Nucleic Acid Detection Technologies Research Articles

Developing, characterizing and modeling CRISPR-based point-of-use pathogen diagnostics.

Recent years have seen intense interest in the development of point-of-care nucleic acid diagnostic technologies to address the scaling limitations of laboratory-based approaches. Chief among these are combinations of isothermal amplification approaches with CRISPR-based detection and readouts of target products. Here, we contribute to the growing body of rapid, programmable point-of-care pathogen tests by developing and optimizing a one-pot NASBA-Cas13a nucleic acid detection assay. This test uses the isothermal amplification technique NASBA to amplify target viral nucleic acids, followed by Cas13a-based detection of amplified sequences. We first demonstrate an in-house formulation of NASBA that enables optimization of individual NASBA components. We then present design rules for NASBA primer sets and LbuCas13a guide RNAs for fast and sensitive detection of SARS-CoV-2 viral RNA fragments, resulting in 20 - 200 aM sensitivity without any specialized equipment. Finally, we explore the combination of high-throughput assay condition screening with mechanistic ordinary differential equation modeling of the reaction scheme to gain a deeper understanding of the NASBA-Cas13a system. This work presents a framework for developing a mechanistic understanding of reaction performance and optimization that uses both experiments and modeling, which we anticipate will be useful in developing future nucleic acid detection technologies.

Polymerase Chain Reaction Cleaner with Antibacterial Effect and Faster Elimination of Nucleic Acid Aerosol Pollution

With advancement of nucleic acid detection technology, most universities, biological testing companies, and hospitals have Polymerase Chain Reaction (PCR) laboratories. PCR detection technology is the core technology for nucleic acid detection. When nucleic acid detection is performed in a PCR laboratory, nucleic acid aerosol samples are often dispersed to the environment in the form of aerosols. At this time, there will be some nucleic acid contamination in the PCR laboratory, resulting in false positive samples. The purpose of this paper is to propose a new type of nucleic acid pollution scavenger called PCR Cleaner. Firstly, the best ratio of PCR Cleaner was obtained by a control experiment, and then the antibacterial test for the PCR Cleaner was carried by comparing the nucleic acid pollution removal efficiency of different ratios of PCR Cleaner and common nucleic acid pollution scavenger on the surface and in the air. Experiment results showed that, the removal efficiency of PCR Cleaner on the surface of nucleic acid was much higher than that of alcohol and aqueous solution. Its effect was good enough when compared to the two commonly used nucleic acid pollution scavengers (DNA/RNA-ExitusPlus and PCR clean). The antibacterial and bacteriostatic PCR Cleaner can significantly inhibit the growth of high concentration of E. coli, and can also completely inhibit the low concentration of E. coli.

Preamplification-free ultra-fast and ultra-sensitive point-of-care testing via LwaCas13a

CRISPR based nucleic acid detection technology provides a deployable approach to point of care testing. While, there remain challenges limiting its practical applications, such as the need for pre-amplification and the long turnaround time. Here, we present a self-cascade signal amplification method with LwaCas13a and an artificially designed “U” rich RNA of stem-loop structure (URH) for pre-amplification-free ultra-fast and ultra-sensitive point-of-care testing (PASSPORT). The PASSPORT system contains: URH, crRNA targeted the URH, crRNA targeted the interesting RNA, fluorescent RNA reporter and LwaCas13a. The assay realized the detection of 100 copies/mL, within 5 min. The PASSPORT platform was further adopted for the detection of marker gene from SASR-CoV-2 and Severe fever with thrombocytopenia syndrome virus (SFTSV), respectively, and 100% accuracy for the analysis of clinical specimens (100 SASR-CoV-2 specimens and 16 SFTSV specimens) was obtained. Integrated with a lateral flow assay device, this assay could provide an alternative platform for the development of point of care testing (POCT) biosensors. PASSPORT has the potential to enable sensitive, specific, user-friendly, rapid, affordable, equipment-free and point-of-care testing for the purpose of large-scale screening and in case of epidemic outbreak.

A Fast and Sensitive One-Tube SARS-CoV-2 Detection Platform Based on RTX-PCR and Pyrococcus furiosus Argonaute.

Since SARS-CoV-2 is a highly transmissible virus, alternative reliable, fast, and cost-effective methods are still needed to prevent virus spread that can be applied in the laboratory and for point-of-care testing. Reverse transcription real-time fluorescence quantitative PCR (RT-qPCR) is currently the gold criteria for detecting RNA viruses, which requires reverse transcriptase to reverse transcribe viral RNA into cDNA, and fluorescence quantitative PCR detection was subsequently performed. The frequently used reverse transcriptase is thermolabile; the detection process is composed of two steps: the reverse transcription reaction at a relatively low temperature, and the qPCR performed at a relatively high temperature, moreover, the RNA to be detected needs to pretreated if they had advanced structure. Here, we develop a fast and sensitive one-tube SARS-CoV-2 detection platform based on Ultra-fast RTX-PCR and Pyrococcus furiosus Argonaute-mediated Nucleic acid Detection (PAND) technology (URPAND). URPAND was achieved ultra-fast RTX-PCR process based on a thermostable RTX (exo-) with both reverse transcriptase and DNA polymerase activity. The URPAND can be completed RT-PCR and PAND to detect nucleic acid in one tube within 30 min. This method can specifically detect SARS-CoV-2 with a low detection limit of 100 copies/mL. The diagnostic results of clinical samples with one-tube URPAND displayed 100% consistence with RT-qPCR test. Moreover, URPAND was also applied to identify SARS-CoV-2 D614G mutant due to its single-nucleotide specificity. The URPAND platform is rapid, accurate, tube closed, one-tube, easy-to-operate and free of large instruments, which provides a new strategy to the detection of SARS-CoV-2 and other RNA viruses.

Self-correction of cycle threshold values by a normal distribution-based process to improve accuracy of quantification in real-time digital PCR.

Thedigital polymerase chain reaction (dPCR) is a new and developing nucleic acid detection technology with high sensitivity that can realize the absolute quantitative analysis of samples. In order to improve the accuracy of quantitative results, real-time digital PCR emphasizes the kinetic information during amplification to identify prominent abnormal data. However, it is challenging to use a unified standard to accurately classify the amplification curve of each well as negative and positive, due to the interference caused by various factors in the experiment. In this work, a normal distribution-based cycle threshold value self-correcting model (NCSM) was established, which focused on the feature of the cycle threshold values in amplification curves and conducted continuous detection and correction on the whole. The cycle threshold value distribution was closer to the ideal normal distribution to avoid the influence of interference. Thus, the model achieves a more accurate classification between positive and negative results. The corrective process was applied to plasmid samples and resulted in an accuracy improvement from 92 to 99%. The coefficient of variation was below 5% when considering the quantitation of a range between 100 and 10,000 copies. At the same time, by utilizing this model, the distribution of cycle threshold values at the endpoint can be predicted with fewer thermal cycles, which can reduce the cycling time by around 25% while maintaining a consistency of more than 98%. Therefore, using the NCSM can effectively enhance the quantitative accuracy and increase the detection efficiency based on the real-time dPCR platform.

A Universal Strategy for Enhancing the Circulating miRNAs' Detection Performance of Rolling Circle Amplification by Using a Dual-Terminal Stem-Loop Padlock.

Rolling circle amplification (RCA) is one of the most promising nucleic acid detection technologies and has been widely used in the molecular diagnosis of disease. Padlock probes are often used to form circular templates, which are the core of RCA. However, RCA often suffers from insufficient specificity and sensitivity. Here we report a reconstruction strategy for conventional padlock probes to promote their overall performance in nucleic acid detection while maintaining probe functions uncompromised. When two rationally designed stem-loops were strategically placed at the two terminals of linear padlock probes, the specificity of target recognition was enhanced and the negative signal was significantly delayed. Our design achieved the best single-base discrimination compared with other structures and over a 1000-fold higher sensitivity than that of the conventional padlock probe, validating the effectiveness of this reconstruction. In addition, the underlying mechanisms of our design were elucidated through molecular dynamics simulations, and the versatility was validated with longer and shorter padlocks targeting the same target, as well as five additional targets (four miRNAs and dengue virus - 2 RNA mimic (DENV-2)). Finally, clinical applicability in multiplex detection was demonstrated by testing real plasma samples. Our exploration of the structures of nucleic acids provided another perspective for developing high-performance detection systems, improving the efficacy of practical detection strategies, and advancing clinical diagnostic research.

RT-RPA-assisted CRISPR/Cas12a for rapid and multiplex detection of respiratory infectious viruses based on centrifugal microfluidics

Clustered regularly interspaced short palindromic repeats (CRISPR)-based nucleic acid detection technology combined with recombinase polymerase amplification (RPA) allows for convenient, rapid, and highly sensitive nucleic acid detection. However, the non-specific cleavage characteristics of Cas12a after activation during the CRISPR nucleic acid detection process limit the detection of multiple targets. In this study, we designed a pressure-assisted and centrifugal-driven microfluidic chip for detecting respiratory pathogens. This chip achieved a high-sensitivity detection of 14 targets within 30 min. By freeze-drying, the detection reagents can be stored in the natural environment for one week and then stored at low temperature with little influence on biological activity, which reduces the difficulty of storage and transportation. The chip had independent amplification chambers to avoid competitive reactions, separating the amplification and detection chambers prevented mutual interference between the RPA and the CRISPR detection processes. Thus, this design exhibited a sensitivity of 2 copies/μL and 14 targets nucleic acids can be detected simultaneously. Compared with real-time fluorescence quantitative polymerase chain reaction (PCR) using real clinical samples of SARS-CoV-2, the new method could accurately detect the SARS-CoV-2 virus and identify the virus subtypes, which is important for infectious disease prevention and control.

An ultrafast and portable nucleic acid detection system based on photothermal PCR and real-time fluorescence detection

The nucleic acid detection technology based on polymerase chain reaction (PCR) is widely used in infectious disease detection due to its excellent sensitivity and specificity. However, the drawbacks of this method include bulky instrumentation in centralized laboratories and an assay time of 1–2 h. Here, we show a portable nucleic acid detection system, successfully performing ultrafast thermocycling and real-time fluorescence detection in a glass microtubule. By using plasmonic nanoparticles, the system can achieve 30 thermocycles in ∼ 2.5 min. The nucleic acid detection efficiency of the system is comparable to that of the conventional thermocycler. The system correctly distinguished the positive and negative samples of the clinical hepatitis B virus (HBV). Overall, this study extends PCR technology to point-of-care use and provides ideas for developing new virus detection systems.

CRISPR/Cas12a-assisted visible fluorescence for pseudo dual nucleic acid detection based on an integrated chip

BackgroundA false negative result is one of the major problems in nucleic acid detection. Failure to screen positive samples for pathogens or viruses poses a risk to public health. This situation will lead to more serious consequences for infectious pathogens or viruses. At present, the common solution is to introduce exogenous or endogenous internal control. Because it amplifies and is detected separately from the target gene, it cannot avoid false negative results caused by DNA extraction failure or reagent inactivation. There is an urgent need for a simple and reliable method to solve the false negative problem of nucleic acid detection. ResultsWe established a chip and an on-chip detection method for the integrated detection of target genes and internal control using the CRISPR system in LAMP amplification products. The chip is processed from a low-cost PMMA board and has three chambers and some channels. After adding the sample, the chip only needs to be rotated twice, and the sample enters three chambers successively depending on its gravity for dual LAMP reaction and CRISPR detections. With a portable LED blue light exciter, visual fluorescence detection is realized. Whether the detection result is positive, negative, or invalid can be determined according to the fluorescence in the CRISPR chamber for target gene and CRISPR chamber for internal control. In this study, the detection of Salmonella enterica in Fenneropenaeus chinensis was taken as an example. The results showed good specificity and sensitivity. It could detect as low as 15 copies/μL of Salmonella enterica. SignificanceThe on-chip detection solves the problem of aerosol contamination and false negative results. It has the advantages of high sensitivity, high specificity, high accuracy, and low cost. This research will advance the development of nucleic acid detection technology, providing a new and reliable strategy for POCT detection of pathogenic bacteria and viruses.

Advances in Cas12a-Based Amplification-Free Nucleic Acid Detection.

In biomedicine, rapid and sensitive nucleic acid detection technology plays an important role in the early detection of infectious diseases. However, most traditional nucleic acid detection methods require the amplification of nucleic acids, resulting in problems such as long detection time, complex operation, and false-positive results. In recent years, clustered regularly interspaced short palindromic repeats (CRISPR) systems have been widely used in nucleic acid detection, especially the CRISPR-Cas12a system, which can trans cleave single-stranded DNA and can realize the detection of DNA targets. But, amplification of nucleic acids is still required to further improve detection sensitivity, which makes Cas12a-based amplification-free nucleic acid detection methods a great challenge. This article reviews the recent progress of Cas12a-based amplification-free detection methods for nucleic acids. These detection methods apply electrochemical detection methods, fluorescence detection methods, noble metal nanomaterial detection methods, and lateral flow assay. Under various optimization strategies, unamplified nucleic acids have the same sensitivity as amplified nucleic acids. At the same time, the article discusses the advantages and disadvantages of each method and further discusses the current challenges such as off-target effects and the ability to achieve high-throughput detection. Amplification-free nucleic acid detection technology based on CRISPR-Cas12a has great potential in the biomedical field.

Ultrasensitive detection porcine transmissible gastroenteritis virus based on the dual-photoelectrode photofuel cell with in-situ isothermal amplification

Loop-mediated isothermal amplification (LAMP) as a nucleic acid detection technology has many advantages than conventional polymerase chain reaction in terms of sensitivity, specificity, and equipment requirements. And combining photocatalytic fuel cell (PFC) power density readout with high sensitivity, we developed a LAMP-based dual-photoelectrode photocatalytic fuel cell based self-powered biosensors (LAMP PFC-SPBs) for the ultra-sensitive detection of transmissible gastroenteritis virus (TGEV). In this sensor, the specially designed primers were immobilized on the photosensitive material CdS-modified sensing substrate with a distinct electrochemical signal and amplified TGEV within 1 h. A wide detection range of the LAMP PFC-SPBs was demonstrated, ranging from 0.075 fg μL−1 to 7.5 ng μL−1, with a lower detection limit of 0.025 fg μL−1. In-situ LAMP PFC-SPBS had great merits in simplicity (no complex thermal cycles, no need for expensive instruments), sensitivity, specificity, and provides a new perspective on the combination of nucleic acid amplification and electrochemistry.

Argonaute protein-based nucleic acid detection technology.

It is vital to diagnose pathogens quickly and effectively in the research and treatment of disease. Argonaute (Ago) proteins are recently discovered nucleases with nucleic acid shearing activity that exhibit specific recognition properties beyond CRISPR-Cas nucleases, which are highly researched but restricted PAM sequence recognition. Therefore, research on Ago protein-mediated nucleic acid detection technology has attracted significant attention from researchers in recent years. Using Ago proteins in developing nucleic acid detection platforms can enable efficient, convenient, and rapid nucleic acid detection and pathogen diagnosis, which is of great importance for human life and health and technological development. In this article, we introduce the structure and function of Argonaute proteins and discuss the latest advances in their use in nucleic acid detection.

Research Progress of Nucleic Acid Detection Technology for Genetically Modified Maize.

Genetically modified (GM) maize is one of the earliest GM crops to have achieved large-scale commercial cultivation globally, and it is of great significance to excel in the development and implementation of safety policy regarding GM, and in its technical oversight. This article describes the general situation regarding genetically modified maize, including its varieties, applications, relevant laws and regulations, and so on. From a technical point of view, we summarize and critically analyze the existing methods for detecting nucleic acid levels in genetically modified maize. The nucleic acid extraction technology used for maize is explained, and the introduction of traditional detection techniques, which cover variable-temperature and isothermal amplification detection technology and gene chip technology, applications in maize are described. Moreover, new technologies are proposed, with special attention paid to nucleic acid detection methods using sensors. Finally, we review the current limitations and challenges of GM maize nucleic acid testing and share our vision for the future direction of this field.

Research progress of CRISPR/Cas systems in nucleic acid detection of infectious diseases

AbstractInfectious diseases are a serious threat to human health, and accurate, rapid and convenient early detection of pathogens is the first step of active treatment. Technologies that detect pathogens have advanced significantly because of the development of fundamental disciplines and the integration of multidisciplinary fields. Among these technologies, nucleic acid detection technology is preferred because of its rapid measurement, accuracy and high sensitivity. The CRISPR/Cas system, consisting of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR‐associated (Cas), is an adaptive immune system that specifically recognizes, binds and cleaves exogenous invasive nucleic acids. The CRISPR/Cas system is widely found in bacteria and archaea. Researchers have developed nucleic acid detection technologies with single‐molecule sensitivity, single‐base precision specificity, portability and low cost based on the specific cleavage and trans‐cleavage activities of the CRISPR/Cas system. The next generation of in‐vitro diagnostics is shifting to nucleic acid technology because this technology shows promise in a wide range of applications in resource‐constrained environments. In this review, the development and mechanism of the CRISPR/Cas system are presented together with representative CRISPR/Cas applications in nucleic acid detection. Additionally, the review summarizes future perspectives and trends of the CRISPR/Cas system in nucleic acid detection.

An Integrated Amplification-Free Digital CRISPR/Cas-Assisted Assay for Single Molecule Detection of RNA.

Conventional nucleic acid detection technologies usually rely on amplification to improve sensitivity, which has drawbacks, such as amplification bias, complicated operation, high requirements for complex instruments, and aerosol pollution. To address these concerns, we developed an integrated assay for the enrichment and single molecule digital detection of nucleic acid based on a CRISPR/Cas13a and microwell array. In our design, magnetic beads capture and concentrate the target from a large volume of sample, which is 100 times larger than reported earlier. The target-induced CRISPR/Cas13a cutting reaction was then dispersed and limited to a million individual femtoliter-sized microwells, thereby enhancing the local signal intensity to achieve single-molecule detection. The limit of this assay for amplification-free detection of SARS-CoV-2 is 2 aM. The implementation of this study will establish a "sample-in-answer-out" single-RNA detection technology without amplification and improve the sensitivity and specificity while shortening the detection time. This research has broad prospects in clinical application.

Genotyping SARS-CoV-2 Variants Using Ratiometric Nucleic Acid Barcode Panels.

Designing diagnostic assays to genotype rapidly mutating viruses remains a challenge despite the overall improvements in nucleic acid detection technologies. RT-PCR and next-generation sequencing are unsuitable for genotyping during outbreaks or in point-of-care detection due to their infrastructure requirements and longer turnaround times. We developed a quantum dot barcode multiplexing system to genotype mutated viruses. We designed multiple quantum dot barcodes to target conserved, wildtype, and mutated regions of SARS-CoV-2. We calculated ratios of the signal output from different barcodes that enabled SARS-CoV-2 detection and identified SARS-CoV-2 variant strains from a sample. We detected different sequence types, including conserved genes, nucleotide deletions, and single nucleotide substitutions. Our system detected SARS-CoV-2 patient specimens with 98% sensitivity and 94% specificity across 91 patient samples. Further, we leveraged our barcoding and ratio system to track the emergence of the N501Y SARS-CoV-2 mutation from December 2020 to May 2021 and demonstrated that the more transmissible N501Y mutation started to dominate infections by April 2021. Our barcoding and signal ratio approach can genotype viruses and track the emergence of viral mutations in a single diagnostic test. This technology can be extended to tracking other viruses. Combined with smartphone detection technologies, this assay can be adapted for point-of-care tracking of viral mutations in real time.

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.

CRISPR-based molecular diagnostics: a review

Rapid and accurate detection technologies are crucial for disease prevention and control. In particular, the COVID-19 pandemic has posed a great threat to our society, highlighting the importance of rapid and highly sensitive detection techniques. In recent years, CRISPR/Cas-based gene editing technique has brought revolutionary advances in biotechnology. Due to its fast, accurate, sensitive, and cost-effective characteristics, the CRISPR-based nucleic acid detection technology is revolutionizing molecular diagnosis. CRISPR-based diagnostics has been applied in many fields, such as detection of infectious diseases, genetic diseases, cancer mutation, and food safety. This review summarized the advances in CRISPR-based nucleic acid detection systems and its applications. Perspectives on intelligent diagnostics with CRISPR-based nucleic acid detection and artificial intelligence were also provided.

Deep Learning-Assisted Droplet Digital PCR for Quantitative Detection of Human Coronavirus.

Since coronavirus disease 2019 (COVID-19) pandemic rapidly spread worldwide, there is an urgent demand for accurate and suitable nucleic acid detection technology. Although the conventional threshold-based algorithms have been used for processing images of droplet digital polymerase chain reaction (ddPCR), there are still challenges from noise and irregular size of droplets. Here, we present a combined method of the mask region convolutional neural network (Mask R-CNN)-based image detection algorithm and Gaussian mixture model (GMM)-based thresholding algorithm. This novel approach significantly reduces false detection rate and achieves highly accurate prediction model in a ddPCR image processing. We demonstrated that how deep learning improved the overall performance in a ddPCR image processing. Therefore, our study could be a promising method in nucleic acid detection technology.

A wash-free, elution-free and low protein adsorption paper-based material for nucleic acid extraction.

Nucleic acid detection technologies have been widely utilized for various diseases. Conventional laboratory tests are less suitable for use in resource-limited settings as they are time-consuming, high-cost, complex, and heavily dependent on benchtop equipment. Rapid nucleic acid detection methods that consist of rapid nucleic acid extraction steps could overcome these challenges. A paper-based platform has been utilized to develop various rapid nucleic acid extraction methods owing to its cost-effectiveness, portability, and easy-modification. However, the existing paper-based nucleic acid extraction technologies mainly focus on improving the adsorption capacity of nucleic acids without reducing the non-specific adsorption capacity of proteins. In this study, paper-based nucleic acid extraction technology with wash-free, elution-free, and low protein adsorption was developed. The fabrication of paper involves the mixing of polyethylene glycol (PEG)-modified cotton fiber, chitosan (COS)-modified cotton fiber, and cotton fiber to form PEG-modified cotton fiber/chitosan-modified cotton fiber/cotton fiber (PEG-CF/COS-CF/CF) paper by the wet molding method. The result showed that PEG-CF/COS-CF/CF paper has a desirable pore size (23.9 ± 4.03 μm), good mechanical strength (dry: 9.37 Mpa and wet: 0.28 Mpa), and hydrophilicity (contact angle: 42.6° ± 0.36°). NH3+ groups of COS and OH- groups of PEG were observed on its surface and the adsorption efficiency of nucleic acid in TE buffer was 42.48% ± 0.30%. The limit of detection of pure DNA with this PEG-CF/COS-CF/CF paper by qPCR was as low as 25 ng. Additionally, this platform could successfully extract nucleic acid from 30 μL of a saliva sample, highlighting its potential use for clinical sample testing. The proposed paper-based nucleic acid extraction platform shows tremendous potential for disease diagnosis in resource-limited settings.