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.

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%).

Rapid detection of the rice false smut fungus Ustilaginoidea virens by lateral flow strip-based recombinase polymerase amplification assay

Rice false smut,caused by Ustilaginoidea virens,is a devastating disease that greatly reduces rice yield and quality.However,controlling rice false smut disease is challenging due to the unique infection mode of U.virens.Therefore,there is a need for early diagnosis and monitoring techniques to prevent the spread of this disease.Lateral flow strip-based recombinase polymerase amplification(LF-RPA)overcomes the limitations of current U.virens detection technologies,which are time-consuming,require delicate equipment,and have a high false-positive rate.In this study,we used a comparative genomics approach to identify Uv_3611,a specific gene of U.virens,as the target for the LF-RPA assay.The designed primers and probe efffectively detected the genomic DNA(gDNA)of U.virens and demonstrated no cross-reactivity with related pathogens.Under optimal conditions,the LF-RPA assay demonstrated a sensitivity of 10 pg of U.virens gDNA.Additionally,by incorporating a simplified PEG-NaOH method for plant DNA extraction,the LF-RPA assay enabled the detection of U.virens in rice spikelets within 30 min,without the need for specialized equipment.Furthermore,the LF-RPA assay successfully detected U.virens in naturally infected rice and seed samples in the field.Therefore,the LF-RPA assay is sensitive,efficient,and convenient,and could be developed as a kit for monitoring rice false smut disease in the field.

Development of a multiplex recombinase polymerase amplification coupled with lateral flow dipsticks for the simultaneous rapid detection of Salmonella spp.,Salmonella Typhimurium and Salmonella Enteritidis

Objectives:Salmonella spp.is a world-leading foodborne pathogen and its rapid detection is essential for ensuring food safety.Conventional methods require expensive instruments,considerable operational skills and cannot provide fast mobile on-site systems to detect Salmonella in food.Materials and Methods:A visual method was established based on multiple recombinase polymerase amplification(RPA)coupled with lateral flow dipsticks(LFD)for the simultaneous detection of Salmonella spp.,Salmonella Enteritidis and Salmonella Typhimurium in vitro and food.Results:The optimal volume and temperature for the multiplex RPA-LFD method were determined to be 25μL and 38°C,respectively.The reaction process was completed within 25 min and the results were observed visually.The limits of detection(LODs)were 2.8×10^(2),5.9×10^(2),and 7.6×10^(2) CFU/mL for Salmonella spp.,S.Enteritidis and S.Typhimurium,respectively.Meanwhile,the results of the established method showed no cross-reactivity between the Salmonella cells and other common foodborne bacteria,which was highly specific for Salmonella.More importantly,the developed method exhibited good performance in artificially contaminated chicken samples with the LODs of 2.8×10^(3),5.9×10^(3),and 7.6×10^(3) CFU/mL for Salmonella spp.,S.Enteritidis,and S.Typhimurium,respectively.Finally,the application of the multiple RPA-LFD methods in retailed food samples displayed that this method was effective and practical for the detection of Salmonella spp.in food.Conclusion:The developed multiplex RPA-LFD method provides a new sensitive and rapid alternative for the specific detection of Salmonella spp.and its important serovars in food.

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.

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.

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.

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.

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.

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.

Designing Primers for H5 and H7 Subtypes of Avian Influenza Virus and Multiplex RT-PCR Amplification

[Objective] The research aimed to design primers that are suitable for detecting H5 and H7 subtypes of avian influenza virus （AIV） ; [Method] DNAStar was used to analyze the homology of the sequences of H5 and H7 subtypes of AIV accessed in GenBank, and design primers（ by Primer Premier 5.0） on high homologous region of these sequences, and then amplified by RT-PCR. [Result] The multiplex RT-PCR amplification, agarose gel electrophoresis and sequencing results showed that the self-designed primers are successful for detecting AIV. [Conclusion] It is feasible to rapidly diagnose AIV through this method.

Application of Abnormal Touchdown PCR with High Degeneracy Primer in Amplification of Large-Family Genes

[Objective] The aim was to explore the special methods for amplification of large-family genes by using primers with high degeneracy.[Method] By using the primers with high degeneracy,conventional PCR,conventional touchdown PCR and the optimized abnormal touchdown PCR were respectively carried out to amplify the genomic DNA of Cyprinus carpio.[Result] Only one evident electrophoretic band and a few Sox genes were obtained by using normal PCR;no obvious electrophoretic band but dispersive product was obtained by normal touchdown PCR;ideal result was obtained by the abnormal touchdown PCR that three evident electrophoretic bands and much more Sox genes were amplified.[Conclusion] The research provided theoretical basis for the optimization and selection of PCR amplification conditions of the large-family genes.

Development of an One-step Reverse Transcription Loop-mediated Isothermal Amplification Method for Rapid Detection of Bovine Viral Diarrhea Virus

Objective] This study aimed to develop a reverse transcription loop-medi-ated isothermal amplification （RT-LAMP） method for detecting BVDV. [Method] Since gp48 gene of BVDV is among the most conserved regions, a set of four primers was designed to amplify six target sequences at the gp48 gene region for the RT-LAMP assay. The optimization of the RT-LAMP reaction was performed by evaluat-ing reaction temperature and reaction time. [Result] The RT-LAMP aasay was suc-cessful y conducted at 56 ℃ within 40 min under isothermal conditions, and the re-sults could be detected as ladder-like bands using agarose gel electrophoresis. The RT-LAMP assay is highly sensitive and able to detect 3.74 ×100 copies/μl of BVDV RNA, as no cross-reaction was observed with other viruses. [Conclusion] Overal , the newly established RT-LAMP assay indicates the potential application in both clinical diagnosis and field surveil ance of BVDV.

Visual Detection of Vibrio parahaemolyticus using Combined CRISPR/Cas12a and Recombinase Polymerase Amplification

Objective To establish an ultra-sensitive,ultra-fast,visible detection method for Vibrio parahaemolyticus(VP).Methods We established a new method for detecting the tdh and trh genes of VP using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 12a(CRISPR/Cas12a)combined with recombinase polymerase amplification and visual detection(CRISPR/Cas12a-VD).Results CRISPR/Cas12a-VD accurately detected target DNA at concentrations as low as 10^(-18)M(single molecule detection)within 30 min without cross-reactivity against other bacteria.When detecting pure cultures of VP,the consistency of results reached 100%compared with real-time PCR.The method accurately analysed pure cultures and spiked shrimp samples at concentrations as low as 10^(2)CFU/g.Conclusion The novel CRISPR/Cas12a-VD method for detecting VP performed better than traditional detection methods,such as real-time PCR,and has great potential for preventing the spread of pathogens.

One New Method of Nucleic Acid Amplification-Loop-mediated Isothermal Amplification of DNA

Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method, which amplifies DNA with high specificity, sensitivity, rapidity and efficiency under isothermal conditions using a set of four specially designed primers and a Bst DNA polymerase with strand displacement activity. The basic principle, characteristics, development of LAMP and its applications are summarized in this article.

Rapid detection of Pseudomonas aeruginosa by cross priming amplification

Pseudomonas aeruginosa(PA)is an opportunistic pathogen of humans and animals and a common source of nosocomial infections especially of the respiratory tract.Pseudomonas aeruginosa is also a major bacterial disease of poultry and in particular,eggs and newly hatched chicks.In this study,we developed a simple,accurate and rapid molecular detection method using cross priming amplification(CPA)with a nucleic acid test strip to detect P.aeruginosa.The assay efficiently amplified the target gene within 45 min at 62℃only using a simple water bath.The detection limit of the method was 1.18x 102 copiesμL^-1 for plasmid DNA and 4.4 CFU mL^-1 for bacteria in pure culture,and was 100 times more sensitive than conventional PCR.We screened 83 clinical samples from yellow-feather broiler breeder chickens and hospitalized/treated dogs and cats using CPA,PCR and traditional culture methods.The positive sample ratios were 15.3%(13/83)by CPA,13.3%(11/83)by PCR and 12.1%(10/83)by the culture method.The established CPA method has significant advantages for detecting P.aeruginosa.The method is easy to use and possesses high specificity and sensitivity without the requirements of complicated experimental equipment.The PA-CPA assay is especially fit for outdoor and primary medical units and is an ideal system for the rapid detection and monitoring of P.aeruginosa.

Changes in polar amplification in response to increasing warming in CMIP6

The climate in polar regions has experienced an obvious warming amplification due to global warming.In this study,the changes in polar amplification are analyzed in response to feedback mechanisms(including Planck,lapse rate,cloud,water vapor,albedo feedback,CO_(2) radiative forcing,ocean heat uptake,and atmospheric heat transport)under three warming scenarios in CMIP6—namely,SSP1-2.6,SSP2-4.5,and SSP5-8.5.The results show that,by quantifying the warming contribution of different feedback mechanisms to surface air temperature with the“radiative kernel”method,Arctic amplification(AA)is stronger than Antarctic amplification(ANA),mostly resulting from the lapse rate feedback,followed by the albedo and Planck feedbacks.Furthermore,ocean heat uptake causes stronger polar warming in winter than in summer.During winter,the lapse rate feedback causes a larger AA than ANA.The intermodel spread for both AA and ANA decrease with increasing strength of global warming from SSP1-2.6 to SSP5-8.5,and the dominant mechanisms are the Planck,lapse rate,albedo,and ocean heat uptake feedbacks.These findings help to enhance our understanding of polar regions’responses to different strengths of global warming.

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.

Establishment of Reverse-transcription Loopmediated Isothermal Amplification Method for Detection of Wheat Streak Mosaic Virus

A reverse-transcription loop-mediated isothermal amplification （RT-LAMP） method was established for the detection of wheat streak mosaic virus （WSMV）. Ac-cording to the conservative regions of the genes that encode the coat protein of WSMV, 2 pairs of primers were designed. Final y, the 1st pair of primers was select-ed through the specificity test. The sensitivity test showed the sensitivity of RT-LAMP method was 10 times higher than that of RT-PCR. In addition, the amplifica-tion of target gene could be judged visual y from the presence of fluorescence （cal-cein） in the final reaction system. The RT-LAMP method, established in this study, was rapid, easy, specific and sensitive. Moreover, it did not require sophisticated equip-ment. The RT-LAMP was suitable for the rapid detection of WSMV.

Chromosomal Localization of Genes bz1,bz2 in Maize by Using Ultra-sensitive FISH with Tyramide Signal Amplification(TSA-FISH)

It has been reported that endosperm undergoes programmed cell death (PCD) during maize kernel development.Both bz1 (bronze ) and bz2 are anthocyanin biosynthetic genes,and related to development of aleuronic layer of maize seeds.Tyramide signal amplification fluorescence in situ hybridization (TSA FISH) is a novel and high sensitive FISH technique,which is suitable for routine application in plant cytogenetic research.Using this technique,we physically mapped the bz1 gene onto the short arm of chromosome 9 and the long arm of chromosome 1;the percentage distances from centromere to hybridization site were 40.2,75.4 respectively,and the bz2 onto the long arm of chromosome 1 and the short arm of chromosome 5;the percentage distances from centromere to hybridization site were 21.6,15.3 separately.The TSA FISH techniques of small low copy DNA sequences for plants are discussed.