Nucleic Acid Amplification Strategies Research Articles

Portable, and ultrasensitive HR-HPV tests based on nucleic acid biosensors.

Cervical cancer is the third most common cancer threatening women's health globally, and high-risk human papillomavirus (HR-HPV) infection is the main cause of cervical cancer worldwide. Given the recurrent nature of HR-HPV infection, accurate screening is essential for its control. Since the commonly used polymerase chain reaction (PCR) technique is limited by professional equipment and personnel, convenient and ultrasensitive detection methods for HR-HPV are still highly needed. As new molecular detection methods, nucleic acid amplification-based biosensors have the advantages of high sensitivity, rapid operation, and portability, which are helpful for point-of-care testing in rural and remote areas. This review summarized nucleic acid biosensors for HR-HPV screening based on a variety of nucleic acid amplification strategies involved in improved PCR, loop-mediated isothermal amplification, recombinase polymerase amplification, hybridization chain reaction, catalyzed hairpin assembly, and CRISPR/Cas systems. In combination with microfluidic technology, lateral flow assays, electrochemical analysis and other sensing technologies, HR-HPV nucleic acid biosensors have the advantages of high throughput, short response time, high sensitivity and easy operation in the field. Although there are still shortcomings, such as high cost and poor reproducibility, this approach will be suitable for on-site screening of HR-HPV infection or cervical cancer and for auxiliary clinical diagnosis in complex environments and poor areas in the future.

Solid-State Nanopore Sensors with Enhanced Sensitivity through Nucleic Acid Amplification.

Solid-state nanopores have wide applications in DNA sequencing, energy conversion and storage, seawater desalination, sensors, and reactors due to their high stability, controllable geometry, and a variety of pore-forming materials. Solid-state nanopore sensors can be used for qualitative and quantitative analyses of ions, small molecules, proteins, and nucleic acids. The combination of nucleic acid amplification and solid-state nanopores to achieve trace detection of analytes is gradually attracting attention. This review outlines nucleic acid amplification strategies for enhancing the sensitivity of solid-state nanopore sensors by summarizing the articles published in the past 10 years. The future development prospects and challenges of nucleic acid amplification in solid-state nanopore sensors are discussed. This review helps readers better understand the field of solid-state nanopore sensors. We believe that solid-state nanopore sensors will break through the bottleneck of traditional detection and become a powerful single-molecule detection platform.

Smart Target-Initiated Catalytic DNA Junction Circuit Amplification Strategy for the Ultrasensitive Electrochemiluminescence Detection of MicroRNA.

One of the highly attractive research directions in the electrochemiluminescence (ECL) field is how to regulate and improve ECL efficiency. Quantum dots (QDs) are highly promising ECL materials due to their adjustable luminescence size and strong luminous efficiency. MoS2 NSs@QDs, an ECL emitter, is synthesized via hydrothermal methods, and its ECL mechanism is investigated using cyclic voltammetry and ECL-potential curves. Then, a stable and vertical attachment of a triplex DNA (tsDNA) probe to the MoS2 nanosheets (NSs) is applied to the electrode. Next, an innovative ECL sensor is courageously empoldered for precise and ultrasensitive detection of target miRNA-199a through the agency of ECL-resonance energy transfer (RET) strategy and a dextrous target-initiated catalytic three-arm DNA junction assembly (CTDJA) based on a toehold strand displacement reaction (TSDR) signal amplification approach. Impressively, the ingenious system not only precisely regulates the distance between energy donor-acceptor pairs leave energy less loss and more ECL-RET efficiency, but also simplifies the operational procedure and verifies the feasibility of this self-assembly process without human intervention. This study can expand MoS2 NSs@QDs utilization in ECL biosensing applications, and the proposed nucleic acid amplification strategy can become a miracle cure for ultrasensitive detecting diverse biomarkers, which helps researchers to better study the tumor mechanism, thereby unambiguously increasing cancer cure rates and reducing the risk of recurrence.

CPA-Cas12a-based lateral flow strip for portable assay of Methicillin-resistant Staphylococcus aureus in clinical sample

The rapid and accurate identification of methicillin-resistant Staphylococcus aureus at an early antibiotic therapy stage would be benefit to disease diagnosis and antibiotic selection. Herein, we integrated cross-priming amplification (CPA) and CRISPR/Cas 12a (designated as CPA-Cas 12a) systems to establish a sensitive and efficient lateral flow assay to detect methicillin-resistant Staphylococcus aureus. This assay relies on the CPA isothermal nucleic acid amplification strategy which can amplify the DNA extracted from Staphylococcus aureus and accompanying the indiscriminately trans-cleavage process of Cas 12a/CrRNA duplex after recognizing specific sequence. Taking the advantage of reporter and high turnover Cas 12a activity, a dramatic change in response was achieved to produce a significant increase in the analytical sensitivity. The signal conversion and output were realized using a lateral flow strip to achieve field-deployable detection. Furthermore, this bioassay was accommodated with a microfluidic device to realize automatically portable detection. This proposed assay completed within 30 min with the detection limit of 5 CFU mL-1, was verified by testing bacterial suspension and 202 clinical samples. Given the high sensitivity, specificity and efficiency, this colorimetric readout assay through strip could be further promoted to the clinical diagnosis, clinical medication of multidrug-resistant bacteria.

Advances in Multiplexed Microfluidics for Infectious Disease Detection

Abstract Microfluidics enables miniaturization, functionality, high throughput and reproducibility of multipathogen detection. Multiplexed microfluidic devices are electrochemical sensor–based, optical sensor–based, immunosensor-based and paper-based multiplexed microfluidics. However, the simultaneous detection of multiple pathogens is limited because of the complexity and diversity of infectious disease sources and mutual interference among analytes. This review provides an overview of recent advances in developing multiplex diagnostic microfluidic devices for detecting infectious diseases and discusses practical issues and perspectives. This review also covers microfluidic nucleic acid amplification strategies to improve detection sensitivity. Finally, we discuss the limitations and challenges in the design of multiplexed microfluidics.

In situ reduction of gold nanoparticles-decorated MXenes-based electrochemical sensing platform for KRAS gene detection.

In this work, gold nanoparticles@Ti3C2 MXenes nanocomposites with excellent properties were combined with toehold-mediated DNA strand displacement reaction to construct an electrochemical circulating tumor DNA biosensor. The gold nanoparticles were synthesized in situ on the surface of Ti3C2 MXenes as a reducing and stabilizing agent. The good electrical conductivity of the gold nanoparticles@Ti3C2 MXenes composite and the nucleic acid amplification strategy of enzyme-free toehold-mediated DNA strand displacement reaction can be used to efficiently and specifically detect the non-small cell cancer biomarker circulating tumor DNA KRAS gene. The biosensor has a linear detection range of 10fM -10nM and a detection limit of 0.38fM, and also efficiently distinguishes single base mismatched DNA sequences. The biosensor has been successfully used for the sensitive detection of KRAS gene G12D, which has excellent potential for clinical analysis and provides a new idea for the preparation of novel MXenes-based two-dimensional composites and their application in electrochemical DNA biosensors.

Nucleic acid and nanomaterial-assisted signal-amplified strategies in fluorescent analysis of circulating tumor cells and small extracellular vesicles.

As two main types of liquid biopsy markers, both circulating tumor cells (CTCs) and small extracellular vesicles (sEVs) play important roles in the diagnosis and prognosis of cancers. CTCs are malignant cells that detach from the original tumor tissue and enter the circulation of body fluids. sEVs are nanoscale vesicles secreted by normal cells or pathological cells. However, CTCs and sEVs in body fluids are scarce, leading to great difficulties in the accurate analysis of related diseases. For the sensitive detection of CTCs and sEVs in body fluids, various types of nucleic acid and nanomaterial-assisted signal amplification strategies have been developed. In this review, we summarize the recent advances in fluorescent detection of CTCs and sEVs in liquid biopsy based on nucleic acid and nanomaterial-assisted signal amplification strategies. We also discuss their advantages, challenges, and future prospects.

A metal ion-coordinated DNA probe for sensitive fluorescence detection of metallothionein via a dual nucleic acid amplification strategy.

Sensitively monitoring metallothionein (MT), a heavy metal-binding protein with substantial cysteine content, is of significance for evaluating heavy metal poisoning in both humans and animals. Based on a new metal ion-coordinated DNA probe and the heavy metal ion binding capability of MT, as well as the substantial signal enhancement of the hybridization chain reaction (HCR) and rolling circle amplification (RCA), we demonstrate a highly sensitive fluorescence MT detection assay. MT binds the metal ions in the hairpin structured, metal ion-coordinated DNA probe to switch its hairpin structure into ssDNA, which triggers subsequent RCA reactions and HCRs to open plenty of fluorescently quenched signal hairpins to exhibit drastically amplified fluorescence recovery for assaying MT down to 0.58 nM within a dynamic range of 1-320 nM. In addition, the investigation of low contents of MT in diluted human serum by such an assay has also been verified, indicating its promising application potential for diagnosing heavy metal poisoning.

Interference reduction isothermal nucleic acid amplification strategy for COVID-19 variant detection

Common reference methods for COVID-19 variant diagnosis include viral sequencing and PCR-based methods. However, sequencing is tedious, expensive, and time-consuming, while PCR-based methods have high risk of insensitive detection in variant-prone regions and are susceptible to potential background signal interference in biological samples. Here, we report a loop-mediated interference reduction isothermal nucleic acid amplification (LM-IR-INA) strategy for highly sensitive single-base mutation detection in viral variants. This strategy exploits the advantages of nicking endonuclease-mediated isothermal amplification, luminescent iridium(III) probes, and time-resolved emission spectroscopy (TRES). Using the LM-IR-INA strategy, we established a luminescence platform for diagnosing COVID-19 D796Y single-base substitution detection with a detection limit of 2.01 × 105 copies/μL in a linear range of 6.01 × 105 to 3.76 × 108 copies/μL and an excellent specificity with a variant/wild-type ratio of significantly less than 0.0625%. The developed TRES-based method was also successfully applied to detect D796Y single-base substitution sequence in complicated biological samples, including throat and blood, and was a superior to steady-state technique. LM-IR-INA was also demonstrated for detecting the single-base substitution D614G as well as the multiple-base mutation H69/V70del without mutual interference, indicating that this approach has the potential to be used as a universal viral variant detection strategy.

The dual nucleic acid amplification with dynamic light scattering strategy for ultrasensitive detection of Salmonella in milk

Rapid detection of Salmonella in food has always been a research hotspot, which can contaminate milk. In this study, a novel method that combines streptavidin (SA)-modified gold nanoparticles (AuNPs) and dynamic light scattering (DLS) technology for ultrasensitive detection of Salmonella was proposed. First, dual nucleic acid amplification strategies, namely, polymerase chain reaction (PCR) and hybridization chain reaction (HCR), PCR was used to amplify the invA gene from Salmonella to produce the target single-stranded DNA to trigger HCR, whereas HCR was used to produce a long biotin-containing double-strand DNA. After adding the SA-AuNPs probe, the diameter change was measured by DLS. And different characterization methods were also used to verify the feasibility of the established PCR-HCR-DLS strategy. Moreover, under optimal experimental conditions, the developed method showed a limit of detection (LOD) at 100 CFU/mL in both pure culture and spiked skimmed milk. Therefore, the biosensor based on SA-AuNPs and PCR-HCR-DLS showed potential application in detecting Salmonella in milk.

Ultrasensitive Fluorescence Detection and Imaging of MicroRNA in Cells Based on a Hyperbranched RCA-Assisted Multiposition SDR Signal Amplification Strategy.

Herein, an innovative fluorescent sensor was courageously empoldered for precise and ultrasensitive detection and imaging of target miRNA-21 through the agency of a dextrous target-motivated polymerization/nicking DNA nanomachineries based on a hyperbranched rolling circle amplification (HB-RCA)-assisted multiposition strand displacement reaction (SDR) signal amplification approach. Impressively, the ingenious technique not only realized target recycling via polymerization/nicking DNA nanomachineries but also involved HB-RCA amplification induced by the released transformation target as the repeated signal amplification. Most importantly, HB-RCA was firstly exploited to remarkably increase the local concentration and collision efficiency of the templates and primers, which could simultaneously generate multiple repeated DNA sequences as initiators to supply substantial banding positions for SDR, removing the massive fluorescence-resonance-energy-transfer (FRET) DNA duplexes from the repeated DNA sequences to remarkably avert the self-quenching of the fluorescence signal due to self-aggregation caused by the winding of the HB-RCA products, thereby leading to a conspicuously improved signal amplification multiplier. As proof of concept, an ingenious technique effectively and accurately distinguished target miRNA-21 even with a tiny change in cells compared to the conventional fluorescence in situ hybridization (FISH) approach. Moreover, the proposed fluorescent method apparently discriminated drug-manipulative miRNA expression level abnormities. Therefore, the proposed cascade nucleic acid amplification strategy could provide an epigamic avenue for ultrasensitive imaging of diverse biomarkers, which help researchers to better study the tumor mechanism, thereby unambiguously increasing cancer cure rates and reducing the risk of recurrence.

Framework and Spherical Nucleic Acids Synergistically Enhanced Electrochemiluminescence Nanosensors for Rapidly Diagnosing Acute Myocardial Infarction Based on Circulating MicroRNA Levels.

Acute myocardial infarction (AMI)-related microRNAs (miRNAs) in circulating blood have been proved as promising biomarkers for AMI diagnosis. The detection of these miRNAs at ultralow levels usually requires nucleic acid amplification strategies to improve the sensitivity at the cost of time. Given that the first hour after an AMI attack is the golden time for saving AMI patients' lives, shortening the time of ultrasensitive miRNA analysis is of great significance for clinical AMI diagnosis. Toward this goal, here we present a direct electrochemiluminescence (ECL) sensing strategy for fast and ultrasensitive miRNA detection, circumventing the time-consuming signal amplification steps. Target miRNAs are directly hybridized with two probe strands that are attached to a covalently hemin-modified spherical nucleic acid enzyme (SNAzyme) and a truncated triangular pyramid DNA nanoplatform on the electrode, respectively. Both of them improve the ECL signal and meanwhile reduce the background, thereby remarkably promoting the detection sensitivity of target miRNAs. It enables the rapid detection of an AMI-related miRNA (miR-499) at 10 aM in human serum within 30 min using the SNAzyme-catalyzed luminol-H2O2 ECL reaction. This sensing strategy is then utilized for AMI diagnosis via probing endogenous miR-499 in patients' circulating blood with endogenous miR-16 as an intrinsic reference, showing a significant difference (P < 0.001) between the miR-499 levels of patients and the healthy.

Oriented interfacial self-assembled SERS platform with dual nucleic acid amplification for detection of MiRNA 21

Liquid‒liquid interface self-assembly was recently proposed to be an efficient strategy for surface-enhanced Raman scattering (SERS) detection. Traditionally, most of the research focuses on the self-assembly of single morphology and homogeneous materials, which are susceptible to the rotation of materials in the liquid phase due to the interference of Brownian motion and the external environment, causing poor stability. In this work, we introduced ternary Ag–Au–SiO2 nanoparticles to a liquid‒liquid self-assembled SERS platform, utilizing the differences in hydrophilicity of the materials to achieve oriented interfacial arrays, improving the stability of the self-assembled SERS substrate. To enhance the detection efficiency, a dual nucleic acid amplification strategy was designed to reach ultrasensitive detection of target microRNA 21 with a detection limit of (LOD) of 1.41 aM, providing a foresight for the practicability of the two-phase interfacial SERS platform.

Three-Dimensional Printed Microdevice to Enhance Headspace Microextraction for Enrichment of Histamine in Milk.

In this work, a three-dimensional (3D) printed microdevice was designed to fix a drop of extractant that was applied to the enrichment of the most toxic biogenic amine, histamine, by headspace single-drop microextraction (HS-SDME). Concomitantly, based on the hybridization chain reaction of the histamine aptamer isothermal nucleic acid amplification strategy, a new fluorescence sensing method was developed to realize the highly sensitive detection of histamine. This is the first application of a 3D-printed microdevice to realize the HS-SDME process, which, among other advantages, effectively solves the problem of unstable and variable drop volumes that can plague traditional SDME and ensures the accuracy and repeatability of the extraction process. The calibration linear range of this SDME-fluorescence method was from 10 pM to 5 μM (R2 > 0.98), and the limit of detection was as low as 3 pM. In addition, the method was successfully demonstrated to determine histamine spiked in milk, with recoveries of between 93% and 104%, and relative standard deviations of less than 5%. The method established in this study has important practical significance for food safety monitoring and human health and provides new ideas and solutions for the design and application of biosensors.

A Comparison of Two Methods for Detection of Norovirus RNA in Environmental Swab Samples

Standardised molecular methods are available for the detection of norovirus from water and specific food items. Detection of norovirus from stool samples also relies on molecular methods, but differences exist between nucleic acid extraction, reverse transcription, and amplification strategies recommended by the ISO 15216-1:2017, and those employed in clinical laboratories. Here, we conduct a direct comparison of two methods for the detection and quantitation of norovirus from a stool sample and from artificially contaminated swabs. We also compare use of linear dsDNA standards as recommended in ISO 15216:2017 against an in vitro-transcribed single-stranded RNA (ssRNA) for estimation of norovirus genome copy number. Our results show that the two methods have comparable sensitivity for the detection of norovirus RNA from a clinical sample or swab. The use of a ssRNA standard revealed that quantitation performed against a linear dsDNA standard consistently underestimated the genome copy numbers by 1.5 to 2 log due to the relative inefficiency of the reverse transcription step. This has important implications for the estimation of the sensitivity of norovirus detection methods, comparability of results across sites, and assessment of viral loads that may be clinically significant or estimated to constitute infectious doses.

Advances in Nucleic Acid Amplification-Based Microfluidic Devices for Clinical Microbial Detection

Accurate and timely detection of infectious pathogens is urgently needed for disease treatment and control of possible outbreaks worldwide. Conventional methods for pathogen detection are usually time-consuming and labor-intensive. Novel strategies for the identification of pathogenic nucleic acids are necessary for practical application. The advent of microfluidic technology and microfluidic devices has offered advanced and miniaturized tools to rapidly screen microorganisms, improving many drawbacks of conventional nucleic acid amplification-based methods. In this review, we summarize advances in the microfluidic approach to detect pathogens based on nucleic acid amplification. We survey microfluidic platforms performing two major types of nucleic acid amplification strategies, namely, polymerase chain reaction (PCR) and isothermal nucleic acid amplification. We also provide an overview of nucleic acid amplification-based platforms including studies and commercialized products for SARS-CoV-2 detection. Technologically, we focus on the design of the microfluidic devices, the selected methods for sample preparation, nucleic acid amplification techniques, and endpoint analysis. We also compare features such as analysis time, sensitivity, and specificity of different platforms. The first section of the review discusses methods used in microfluidic devices for upstream clinical sample preparation. The second section covers the design, operation, and applications of PCR-based microfluidic devices. The third section reviews two common types of isothermal nucleic acid amplification methods (loop-mediated isothermal amplification and recombinase polymerase amplification) performed in microfluidic systems. The fourth section introduces microfluidic applications for nucleic acid amplification-based detection of SARS-CoV-2. Finally, the review concludes with the importance of full integration and quantitative analysis for clinical microbial identification.

HOGG1-assisted DNA methylation analysis via a sensitive lanthanide labelling strategy

The assessment of DNA methylation level is an important indicator for the diagnosis and treatment of some diseases. DNA methylation assays are usually based on nucleic acid amplification strategies, which are time-consuming and complicated in operation procedures. Herein, we proposed a sensitive lanthanide-labelled ICP-MS method for DNA methylation analysis that exploited the feature of Human 8-oxoGuanine DNA Glycosylase (hOGG1), which specifically recognizes 8-oxo-G/5mC base pairs to effectively distinguish methylated DNA. A low limit of detection of 84 pM was achieved, and a 0.1% methylation level can be discriminated in the mixture, without any amplification procedure. Compared with commonly used nucleic acid amplification strategies, this proposed method is time-saving and low probability of false positive. Moreover, this work has been successfully validated in human serum samples, the recovery rates is between 96.7% and 105%, and the relative standard deviation (RSD) is in the range of 3.0%–3.5%, indicating that this method has the potential to be applied in clinical and biological samples quantitative analysis.

Simultaneous detection of trace Hg2+ and Ag+ by SERS aptasensor based on a novel cascade amplification in environmental water

Mercury ions and silver ions will be doped in environment during the entire process of water circulation, which is harmful to ecological balance and human body. Surface Enhanced Raman Scattering (SERS) acts as an effective method in detecting heavy metal ions, due to its easy processing, rapid detection, and fingerprintidentification. At present, it is still a challenge to simultaneously detect multiple heavy metal ions based on SERS sensor. Herein, we developed a new SERS aptasensor, applying cascade nucleic acid amplification strategy to detect Hg2+ and Ag+, which greatly improved the sensitivity. Meanwhile, the use of aptamers can transform the concentrations of Hg2+ and Ag+ into DNA signal. And thymine-Hg2+-thymine and cytosine-Ag+-cytosine pairs were formed to simultaneously identify Hg2+ and Ag+. Multiple analytical strategy of this SERS aptasensor can not only avoid the inconvenience caused by different techniques or experiment conditions, but also reduce cost, time, and difficulty. In consequence, we obtained a lower detection limitation (LOD Hg2+=4.40 aM, LOD Ag+=9.97 aM) in the linear range of 0.2–125 fM. The results demonstrate that this SERS aptasensor is capable of being widely applied to simultaneously detect multiple heavy metal ions or other toxic substance in environmental water.

A rapid and label-free DNA-based interference reduction nucleic acid amplification strategy for viral RNA detection

Common reference methods for COVID-19 diagnosis include thermal cycling amplification (e.g. RT-PCR) and isothermal amplification methods (e.g. LAMP and RPA). However, they may not be suitable for direct detection in environmental and biological samples due to background signal interference. Here, we report a rapid and label-free interference reduction nucleic acid amplification strategy (IR-NAAS) that exploits the advantages of luminescent iridium(III) probes, time-resolved emission spectroscopy (TRES) and multi-branch rolling circle amplification (mbRCA). Using IR-NAAS, we established a luminescence approach for diagnosing COVID-19 RNAs sequences RdRp, ORF1ab and N with a linear range of 0.06–6.0 × 105 copies/mL and a detection limit of down to 7.3 × 104 copies/mL. Moreover, the developed method was successfully applied to detect COVID-19 RNA sequences from various environmental and biological samples, such as domestic sewage, and mice urine, blood, feces, lung tissue, throat and nasal secretions. Apart from COVID-19 diagnosis, IR-NAAS was also demonstrated for detecting other RNA viruses, such as H1N1 and CVA10, indicating that this approach has great potential approach for routine preliminary viral detection.

Contemporary electrochemical sensing and affinity biosensing to assist traces metal ions determination in clinical samples

AbstractThe determination of metals in clinical samples is an important challenge since their levels are relevant to ensure personal exposure to safe environments, as well as to assist in the prevention and treatment of disease. The low concentrations of metal ions in clinical samples and their coexistence with other metals and/or molecules of diverse nature implies the need for sensitive and selective analytical methodologies, which can be provided by conventional techniques. However, most of these techniques do not satisfy other highly demanded requirements such as high sampling frequency, multiplexed ability, and in‐field analysis. In this context, the simplicity, speed of response, and compatibility with single or multiplexed point‐of‐care determinations have led to the successful incursion of electrochemical (bio)sensing into the clinical field for the determination of these analytes. Electrochemical (bio)devices have come on strong by exploiting and combining the unique attributes provided by electrochemical techniques (primarily voltammetry but also giving way to impedimetry in electrochemical affinity biosensing), electrode materials (Hg, Bi, Sb, Au, GCE, CPE, ITO, FTO), and substrates (conventional, screen‐printed, flexible, paper, wearable or tattoo‐type), suitable modifiers (nanomaterials, polymers, mesoporous films), bioreceptors (aptamers, peptides, biomolecular switches, DNAzymes, and DNA molecules), and nucleic acid amplification strategies (rolling circle and exonucleases‐assisted target recycling). Therefore, the purpose of this review is to provide a bird's eye view of the state of the art, discussing the remarkable opportunities and potentialities demonstrated in the last decade by electrochemical sensors and electrochemical affinity biosensors for the analysis of metal ions in challenging clinical samples and forecasting future directions in this exciting field.