Multiple-bud regeneration, i.e., multiple amplification, has been shown to exist in peripheral nerve regeneration. Multiple buds grow towards the distal nerve stump during proximal nerve fiber regeneration. Our previo...Multiple-bud regeneration, i.e., multiple amplification, has been shown to exist in peripheral nerve regeneration. Multiple buds grow towards the distal nerve stump during proximal nerve fiber regeneration. Our previous studies have verified the limit and validity of multiple ampli- fication of peripheral nerve regeneration using small gap sleeve bridging of small donor nerves to repair large receptor nerves in rodents. The present study sought to observe multiple ampli- fication of myelinated nerve fiber regeneration in the primate peripheral nerve. Rhesus monkey models of distal ulnar nerve defects were established and repaired using muscular branches of the right forearm pronator teres. Proximal muscular branches of the pronator teres were su- tured into the distal ulnar nerve using the small gap sleeve bridging method. At 6 months after suture, two-finger flexion and mild wrist flexion were restored in the ulnar-sided injured limbs of rhesus monkey. Neurophysiological examination showed that motor nerve conduction veloc- ity reached 22.63 _＋ 6.34 m/s on the affected side of rhesus monkey. Osmium tetroxide staining demonstrated that the number of myelinated nerve fibers was 1,657 ＋ 652 in the branches of pronator teres of donor, and 2,661 ~ 843 in the repaired ulnar nerve. The rate of multiple amplification of regenerating myelinated nerve fibers was 1.61. These data showed that when muscular branches of the pronator teres were used to repair ulnar nerve in primates, effective regeneration was observed in regenerating nerve fibers, and functions of the injured ulnar nerve were restored to a certain extent. Moreover, multiple amplification was subsequently detected in ulnar nerve axons.展开更多
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 mult...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.展开更多
Due to the fact that most microRNAs are small in size,low abundance in biological samples,homologous sequence among family members,and protein enzymes-based strategies display limited practical applications,therefore,...Due to the fact that most microRNAs are small in size,low abundance in biological samples,homologous sequence among family members,and protein enzymes-based strategies display limited practical applications,therefore,we reported a simple enzyme-free DNA sensor for microRNA detection utilizing a multiple signal amplification strategy.The sensing system termed as C-CHA-HCR includes six hairpin DNA reactants that are metastable on account of intramolecular hybridization.The DNA hairpin reactants are opened and hybridized with the corresponding complementary DNA strand in the presence of miR-21 via toehold-mediated CHA,HCR reaction,and circulation between CHA and HCR,resulting in a hugely amplifying signal output.Without introducing external protein enzymes,this sensing system showed highly sensitive and selective on the detection of miR-21.A linear response range of miR-21 from 25 pmol/L to 1 nmol/L with a limit of detection(LOD)of 1.8 pmol/L was obtained.This promising biosensor was successfully applied to the detection of microRNA in human serum samples with acceptable recovery rates,suggesting the potential applications in disease diagnosis,treatment,and prognosis.展开更多
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 analy...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.展开更多
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 dis...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.展开更多
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 me...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.展开更多
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 f...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.展开更多
基金supported by grants from the National Program on Key Basic Research Project of China(973 Program),No.2014CB542200the National Natural Science Foundation of China,No.31271284,81171146,31100860+1 种基金Program for Innovative Research Team in University of Ministry of Education of China,No.IRT1201the Natural Science Foundation of Beijing of China,No.7142164
文摘Multiple-bud regeneration, i.e., multiple amplification, has been shown to exist in peripheral nerve regeneration. Multiple buds grow towards the distal nerve stump during proximal nerve fiber regeneration. Our previous studies have verified the limit and validity of multiple ampli- fication of peripheral nerve regeneration using small gap sleeve bridging of small donor nerves to repair large receptor nerves in rodents. The present study sought to observe multiple ampli- fication of myelinated nerve fiber regeneration in the primate peripheral nerve. Rhesus monkey models of distal ulnar nerve defects were established and repaired using muscular branches of the right forearm pronator teres. Proximal muscular branches of the pronator teres were su- tured into the distal ulnar nerve using the small gap sleeve bridging method. At 6 months after suture, two-finger flexion and mild wrist flexion were restored in the ulnar-sided injured limbs of rhesus monkey. Neurophysiological examination showed that motor nerve conduction veloc- ity reached 22.63 _＋ 6.34 m/s on the affected side of rhesus monkey. Osmium tetroxide staining demonstrated that the number of myelinated nerve fibers was 1,657 ＋ 652 in the branches of pronator teres of donor, and 2,661 ~ 843 in the repaired ulnar nerve. The rate of multiple amplification of regenerating myelinated nerve fibers was 1.61. These data showed that when muscular branches of the pronator teres were used to repair ulnar nerve in primates, effective regeneration was observed in regenerating nerve fibers, and functions of the injured ulnar nerve were restored to a certain extent. Moreover, multiple amplification was subsequently detected in ulnar nerve axons.
文摘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.
基金supported by the National Natural Science Foundation of China(21874042,21974042).
文摘Due to the fact that most microRNAs are small in size,low abundance in biological samples,homologous sequence among family members,and protein enzymes-based strategies display limited practical applications,therefore,we reported a simple enzyme-free DNA sensor for microRNA detection utilizing a multiple signal amplification strategy.The sensing system termed as C-CHA-HCR includes six hairpin DNA reactants that are metastable on account of intramolecular hybridization.The DNA hairpin reactants are opened and hybridized with the corresponding complementary DNA strand in the presence of miR-21 via toehold-mediated CHA,HCR reaction,and circulation between CHA and HCR,resulting in a hugely amplifying signal output.Without introducing external protein enzymes,this sensing system showed highly sensitive and selective on the detection of miR-21.A linear response range of miR-21 from 25 pmol/L to 1 nmol/L with a limit of detection(LOD)of 1.8 pmol/L was obtained.This promising biosensor was successfully applied to the detection of microRNA in human serum samples with acceptable recovery rates,suggesting the potential applications in disease diagnosis,treatment,and prognosis.
基金project 61971125 of the National Natural Science Foundation of China and the Fundamental Research Funds for the Central Universities of China.
文摘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.
基金Project supported by the National Basic Research Program (973) of China (No.2007CB948104)the Natural Science Foundation of Zhejiang Province,China (No.Z207021)
文摘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.
基金We thank Angus Sidore and Freeman Lan for helpful scientific discussions.This work was supported by the UCSF Division of Hematology-Oncology Perkins Philanthropy(PLP)the National Science Foundation CAREER Award(Grant Number DBI-1253293)+2 种基金the National Institutes of Health(NIH)(Grant Numbers HG007233-01,R01-EB019453-01,and DP2-AR068129-01)the Defense Advanced Research Projects Agency Living Foundries Program(Contract Numbers HR0011-12-C-0065,N66001-12-C-4211,and HR0011-12-C-0066)Fold F(x)Program(Contract Number DE-AC02-05CH11231).
文摘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.
文摘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.
