Development of a Recombinase-aided Amplification Combined With Lateral Flow Dipstick Assay for the Rapid Detection of the African Swine Fever Virus

Objective To establish a sensitive,simple and rapid detection method for African swine fever virus(ASFV)B646L gene.Methods A recombinase-aided amplification-lateral flow dipstick(RAA-LFD)assay was developed in this study.Recombinase-aided amplification(RAA)is used to amplify template DNA,and lateral flow dipstick(LFD)is used to interpret the results after the amplification is completed.The lower limits of detection and specificity of the RAA assay were verified using recombinant plasmid and pathogenic nucleic acid.In addition,30 clinical samples were tested to evaluate the performance of the RAA assay.Results The RAA-LFD assay was completed within 15 min at 37°C,including 10 min for nucleic acid amplification and 5 minutes for LFD reading results.The detection limit of this assay was found to be 200 copies per reaction.And there was no cross-reactivity with other swine viruses.Conclusion A highly sensitive,specific,and simple RAA-LFD method was developed for the rapid detection of the ASFV.

Establishment of a Multiplex Detection Method for Common Bacteria in Blood Based on Human Mannan-Binding Lectin Protein-Conjugated Magnetic Bead Enrichment Combined with Recombinase-Aided PCR Technology

Objective Recombinase-aided polymerase chain reaction(RAP)is a sensitive,single-tube,two-stage nucleic acid amplification method.This study aimed to develop an assay that can be used for the early diagnosis of three types of bacteremia caused by Staphylococcus aureus(SA),Pseudomonas aeruginosa(PA),and Acinetobacter baumannii(AB)in the bloodstream based on recombinant human mannanbinding lectin protein(M1 protein)-conjugated magnetic bead(M1 bead)enrichment of pathogens combined with RAP.Methods Recombinant plasmids were used to evaluate the assay sensitivity.Common blood influenza bacteria were used for the specific detection.Simulated and clinical plasma samples were enriched with M1 beads and then subjected to multiple recombinase-aided PCR(M-RAP)and quantitative PCR(qPCR)assays.Kappa analysis was used to evaluate the consistency between the two assays.Results The M-RAP method had sensitivity rates of 1,10,and 1 copies/μL for the detection of SA,PA,and AB plasmids,respectively,without cross-reaction to other bacterial species.The M-RAP assay obtained results for<10 CFU/mL pathogens in the blood within 4 h,with higher sensitivity than qPCR.M-RAP and qPCR for SA,PA,and AB yielded Kappa values of 0.839,0.815,and 0.856,respectively(P<0.05).Conclusion An M-RAP assay for SA,PA,and AB in blood samples utilizing M1 bead enrichment has been developed and can be potentially used for the early detection of bacteremia.

Trends and frontiers in signal amplification for aptamer-based tumor detection:A bibliometric analysis

BACKGROUND Malignant tumors are one of the leading causes of death worldwide,imposing a substantial economic and social burden.Early detection is the key to improving cure rates and reducing mortality rates,which requires the development of sensitive early detection technologies.Signal amplification techniques play a crucial role in aptamer-based early detection of tumors and are increasingly garnering attention from researchers.AIM To investigate the current research status,developmental trajectories,and hotspots in signal amplification for aptamer-based tumor detection through bibliometric analysis.METHODS English publications pertaining to signal amplification in aptamer-based tumor detection were retrieved from the Web of Science Core Collection database.VOSviewer and CiteSpace software were employed to analyze various information within this field,including countries,institutions,authors,co-cited authors,journals,co-cited journals,cited references,and keywords.RESULTS A total of 757 publications were included in this study.China accounted for 85.47%of all publications,with Nanjing University(China)emerging as the institution with the highest publication output.The most influential authors and journals were Hasanzadeh M.from Iran and"Biosensors and Bioelectronics",respectively.Exosomes and carcinoembryonic antigen(CEA)stood out as the most researched tumor-related molecules.Currently,the predominant signal amplification technique,nanomaterial,and signal transduction method were identified as hybridization chain reactions,gold nanoparticles,and electrochemical methods,respectively.Over the past 3 years,exosomes,CEA,electrochemical biosensors,and nanosheets have emerged as research hotspots,exhibiting a robust burst of intensity.CONCLUSION This study is the first bibliometric analysis of literature on signal amplification in aptamer-based tumor detection and elucidates the current status,hotspots,and prospective research directions within this realm.Additionally,it provides an important reference for researchers.

A Study of Radiation-Induced Telomere Instability Using Multiplex Ligation-Dependent Probe Amplification (MLPA)

The integrity of the chromosomes for two WIL2-derived lymphoblastoid cell lines (TK6 and WTK1) in the presence and absence of ionizing radiation was analyzed by Multiplex Ligation-Dependent Probe Amplification (MLPA). The TK6 cell line has the native p53 tumor-suppressor gene, whereas WTK1 cells contain a p53 mutation. Each cell line was isolated pre- and post-irradiation (2 and 3 Gy) and analyzed by MLPA. The impact of irradiation on these two cell lines was investigated using probes that target specific regions on chromosomes associated with subtelomeric regions. Results indicate that WTK1 and TK6 are impacted differently after irradiation, and that each cell line presents its own unique MLPA profile. The most notable differences are the appearance of a number of probes in the post-irradiated MLPA profile that are not present in the controls, and two unique probe signals only seen in WTK1 cells. These results build on our previous studies that indicate how different human cell lines can be affected by radiation in significantly different ways depending on the presence or absence of wild type p53.

Modified Sadowski formula-based model for the slope shape amplification effect under multistage slope blasting vibration

Blasting operations,which are crucial to open-pit mine production due to their simplicity and efficiency,require precise control through accurate vibration velocity calculations.The conventional Sadowski formula mainly focuses on blast center distance but neglects the amplification effect of blasting vibration waves by terraced terrain,from which the calculated blasting vibration velocities are smaller than the actual values,affecting the safety of the project.To address this issue,our model introduces the influences of slope and time into Sadowski formula to measure safety through blast vibration displacement.In the northern section of the open-pit quartz mine in Jinchang City,Gansu Province,China,the data of a continuous blasting slope project are referred to.Our findings reveal a noticeable vibration amplification effect during blasting when a multi-stage slope platform undergoes a sudden cross-sectional change near the upper overhanging surface.The amplification vibration coefficient increases with height,while vibration waves within rocks decrease from bottom to top.Conversely,platforms without distinct crosssectional changes exhibit no pronounced amplification during blasting.In addition,the vibration intensity decreases with distance as the rock height difference change propagates.The results obtained by the proposed blast vibration displacement equation incorporating slope shape influence closely agree with real-world scenarios.According to Pearson correlation coefficient(PPMCC)analysis,the average accuracy rate of our model is 88.84%,which exceeds the conventional Sadowski formula(46.92%).

Mesenchymal-epithelial transition factor amplification correlates with adverse pathological features and poor clinical outcome in colorectal cancer

BACKGROUND Colorectal cancer(CRC)is the third most common cancer and the second most common cause of cancer-related mortality worldwide.Mesenchymal-epithelial transition factor(MET)gene participates in multiple tumor biology and shows clinical potential for pharmacological manipulation in tumor treatment.MET amplification has been reported in CRC,but data are very limited.Investigating pathological values of MET in CRC may provide new therapeutic and genetic screening options in future clinical practice.AIM To determine the pathological significance of MET amplification in CRC and to propose a feasible screening strategy.METHODS A number of 205 newly diagnosed CRC patients undergoing surgical resection without any preoperative therapy at Shenzhen Cancer Hospital of Chinese Academy of Medical Sciences were recruited.All patients were without RAS/RAF mutation or microsatellite instability-high.MET amplification and c-MET protein expression were analyzed using fluorescence in situ hybridization(FISH)and immunohistochemistry(IHC),respectively.Correlations between MET aberration and pathological features were detected using the chi-squared test.Progression free survival(PFS)during the two-year follow-up was detected using the Kaplan-Meier method and log rank test.The results of MET FISH and IHC were com pared using one-way ANOVA.RESULTS Polysomy-induced MET amplification was observed in 14.4%of cases,and focal MET amplification was not detected.Polysomy-induced MET amplification was associated with a higher frequency of lymph node metastasis(LNM)(P<0.001)and higher tumor budding grade(P=0.02).In the survival analysis,significant difference was detected between patients with amplified-and non-amplified MET in a two-year follow-up after the first diagnosis(P=0.001).C-MET scores of 0,1+,2+,and 3+were observed in 1.4%,24.9%,54.7%,and 19.0%of tumors,respectively.C-MET overexpression correlated with higher frequency of LNM(P=0.002),but no significant difference of PFS was detected between patients with different protein levels.In terms of concordance between MET FISH and IHC results,MET copy number showed no difference in c-MET IHC 0/1+(3.35±0.18),2+(3.29±0.11)and 3+(3.58±0.22)cohorts,and the MET-to-CEP7 ratio showed no difference in three groups(1.09±0.02,1.10±0.01,and 1.09±0.03).CONCLUSION In CRC,focal MET amplification was a rare event.Polysomy-induced MET amplification correlated with adverse pathological characteristics and poor prognosis.IHC was a poor screening tool for MET amplification.

O(logN) Algorithm for Amplitude Amplification and O(logN) Algorithms for Amplitude Transfer in Grover’s Algorithm

Grovers algorithm is a category of quantum algorithms that can be applied to many problems through the exploitation of quantum parallelism. The Amplitude Amplification in Grovers algorithm is T = O(N). This paper introduces two new algorithms for Amplitude Amplification in Grovers algorithm with a time complexity of T = O(logN), aiming to improve efficiency in quantum computing. The difference between Grovers algorithm and our first algorithm is that the Amplitude Amplification ratio in Grovers algorithm is an arithmetic series and ours, a geometric one. Because our Amplitude Amplification ratios converge much faster, the time complexity is improved significantly. In our second algorithm, we introduced a new concept, Amplitude Transfer where the marked state is transferred to a new set of qubits such that the new qubit state is an eigenstate of measurable variables. When the new qubit quantum state is measured, with high probability, the correct solution will be obtained.

Research and Application of"Soft"Devices for Realizing Servo Amplification

This article analyzes and discusses the working principle and problems encountered by various servo amplification devices used in the on-site continuous adjustment system,analyzes and discusses the application of the servo mechanism,and analyzes the mechanism of the servo device's implementation of the"positioning"func-tion on the control device.Intended to guide the continuous adjustment process in controlling the function/accuracy of actuator equipment and application debugging,ensuring the safe and stable operation of production equipment and facilities.

Current trends in nanomaterials-mediated biosensing platforms and signal amplification strategies for antibiotics detection in dairy products

Dairy products have become one of the most prevalent daily foods worldwide,but safety concerns are rising.In dairy farming,unscrupulous traders misuse antibiotics to treat some diseases such as mastitis in cows,leading to antibiotic residues in dairy products.Rapid,sensitive,and simple detection methods for antibiotic residues are particularly important for food safety in dairy products.Traditional detection technology can effectively detect antibiotics,but there are defects such as complicated pre-treatment and high cost.Biosensors are widely used in food safety due to fast detection speed,low detection cost,strong anti-interference ability,and suitability for the field application.Nevertheless,these sensors often fail to trigger the signal conversion output due to low target concentration.To cope with this issue,some high-efficiency signal amplification systems can be introduced to improve the detection sensitivity and linear range of biosensors.In this review,we focused on:(i)Sources and toxicity of major antibiotics in animal-derived foods.(ii)Nanomaterial-mediated biosensors for real-time detection of target antibiotics in animal-derived foods.(iii)Signal amplification techniques to increase the sensitivity of biosensors.Finally,future prospects and challenges in this research field are discussed.

Enhancement of a recombinase-aided amplification assay using betaine and pullulan

Background:Nucleic acid amplification enhancers suitable for use in a recombinase-aided amplification(RAA)assay were studied for the first time,and amplification of a long-fragment(509 bp)was initially explored.Methods:Using recombinant plasmids and clinical samples,RAA fluorescence and basic methods were used to evaluate the efficacy.The fluorescence method was evaluated by threshold time and fluorescence value,and the basic method was characterized by 2%agarose gel electrophoresis.Results:Taking a previously established RAA assay for HPV18 as an example,we demonstrated that the addition of 0.2 M,0.4 M,and 0.6 M betaine and 10%pullulan could enhance the RAA.The new RAA assays with betaine and pullulan were named B-RAA and P-RAA,respectively.Using the B-RAA and P-RAA fluorescence methods,the threshold time values could be shortened by 1.72-2.32 minutes and 2.60 minutes,respectively,and the fluorescence values could be enhanced by 8847.25-9094.37 mv and 5250 mv,respectively.Using the basic method,the sensitivity could be increased 10-fold.We successfully amplified a long-fragment of 509 bp using a P-RAA assay with a sensitivity of 102 copies/μL(compared with 103 copies/μL in the RAA assay).Conclusions:Thus,we concluded that betaine and pullulan are effective additives to enhance the sensitivity of RAA assays.

Assessment of future Antarctic amplification of surface temperature change under different Scenarios from CMIP6

Global warming may result in increased polar amplification,but future temperature changes under different climate change scenarios have not been systematically investigated over Antarctica.An index of Antarctic amplification(AnA)is defined,and the annual and seasonal variations of Antarctic mean temperature are examined from projections of the Coupled Model Intercomparison Project Phase 6(CMIP6)under scenarios SSP119,SSP126,SSP245,SSP370 and SSP585.AnA occurs under all scenarios,and is strongest in the austral summer and autumn,with an AnA index greater than 1.40.Although the warming over Antarctica accelerates with increased anthropogenic forcing,the magnitude of AnA is greatest in SSP126 instead of in SSP585,which may be affected by strong ocean heat uptake in high forcing scenario.Moreover,future AnA shows seasonal difference and regional difference.AnA is most conspicuous in the East Antarctic sector,with the amplification occurring under all scenarios and in all seasons,especially in austral summer when the AnA index is greater than 1.50,and the weakest signal appears in austral winter.Differently,the AnA over West Antarctica is strongest in austral autumn.Under SSP585,the temperature increase over the Antarctic Peninsula exceeds 0.5℃when the global average warming increases from 1.5℃to 2.0℃above preindustrial levels,except in the austral summer,and the AnA index in this region is strong in the austral autumn and winter.The projections suggest that the warming rate under different scenarios might make a large difference to the future AnA.

Research progress and prospects of nucleic acid isothermal amplification technology

Nucleic acid(DNA and RNA)detection and quantification methods play vital roles in molecular biology.With the development of molecular biology,isothermal amplification of DNA/RNA,as a new molecular biology technology,can be amplified under isothermal condition,it has the advantages of high sensitivity,high specificity,and high efficiency,and has been applied in various fields of biotechnology,including disease diagnosis,pathogen detection,food hygiene and safety detection and so on.This paper introduces the progress of isothermal amplification technology,including rolling circle amplification(RCA),nucleic acid sequence-dependent amplification(NASBA),strand displacement amplification(SDA),loop-mediated isothermal amplification(LAMP),helicase-dependent amplification(HDA),recombinase polymerase amplification(RPA),cross-priming amplification(CPA),and its principle,advantages and disadvantages,and application development are briefly summarized.

Influence of Surface Types on the Seasonality and Inter-Model Spread of Arctic Amplification in CMIP6

A robust phenomenon termed the Arctic Amplification(AA)refers to the stronger warming taking place over the Arctic compared to the global mean.The AA can be confirmed through observations and reproduced in climate model simulations and shows significant seasonality and inter-model spread.This study focuses on the influence of surface type on the seasonality of AA and its inter-model spread by dividing the Arctic region into four surface types:ice-covered,ice-retreat,ice-free,and land.The magnitude and inter-model spread of Arctic surface warming are calculated from the difference between the abrupt-4×CO_(2)and pre-industrial experiments of 17 CMIP6 models.The change of effective thermal inertia(ETI)in response to the quadrupling of CO_(2) forcing is the leading mechanism for the seasonal energy transfer mechanism,which acts to store heat temporarily in summer and then release it in winter.The ETI change is strongest over the ice-retreat region,which is also responsible for the strongest AA among the four surface types.The lack of ETI change explains the nearly uniform warming pattern across seasons over the ice-free(ocean)region.Compared to other regions,the ice-covered region shows the maximum inter-model spread in JFM,resulting from a stronger inter-model spread in the oceanic heat storage term.However,the weaker upward surface turbulent sensible and latent heat fluxes tend to suppress the inter-model spread.The relatively small inter-model spread during summer is caused by the cancellation of the inter-model spread in ice-albedo feedback with that in the oceanic heat storage term.

Nonlinear optical imaging by detection with optical parametric amplification(invited paper)

Nonlinear optical imaging is a versatile tool that has been proven to be exceptionally useful in various research fields.However,due to the use of photomultiplier tubes(PMTs),the wide application of nonlinear optical imaging is limited by the incapability of imaging under am-bient light.In this paper,we propose and demonstrate a new optical imaging detection method based on optical parametric amplification(OPA).As a nonlinear optical process,OPA in-trinsically rejects ambient light photons by coherence gating.Periodical poled lithium niobate(PPLN)crystals are used in this study as the media for OPA.Compared to bulk nonlinear optical crystals,PPLN crystals support the generation of OPA signal with lower pump power.Therefore,this characteristic of PPLN crystals is particularly beneficial when using high-repetition-rate lasers,which facilitate high-speed optical signal detection,such as in spec-troscopy and imaging.A PPLN-based OPA system was built to amplify the emitted imaging signal from second harmonic generation(SHG)and coherent anti-Stokes Raman scattering(CARS)microscopy imaging,and the amplified optical signal was strong enough to be detected by a biased photodiode under ordinary room light conditions.With OPA detection,ambient-light-on SHG and CARS imaging becomes possible,and achieves a similar result as PMT detection under strictly dark environments.These results demonstrate that OPA can be used as a substitute for PMTs in nonlinear optical imaging to adapt it to various applications with complex.light ing conditions.

Personal glucose meters coupled with signal amplification technologies for quantitative detection of non-glucose targets:Recent progress and challenges in food safety hazards analysis

Ensuring food safety is paramount worldwide.Developing effective detection methods to ensure food safety can be challenging owing to trace hazards,long detection time,and resource-poor sites,in addition to the matrix effects of food.Personal glucose meter(PGM),a classic point-of-care testing device,possesses unique application advantages,demonstrating promise in food safety.Currently,many studies have used PGM-based biosensors and signal amplification technologies to achieve sensitive and specific detection of food hazards.Signal amplification technologies have the potential to greatly improve the analytical performance and integration of PGMs with biosensors,which is crucial for solving the challenges associated with the use of PGMs for food safety analysis.This review introduces the basic detection principle of a PGM-based sensing strategy,which consists of three key factors:target recognition,signal transduction,and signal output.Representative studies of existing PGM-based sensing strategies combined with various signal amplification technologies(nanomaterial-loaded multienzyme labeling,nucleic acid reaction,DNAzyme catalysis,responsive nanomaterial encapsulation,and others)in the field of food safety detection are reviewed.Future perspectives and potential opportunities and challenges associated with PGMs in the field of food safety are discussed.Despite the need for complex sample preparation and the lack of standardization in the field,using PGMs in combination with signal amplification technology shows promise as a rapid and cost-effective method for food safety hazard analysis.

Ultrathin metal-organic framework nanosheets (Cu-TCPP)-based isothermal nucleic acid amplification for food allergen detection

The rapid and accurate detection of peanuts and soybeans allergen is important to the food safety. In this study, Cu-TCPP nanosheet, a kind of ultra-thin metal-organic framework(MOF)was synthesized and applied in loop-mediated isothermal amplification(named Cu-TCPP@LAMP), which can inhibit the non-specific amplification by absorbing and precise temperature releasing of single primer. As thus, Cu-TCPP@LAMP can achieve high sensitivity and specific amplification of the target gene. As a result, peanut and soybean allergens genes contained in food were successfully detected with a favorable detection sensitivity(5 ng/μL for peanuts and 10 ng/μL for soybeans)and reliable repeatability(The coefficient of variation was 3.38% for peanuts and 3.33% for soybeans). Moreover, the established method was utilized for detection of several commercial products, and had a high consistency with the standard method. Apart from food allergens, this novel assay can be widely used in other areas, such as pathogen detection, tumor nucleic acid detection and so on.

Enhanced phase sensitive amplification towards improving noise immunity

Quantum states are essential resource for quantum-enhanced applications.Loss incurred in the distribution channel,however,dissipates the high signal-to-noise ratio advantage enjoyed by the squeezed state.Here,we first demonstrate noise immunity enhancement by using phase-sensitive amplifier(PSA)with measurement-based noiseless linear amplifier(MB-NLA).We explore the signal transfer capability with the amplifier in a noisy channel.The MB-NLA enhanced PSA has obvious suppression effect on channel noises,especially it has improvement for the noise contaminated signal.Better performance can be achieved by flexibly adjusting amplifier parameters.With the amplifier,it is promising to overcome the entanglement-distribution loss and show its superiority in squeezing based quantum sensing.

Influence of coupling asymmetry on signal amplification in a three-node motif

The three-node feedforward motif has been revealed to function as a weak signal amplifier. In this motif, two nodes(input nodes) receive a weak input signal and send it unidirectionally to the third node(output node). Here, we change the motif's unidirectional couplings(feedforward) to bidirectional couplings(feedforward and feedback working together).We find that a small asymmetric coupling, in which the feedforward effect is stronger than the feedback effect, may enable the three-node motif to go through two distinct dynamic transitions, giving rise to a double resonant signal response. We present an analytical description of the double resonance, which agrees with the numerical findings.

Development and Characterization of Microsatellite Markers for Harpadon nehereus Based on High-Throughput Sequencing and Cross-Species Amplification in Three Myctophiformes Fishes

Harpadon nehereus is a widespread economical fish found in the coastal seas of China and has important ecological value in the marine ecosystem.H_(o)wever,its germplasm resources have been seriously degraded due to natural factors and anthropogenic activities.In this study,high-throughput sequencing was applied to search for microsatellite loci in H.nehereus transcriptome to provide references for its resource conservation and utilization.Polymorphic loci were developed by non-denaturing polyacrylamide gel electrophoresis,and their cross-species amplified ability was detected in three related species.A total of 5652 microsatellites were identified from 16974320 unigenes.Among the primer pairs designed for 100 SSRs for PCR amplification,80%were successfully amplified,and 26 loci were polymorphic with a high number of alleles from 3 to 11 each.The expected(H_(e))and observed(H_(o))heterozygosities were 0.355–0.885 and 0.375–0.958,respectively.Most of the loci were highly polymorphic(polymorphism information content:0.316–0.852;mean:0.713),and these markers can be applied in the population genetic diversity research of H.nehereus.H_(o)wever,the transferability of these primers was low,probably because of the close relation of the collected species.In follow-up work,simple sequence repeats will be excavated with genome-based technologies,and related species will be gathered to address the present inadequacies.

Dissipation and amplification management in an electrical model of microtubules: Hybrid behavior network

The control of dissipation and amplification of solitary waves in an electrical model of a microtubule is demonstrated.This model consists of a shunt nonlinear resistance–capacitance(J(V)–C(V)) circuit and a series resistance–inductance(R–L) circuit. Through linear dispersion analysis, two features of the network are found, that is, low bandpass and bandpass filter characteristics. The effects of the conductance’s parameter λ on the linear dispersion curve are also analyzed. It appears that an increase of λ induces a decrease(an increase) of the width of the bandpass filter for positive(negative) values of λ. By applying the reductive perturbation method, we derive the equation governing the dynamics of the modulated waves in the system. This equation is the well-known nonlinear Schr?dinger equation extended by a linear term proportional to a hybrid parameter σ, i.e., a dissipation or amplification coefficient. Based on this parameter, we successfully demonstrate the hybrid behavior(dissipation and amplification) of the system. The exact and approximate solitary wave solutions of the obtained equation are derived, and the effects of the coefficient σ on the characteristic parameters of these waves are investigated. Using the analytical solutions found, we show numerically that the waves that are propagated throughout the system can be dissipated, amplified, or remain stable depending on the network parameters. These results are not only in agreement with the analytical predictions, but also with the existing experimental results in the literature.