected in Wuhan, China, spread all over the world, seriously threatening human life. Therefore, accurate and rapid detection of SARS-CoV-2 is very important for controlling the epidemic and preventing its further spread. Currently, nucleic acid detection makes an important contribution to the prevention and control of SARS-CoV-2. In this study, a new and highly sensitive nucleic acid detection method for SARS-CoV-2 has been proposed. The nucleic acid sequences were digitized by Entropy-based mapping technique. Then, the digitized these sequences were divided into 100-unit sections using the sliding window method and given as input to Capsule Networks.10988 segments (5494 SARS-CoV-2, 5494 normal) are classified with capsule nets. With the proposed method, an accuracy performance of 100% was achieved by using capsule networks to identify SARS-CoV-2 from nucleic acid sequences. The results show that the proposed method successfully identifies SARS-CoV-2 from nucleic acid sequences © 2023, Brazilian Archives of Biology and Technology. All rights reserved

Fast In Situ Inactivation of Pathogens in Tubes by Plasma

During the COVID-19 pandemic, the prevailing method for inactivating samples used in nucleic acid detection is a time-consuming process that involves heating the samples in a thermostatic water bath at 56 <inline-formula> <tex-math notation="LaTeX">$