Simple, Rapid and Sensitive Detection of Pseudomonas aeruginesa by Colorimetric Multiple Cross Displacement Amplification

Pseudomonas aeruginosa(P:aeruginosa)is a major opportunistic pathogen in hospital-acquired infections.Thus,carly diagnosis is the best strategy for fighting against these infections.In this report,we incorporated multiple crOsS displacement amplification(MCDA)combined with the malachite green(MG)for rapid,sensitive,specific and visual detection of P.aeruginosa by targeting the oprl gene.The MCDA-MG assay was conducted at 67°C for only 40 min during the amplification stage,and then products were directly detected by using colorimetric indicators(MG),eliminating the use of an electrophoresis instrument or amplicon analysis equipment.The entire process,including specimen processing(35 min),amplification(40 min)and detection(5 min),can be finished within 80 min.All 30 non-P.aeruginosa strains tested negative,indicating the high specificity of the MCDA primers.The analytical sensitivity of the MCDA-MG assay was 100 fg of genomic templates per reaction in pure culture,which was in complete accordance with MCDA by gel clectrophoresis and real-time turbidity.The assay was also successfully applied to detecting P.aeruginosa in stool samples.Therefore,the rapidity,simplicity,and nearly equipment free platform of the MCDA-MG technique make it possible for clinical diagnosis,and more.

Effects of foundation rocking and uplifting on displacement amplification factor

Prediction of displacement demand to assess seismic performance of structures is a necessary step where nonlinear static procedures are followed.While such predictions have been well established in literature for fixed-base structures,fewer bodies of researches have been carried out on the effect of rocking and uplifting of shallow foundations supported by soil,on such prediction.This paper aimed to investigate the effect of soil structure interaction on displacement amplification factor C1 using the beam on nonlinear Winkler foundation concept.A practical range of natural period,force reduction factors,and wide range of anticipated behavior from rocking,uplifting and hinging are considered and using thousands nonlinear time history analysis,displacement amplification factors are evaluated.The results indicate that the suggested equations in current rehabilitation documents underestimate displacement demands in the presence of foundation rocking and uplift.Finally,using regression analyses,new equations are proposed to estimate mean values of C1.

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.

Inelastic displacement ratio of low-to mid-rise BRBFs designed under variable levels of seismicity

Buckling-restrained braces(BRBs)have shown their capability to provide building structures with stiffness,strength,and ductility.Estimating the seismic drifts of buckling-restrained braced frames(BRBFs)is an important design step to control structural and non-structural damage.In current practice of seismic design,the estimation of seismic drifts of BRBFs is performed by using empirical calculations that are independent upon either the type of the structural system or the design level of seismicity.In these empirical calculations,the seismic drifts are estimated by amplifying the reduced elastic drifts obtained under design lateral loading with a displacement amplification factor(DAF).The value of DAF is considered equal to the product of the response modification factor R and the inelastic displacement ratioρ.The goal of the current research is to assess the value ofρfor low-to mid-rise BRBFs designed under low and high levels of seismicity.This goal has been achieved by conducting a series of elastic and inelastic time-history analyses pertaining to an ensemble of earthquake records on 3-,6-and 9-story BRBFs.The results indicate that theρ-ratio increases with an increase in design seismic intensity and an increase in experienced inelasticity.The range ofρfor low seismicity designs ranges from 0.63 to 0.9,while for high seismicity designs this range stretches from 0.83 to 1.29.It has been found that the consideration of a generalρ-ratio of 1.0 is a reasonable estimation for the design of the BRBFs considered in this study.

A primer-initiated strand displacement amplification strategy for sensitive detection of 5-Hydroxymethylcytosine in genomic DNA

5-Hydroxymethylcytosine(5 hmC),an intermediate product of DNA demethylation,is important for the regulation of gene expression during development and even tumorigenesis.The challenges associated with determination of 5 hm C level include its extremely low abundance and high structural similarity with other cytosine derivatives,which resulted in sophisticated treatment with large amount of sample input.Herein,we developed a primer-initiated strand displacement amplification(PISDA)strategy to quantify the global 5 hm C in genomic DNA from mammalian tissues with high sensitivity/selectivity,low input and simple operation.This sensitive fluorescence method is based on 5 hmC-specific glucosylation,primer ligation and DNA amplification.After the primer was labeled on 5 hm C site,DNA polymerase and nicking enzyme will repeatedly act on each primer,causing a significant increase of fluorescence signal to magnify the minor difference of 5 hm C content from other cytosine derivatives.This method enables highly sensitive analysis of 5 hm C with a detection limit of 0.003%in DNA(13.6 fmol,S/N=3)from sample input of only 150 ng,which takes less than 15 min for determination.Further determination of 5 hmC in different tissues not only confirms the widespread presence of 5 hmC but also indicates its significant variation in different tissues and ages.Importantly,this PISDA strategy exhibits distinct advantages of bisulfite-free treatment,mild conditions and simple operation without the involvement of either expensive equipment or large amount of DNA sample.This method can be easily performed in almost all research and medical laboratories,and would provide a promising prospect to detect global 5 hmC in mammalian tissues.

Sequence-specific recognition of HIV-1 DNA based upon nicking-assisted strand displacement amplification and triplex DNA

Nucleic acid amplification test is a reliable method for primary human immunodeficiency virus （HIV） infection diagnosis. Herein, a novel fluorescent method for sequence-specific recognition of DNA fragment of HIV-1 was established based upon nicking-assisted strand displacement amplification （SDA） and triplex DNA. In the presence of target dsDNA, nicking-assisted SDA process generated a lot of ssDNA, which hybridized with molecular beacon to produce signal. The fluorescence intensity was proportional to the concentration of target dsDNA within the range from 5 to 1000 pmol/L, with a detection limit of 1.4 pmol/L. Moreover, it successfully distinguished target dsDNA from the nucleic acid extractive of human blood. Thus this method has the merit of high sensitivity, and it is suitable for sequence-specific recognition of target dsDNA in complex matrices, which made it a potential application in diagnosis of acquired immunodeflciency syndrome （AIDS） in the future.

Recent advances and application in whole-genome multiple displacement amplification

Background:The extremely small amount of DNA in a cell makes it difficult to study the whole genome of single cells,so whole-genome amplification(WGA)is necessary to increase the DNA amount and enable downstream analyses.Multiple displacement amplification(MDA)is the most widely used WGA technique.Results:Compared with amplification methods based on PCR and other methods,MDA renders high-quality DNA products and better genome coverage by using phi29 DNA polymerase.Moreover,recently developed advanced MDA technologies such as microreactor MDA,emulsion MDA,and micro-channel MDA have improved amplification uniformity.Additionally,the development of other novel methods such as TruePrime WGA allows for amplification without primers.Conclusion:Here,we reviewed a selection of recently developed MDA methods,their advantages over other WGA methods,and improved MDA-based technologies,followed by a discussion of future perspectives.With the continuous development of MDA and the successive update of detection technologies,MDA will be applied in increasingly more fields and provide a solid foundation for scientific research.

Review:Whole genome amplification in preimplantation genetic diagnosis

Preimplantation genetic diagnosis (PGD) refers to a procedure for genetically analyzing embryos prior to implantation,improving the chance of conception for patients at high risk of transmitting specific inherited disorders.This method has been widely used for a large number of genetic disorders since the first successful application in the early 1990s.Polymerase chain reaction (PCR) and fluorescent in situ hybridization (FISH) are the two main methods in PGD,but there are some inevitable shortcomings limiting the scope of genetic diagnosis.Fortunately,different whole genome amplification (WGA) techniques have been developed to overcome these problems.Sufficient DNA can be amplified and multiple tasks which need abundant DNA can be performed.Moreover,WGA products can be analyzed as a template for multi-loci and multi-gene during the subsequent DNA analysis.In this review,we will focus on the currently available WGA techniques and their applications,as well as the new technical trends from WGA products.

Simultaneous photoelectrochemical detection of dual microRNAs by capturing CdS quantum dots and methylene blue based on target-initiated strand displaced amplification

Herein,we propose a novel photoelectrochemical(PEC) biosensor for dual microRNAs(miRNAs) highly sensitive and simultaneous biosensing based on strand displaced amplification(SDA) reaction.The recognition of HmiR-21 and Hlet-7 a by microRNA-21 and let-7 a leads to their change in hairpin structures,subsequently initiating the immobilization of abundant CdS quantum dots(CdS QD s) and methylene blue(MB) based on SDA reaction.The immobilized CdS QDs and MB produce both high PEC currents under430 nm light and 627 nm light illumination,respectively,and the generated PEC currents are closely relied on target miRNAs amounts.Thus,highly sensitive and simultaneous detection of microRNA-21 and let-7 a was readily achieved with detection limit at 6.6 fmol/L and 15.4 fmol/L based on 3σ,respectively.Further,this PEC biosensor was applied in simultaneous analysis of miRNA-21 and let-7 a in breast cancer patient’s serum with acceptable results.We expect this biosensor will find more useful application in diagnosis of miRNA-related diseases.

Measurement of copy number variation in single cancer cells using rapid-emulsification digital droplet MDA

Uniform amplification of low-input DNA is important for applications across biology,including single-cell genomics,forensic science,and microbial and viral sequencing.However,the requisite biochemical amplification methods are prone to bias,skewing sequence proportions and obscuring signals relating to copy number.Digital droplet multiple displacement amplification enables uniform amplification but requires expert knowledge of microfluidics to generate monodisperse emulsions.In addition,existing microfluidic methods are tedious and labor intensive for preparing many samples.Here,we introduce rapid-emulsification multiple displacement amplification,a method to generate monodisperse droplets with a hand-held syringe and hierarchical droplet splitter.Although conventional microfluidic devices require >10 min to emulsify a sample,our system requires tens of seconds and yields data of equivalent quality.We demonstrate the approach by using it to accurately measure copy number variation(CNV)in single cancer cells.

Nanoelectromechanical resonant narrow-band amplifiers

This study demonstrates amplification of electrical signals using a very simple nanomechanical device.It is shown that vibration amplitude amplification using a combination of mechanical resonance and thermal-piezoresistive energy pumping,which was previously demonstrated to drive self-sustained mechanical oscillation,can turn the relatively weak piezoresistivity of silicon into a viable electronic amplification mechanism with power gains of 420 dB.Various functionalities ranging from frequency selection and timing to sensing and actuation have been successfully demonstrated for microscale and nanoscale electromechanical systems.Although such capabilities complement solid-state electronics,enabling state-of-the-art compact and high-performance electronics,the amplification of electronic signals is an area where micro-/nanomechanics has not experienced much progress.In contrast to semiconductor devices,the performance of the proposed nanoelectromechanical amplifier improves significantly as the dimensions are reduced to the nanoscale presenting a potential pathway toward deep-nanoscale electronics.The nanoelectromechanical amplifier can also address the need for ultranarrow-band filtering along with the amplification of lowpower signals in wireless communications and certain sensing applications,which is another need that is not efficiently addressable using semiconductor technology.

Antarctic thermolabile uracil-DNA-glycosylase- supplemented multiple cross displacement amplification using a label-based nanoparticle lateral flow biosensor for the simultaneous detection of nucleic acid sequences and elimination of carryover contamination

Here, we report a novel and universal methodology,termed ＂ntarctic thermolabile uracil-DNA-glycosylase （AUDG）-supplemented nucleic acid amplification techniques （NAAs） using a labeled-based nanoparticle lateral flow biosensor （LFB）＂ （AUDG-NAAs-LFB）, which merges enzymatic （AUDG） digestion of contaminant amplicons with different nucleic acid amplification techniques （NAAs）, and uses a lateral flow biosensor （LFB） for the rapid and visual confirmation of the presence of a target nucleic acid sequence. AUDG-NNAs-LFB is a one-pot, closedvessel assay, that can effectively eliminate false-positive signals arising from either carryover contaminants or the interaction between labeled primers. A new LFB was devised for detecting three targets （two amplicons generated from amplification of target sequences, and a chromatography control）, without the need for probe- hybridization or additional incubation steps. As a proof of concept, multiple cross displacement amplification （MCDA）, which is a specific, sensitive, and rapid isothermal amplification method, was selected as the model amplification technique to demonstrate the feasibility of AUDG-NAAs-LFB. As a result, we demonstrate the applicability of the AUDG-MCDA-LFB method for simultaneously detecting high-risk human papillomaviruses genotypes 16 and 18, which are the most and second-most prevalent strains of the virus reported in women worldwide. We also confirm the principle behind the AUDG-MCDA- LFB assay and validate its sensitivity, reproducibility, and specificity using serial dilutions of the type-specific plasmids, as well as clinical samples. This proof- of-concept method （AUDG-MCDA-LFB） can be easily reconfigured to detect various nudeic acid sequences by redesigning the specific MCDA primers.