Soil DNA extraction,such as microbial community analysis and gene drift detection,is an important basis for multiple analyses in different fields.Nevertheless,the soil DNA extraction methods for field detection are st...Soil DNA extraction,such as microbial community analysis and gene drift detection,is an important basis for multiple analyses in different fields.Nevertheless,the soil DNA extraction methods for field detection are still lacking.This study established a rapid soil DNA extraction(RSDE)method that can be used in field detection.In this method,we first utilized the optimized lysate to isolate DNA from soil and then used a filtration membrane and a DNA adsorption membrane to purify the DNA via the column method.Moreover,we used the pressure from the syringe instead of the conventional centrifugal force of the centrifuge to assist the sample filtration,resulting in very low requirements for this method,with an extraction time of less than 20 min.Furthermore,we demonstrated that the RSDE method was applicable for DNA extraction from different types of soils,with the demand for soil samples as low as 0.1 g and that the amount of obtained DNA was,to some extent,greater than that obtained by a commercial kit.Further analysis revealed that this extracted genomic DNA can be used directly for polymerase chain reaction(PCR)analysis,including ordinary PCR,real-time fluorescent quantitative PCR,and recombinase polymerase amplification(RPA)-CRISPR/Cas12a visual assays.In addition,we demonstrated that this method can be used to extract DNA from residual plant roots in addition to soil microbes,which lays a foundation for the comprehensive analysis of soil plants and microorganisms.In summary,the RSDE method proposed in this study may have wide application prospects.展开更多
Liver fibrosis is an important pathological precondition for hepatocellular carcinoma.The degree of hepatic fibrosis is positively correlated with liver cancer.Liver fibrosis is a series of pathological and physiologi...Liver fibrosis is an important pathological precondition for hepatocellular carcinoma.The degree of hepatic fibrosis is positively correlated with liver cancer.Liver fibrosis is a series of pathological and physiological process related to liver cell necrosis and degeneration after chronic liver injury,which finally leads to extracellular matrix and collagen deposition.The early detection and precise staging of fibrosis and cirrhosis are very important for early diagnosis and timely initiation of appropriate therapeutic regimens.The risk of severe liver fibrosis finally progressing to liver carcinoma is＆gt;50%.It is known that biopsy is the gold standard for the diagnosis and staging of liver fibrosis.However,this method has some limitations,such as the potential for pain,sampling variability,and low patient acceptance.Furthermore,the necessity of obtaining a tissue diagnosis of liver fibrosis still remains controversial.An increasing number of reliable non-invasive approaches are now available that are widely applied in clinical practice,mostly in cases of viral hepatitis,resulting in a significantly decreased need for liver biopsy.In fact,the noninvasive detection and evaluation of liver cirrhosis now has good accuracy due to current serum markers,ultrasound imaging,and magnetic resonance imaging quantification techniques.A prominent advantage of the non-invasive detection and assessment of liver fibrosis is that liver fibrosis can be monitored repeatedly and easily in the same patient.Serum biomarkers have the advantages of high applicability（〉95%）and good reproducibility.However,their results can be influenced by different patient conditions because none of these markers are liver-specific.The most promising techniques appear to be transient elastography and magnetic resonance elastography because they provide reliable results for the detection of fibrosis in the advanced stages,and future developments promise to increase the reliability and accuracy of the staging of hepatic fibrosis.This article aims to describe the recent progress in the development of non-invasive assessment methods for the staging of liver fibrosis,with a special emphasize on computer-aided quantitative and deep learning methods.展开更多
Objective:Plasma cell-free DNA(cfDNA)methylation has shown potential in the detection and prognostic testing of multiple cancers.Here,we comprehensively investigate the performance of cfDNA methylation for gastric can...Objective:Plasma cell-free DNA(cfDNA)methylation has shown potential in the detection and prognostic testing of multiple cancers.Here,we comprehensively investigate the performance of cfDNA methylation for gastric cancer(GC)detection and prognosis.Methods:GC-specific differentially methylated regions(DMRs)were identified by sequencing 56 GC tissues and 59 normal adjacent tissues(NATs).We then performed targeted bisulfite sequencing of cfDNA from 294 GC and 446 non-gastric cancer(NGC)plasma samples,identifying 179 DMRs that overlapped with those in tissue samples.The efficacy of plasma cfDNA methylation markers for GC detection and prognosis was evaluated.Results:Based on the 179 DMRs overlapping with those in tissue samples,the random forest(RF)model using28 DMRs achieved an area under the curve(AUC)of 0.998 in the training cohort,whereas further refinement to the top 6 DMRs resulted in an AUC of 0.985.Consistent results were obtained in the validation cohort(28 DMR AUC:0.985;6 DMR AUC:0.988).Support vector machine(SVM)and logistic regression(LR)models also demonstrated robust performance.Additionally,an 11-DMR signature was developed for prognostic prediction,successfully identifying high-risk GC patients with significantly shorter overall survival.Conclusions:Our study highlights the potential utility of cfDNA methylation markers for both the detection and prognostication of GC.展开更多
Detecting lung cancer early is crucial for improving survival rates,yet it remains a significant challenge due to many cases being diagnosed at advanced stages.This review aims to provide advances in epigenetics which...Detecting lung cancer early is crucial for improving survival rates,yet it remains a significant challenge due to many cases being diagnosed at advanced stages.This review aims to provide advances in epigenetics which have highlighted DNA methylation patterns as promising biomarkers for early detection,prognosis,and treatment response in lung cancer.Techniques like bisulfite conversion followed by PCR,digital droplet polymerase chain reaction,and next-generation sequencing are commonly used for detecting these methylation patterns,which occur early in the cancer development process and can be detected in non-invasive samples like blood and sputum.Key genes such as SHOX2 and RASSF1A have demonstrated high sensitivity and specificity in clinical studies,making them crucial for diagnostic purposes.However,several challenges remain to be overcome before these biomarkers can be widely adopted for use in clinical practice.Standardizing the assays and validating their effectiveness are critical steps.Additionally,integrating methylation biomarkers with existing diagnostic tools could significantly enhance the accuracy of lung cancer detection,providing a more comprehensive diagnostic approach.Although progress has been made in understanding and utilizing DNA methylation patterns for lung cancer detection,more research and extensive clinical trials are necessary to fully harness their potential.These efforts will help establish the robustness of methylation patterns as biomarkers and therapeutic targets,ultimately leading to better prevention,diagnosis,and treatment strategies for lung cancer.In conclusion,DNA methylation states represent a promising avenue for advancing early detection,accurate diagnosis,and management of lung cancer.展开更多
DNA repair enzymes are important in the repair of DNA lesions for maintaining the genome stability,and their abnormal expression induced various human cancers.Simultaneous detection of these DNA enzymes could provide ...DNA repair enzymes are important in the repair of DNA lesions for maintaining the genome stability,and their abnormal expression induced various human cancers.Simultaneous detection of these DNA enzymes could provide convincing evidence based on the comparison of the activity of multiple enzymes than on that of single enzyme.Although fluorescence approach has been applied for the simultaneous detection both of DNA repair enzymes,the spectral overlap and multiwavelength excitation severely restrict the number of available fluorophores.Thus,it is difficult to simultaneously detect three enzymes in a single analysis by fluorescence detection.Herein,we developed a method for the simultaneous determination of three DNA repair enzymes including human flap DNA endonuclease 1(FEN1),human alkyladenine DNA glycosylase(hAAG)and uracil DNA glycosylase(UDG)based on the combination of template-free amplification system with capillary electrophoresis-laser induced fluorescence(CE-LIF)detection.The amplification system was adopted to transfer and amplify the enzymatic products into different length DNA fragments which could be separated effectively by CE-LIF without the complicated modification of the capillary inner wall or labeling different tails on signal probes for separation.The method demonstrated a detection limit of 0.07 U/mL(0.08-160 U/mL)for FEN1,2.40 U/mL(2.5-250U/mL)for hAAG and 2.1×10^(-4)U/mL(0.0004-2.5 U/mL)for UDG,the relative standard deviations(RSDs)of peak time and peak area for different analytes were as follows:2.50%-4,37%and 3.24%-7.18%(inter-day);1.37%-2.71%and 1.43%-3.02%(intra-day),4.28%-6.08%and 4.16%-7.57%(column to column),respectively.And it can identify the inhibitor-like drugs,evaluate enzymatic kinetics and achieve the detection of three enzymes in cell extracts,providing a simple and powerful platform for simultaneous detection of more DNA repair enzymes.展开更多
1.Introduction With an estimate of 19,976,499 newly diagnosed cases and 9,743,832 deaths occurred in 2022 worldwide,cancer continues to impose a significant health and economic burden worldwide.1 The development of ca...1.Introduction With an estimate of 19,976,499 newly diagnosed cases and 9,743,832 deaths occurred in 2022 worldwide,cancer continues to impose a significant health and economic burden worldwide.1 The development of cancer is a complex interplay between genetic and environmental factors.2 In addition to genetic modifications,there is a growing body of evidence suggesting that epigenetic changes,which influence gene expression without modifying the DNA sequence,are playing an increasingly significant role in the development of cancer.DNA methylation,a key epigenetic mechanism,has been notably implicated in the early stages of cancer development,positioning it as a potential biomarker for cancer risk assessment.3 Studies have identified a diverse array of DNA methylation biomarkers for the early detection and diagnosis of cancer,utilizing DNA extracted from tissues,blood,stool,urine,and bowel lavage fluid.4 Research of DNA methylation has focused on two primary sources:peripheral blood mononuclear cell or white blood cell(WBC)DNA methylation,5 linked to cancer susceptibility and tumor-derived cell-free DNA(cfDNA)methylation,6 which has gained significant attention in recent years as a promising biomarker for cancer screening and diagnosis.展开更多
An E. coli SOS-EGFP biosensor which expresses enhanced green fluorescent protein as a reporter protein under the control of recA gene promoter in SOS response was constructed for detection of DNA damage and evaluation...An E. coli SOS-EGFP biosensor which expresses enhanced green fluorescent protein as a reporter protein under the control of recA gene promoter in SOS response was constructed for detection of DNA damage and evaluation of DNA damaging chemicals. The chemicals that may cause substantial DNA damage will trigger SOS response in the constructed bacterial biosensor, and then the reporter egfp gene under the control of recA promoter is stimulated to express as a fluorescent protein, allowing fast and sensitive fluorescence detection. Interestingly, this biosensor can be simultaneously applied for evaluation of genotoxicity and cytotoxicity. The SOS-EGFP bacterial biosensor provides a sensitive, specific and simple method for detecting known and potential DNA damaging chemicals.展开更多
Colorectal cancer(CRC)remains a major contributor to the number of cancerrelated deaths that occur annually worldwide.With the development of molecular biology methods,an increasing number of molecular biomarkers have...Colorectal cancer(CRC)remains a major contributor to the number of cancerrelated deaths that occur annually worldwide.With the development of molecular biology methods,an increasing number of molecular biomarkers have been identified and investigated.CRC is believed to result from an accumulation of epigenetic changes,and detecting aberrant DNA methylation patterns is useful for both the early diagnosis and prognosis of CRC.Numerous studies are focusing on the development of DNA methylation detection methods or DNA methylation panels.Thus,this review will discuss the commonly used techniques and technologies to evaluate DNA methylation,their merits and deficiencies as well as the prospects for new methods.展开更多
For efficient and quantitative DNA detection,fluorescence staining is the most often explored approach,which relies on non-covalent binding of dyes with double stranded DNA(dsDNA).Ethidium bromide(EB)is the most class...For efficient and quantitative DNA detection,fluorescence staining is the most often explored approach,which relies on non-covalent binding of dyes with double stranded DNA(dsDNA).Ethidium bromide(EB)is the most classic DNA stain,but suffers from its high carcinogenicity.A series of less toxic alternatives were developed,many of which contain the core structure of the benzothiazole ring.However,the relationship between the structure and the DNA detection performance was not illustrated.Herein,five benzothiazole dyes,namely thiazole orange,SYBR Green I,Pico Green,SYBR Safe,and thioflavine-T,were compared for DNA detection through direct fluorescence and gel electrophoresis,with particular focus on the structure-performance relationship.It turned out that SYBR Green I is currently the best choice for DNA detection.The results in this work may be useful for future DNA-staining dye developments.展开更多
In the present study, a disposable electrochemical DNA nano-biosensor is proposed for the rapid detection of genotoxic compounds and bio-analysis of water pollution. The DNA nano-biosensor is prepared by immobilizing ...In the present study, a disposable electrochemical DNA nano-biosensor is proposed for the rapid detection of genotoxic compounds and bio-analysis of water pollution. The DNA nano-biosensor is prepared by immobilizing DNA on Au nanoparticles and a self-assembled monolayer of cysteamine modified Au electrode. The assembly processes of cysteamine, Au nanoparticles and DNA were characterized by cyclic voltammetry （CV）. The Au nanoparticles enhanced DNA immobilization resulting in an increased guanine signal. The interaction of the analyte with the immobilized DNA was measured through the variation of the electrochemical signal of guanine by square wave voltammetry （SWV）. The biosensor was able to detect the known genotoxic compounds： 2-anthramine, acridine orange and 2- naphthylamine with detection limits of 2, 3 and 50 nmol/L, respectively. The biosensor was also used to test actual water samples to evaluate the contamination level. Additionally, the comparison of results from the classical genotoxiciw bioassay has confirmed the applicability of the method for real samoles.展开更多
In this paper we report on a study of the CMOS image sensor detection of DNA based on self-assembled nano- metallic particles, which are selectively deposited on the surface of the passive image sensor. The nano-metal...In this paper we report on a study of the CMOS image sensor detection of DNA based on self-assembled nano- metallic particles, which are selectively deposited on the surface of the passive image sensor. The nano-metallic particles effectively block the optical radiation in the visible spectrum of ordinary light source. When such a technical method is applied to DNA detection, the requirement for a special UV light source in the most popular fluorescence is eliminated. The DNA detection methodology is tested on a CMOS sensor chip fabricated using a standard 0.5 gm CMOS process. It is demonstrated that the approach is highly selective to detecting even a signal-base mismatched DNA target with an extremely-low-concentration DNA sample down to 10 pM under an ordinary light source.展开更多
The adsorption of DNA bases on a magnetic probe composed of Fe atoms and graphene is studied by using first- principles calculations. The stability of geometry, the electronic structure and magnetic property are inves...The adsorption of DNA bases on a magnetic probe composed of Fe atoms and graphene is studied by using first- principles calculations. The stability of geometry, the electronic structure and magnetic property are investigated. The results indicate that four DNA bases, i.e., adenine, thymine, cytosine and guanine, can all be adsorbed on the probe solidly. However, the magnetic moments of the composite structure can be observed only when adenine adsorbs on the probe. In the cases of the adsorption of the other three bases, the magnetic moments of the composite structure are zero. Based on the significant change of magnetic moment of the composite structure, adenine can be distinguished conveniently from thymine, cytosine and guanine. This work may provide a new way to detect DNA bases.展开更多
Colorectal cancer(CRC) is one of the most prevalent malignancies in the world. CRC-associated morbidity and mortality is continuously increasing, in part due to a lack of early detection. The existing screening tools ...Colorectal cancer(CRC) is one of the most prevalent malignancies in the world. CRC-associated morbidity and mortality is continuously increasing, in part due to a lack of early detection. The existing screening tools such as colonoscopy, are invasive and yet high cost, affecting the willingness of patients to participate in screening programs. In recent years, evidence is accumulating that the interaction of aberrant genetic and epigenetic modifications is the cornerstone for the CRC development and progression by alternating the function of tumor suppressor genes, DNA repair genes and oncogenes of colonic cells. Apart from the understanding of the underlying mechanism(s) of carcinogenesis, the aforementioned interaction has also allowed identification of clinical biomarkers, especially epigenetic, for the early detection and prognosis of cancer patients. One of the ways to detect these epigenetic biomarkers is the cell-free circulating DNA(circ DNA), a blood-based cancer diagnostic test, mainly focusing in the molecular alterations found in tumor cells, such as DNA mutations and DNA methylation.In this brief review, we epitomize the current knowledge on the research in circ DNA biomarkers-mainly focusing on DNA methylation-as potential blood-based tests for early detection of colorectal cancer and the challenges for validation and globally implementation of this emergent technology.展开更多
Rapid,high-throughput and reliable methods are urgently required to accurately detect and monitor harmful algae,which are responsible for algal blooms,such as red and green tides. In this study,we successfully develop...Rapid,high-throughput and reliable methods are urgently required to accurately detect and monitor harmful algae,which are responsible for algal blooms,such as red and green tides. In this study,we successfully developed a multiplex PCR-based DNA microarray method capable of detecting nine harmful algal species simultaneously,namely A lexandrium tamarense,Gyrodinium instriatum,Heterosigma akashiwo,Karenia mikimotoi,Prorocentrum donghaiense,Prorocentrum minimum,Ulva compressa,Ulva ohnoi and Ulva prolifera. This method achieved a limit of detection(LOD) of 0.5 ng of genomic DNA(orders of magnitude of the deci-nanogram range) in the tested algae cultures. Altogether,230 field samples from ship ballast waters and seaport waters were used to evaluate the DNA microarray. The clinical sensitivity and specificity of the DNA microarray assay in detecting field samples were 96.4% and 90.9%,respectively,relative to conventional morphological methods. This indicated that this high-throughput,automatic,and specific method is well suited for the detection of algae in water samples.展开更多
Deoxyribonucleic acid (DNA) damage arising from radiations widely occurred along with the development of nuclear weapons and clinically wide application of computed tomography (CT) scan and nuclear medicine. All ioniz...Deoxyribonucleic acid (DNA) damage arising from radiations widely occurred along with the development of nuclear weapons and clinically wide application of computed tomography (CT) scan and nuclear medicine. All ionizing radiations (X-rays, γ-rays, alpha particles, etc.) and ultraviolet (UV) radiation lead to the DNA damage. Polymerase chain reaction (PCR) is one of the most wildly used techniques for detecting DNA damage as the amplification stops at the site of the damage. Improvements to enhance the efficiency of PCR are always required and remain a great challenge. Here we establish a multiplex PCR assay system (MPAS) that is served as a robust and efficient method for direct detection of target DNA sequences in genomic DNA. The establishment of the system is performed by adding a combination of PCR enhancers to standard PCR buffer. The performance of MPAS was demonstrated by carrying out the direct PCR amplification on 1.2 mm human blood punch using commercially available primer sets which include multiple primer pairs. The optimized PCR system resulted in high quality genotyping results without any inhibitory effect indicated and led to a full-profile success rate of 98.13%. Our studies demonstrate that the MPAS provides an efficient and robust method for obtaining sensitive, reliable and reproducible PCR results from human blood samples.展开更多
BACKGROUND Patients diagnosed with non-small-cell lung cancer with activated epidermal growth factor receptor mutations are more likely to develop leptomeningeal(LM)metastasis than other types of lung cancers and have...BACKGROUND Patients diagnosed with non-small-cell lung cancer with activated epidermal growth factor receptor mutations are more likely to develop leptomeningeal(LM)metastasis than other types of lung cancers and have a poor prognosis.Early diagnosis and effective treatment of leptomeningeal carcinoma can improve the prognosis.CASE SUMMARY A 55-year-old female with a progressive headache and vomiting for one month was admitted to Peking University First Hospital.She was diagnosed with lung adenocarcinoma with osseous metastasis 10 months prior to admittance.epidermal growth factor receptor(EGFR)mutation was detected by genomic examination,so she was first treated with gefitinib for 10 months before acquiring resistance.Cell-free cerebrospinal fluid(CSF)circulating tumor DNA detection by next-generation sequencing was conducted and indicated the EGFR-Thr790Met mutation,while biopsy and cytology from the patient’s CSF and the first enhanced cranial magnetic resonance imaging(MRI)showed no positive findings.A month later,the enhanced MRI showed linear leptomeningeal enhancement,and the cytology and biochemical examination in CSF remained negative.Therefore,osimertinib(80 mg/d)was initiated as a second-line treatment,resulting in a good response within a month.CONCLUSION This report suggests clinical benefit of osimertinib in LM patients with positive detection of the EGFR-Thr790Met mutation in CSF and proposes that the positive findings of CSF circulating tumor DNA as a liquid biopsy technology based on the detection of cancer-associated gene mutations may appear earlier than the imaging and CSF findings and may thus be helpful for therapy.Moreover,the routine screening of chest CT with the novel coronavirus may provide unexpected benefits。展开更多
Non-invasive prenatal gene diagnosis has been developed rapidly in the recent years, and numerous medical researchers are focusing on it. Such techniques could not only achieve prenatal diagnosis accurately, but also ...Non-invasive prenatal gene diagnosis has been developed rapidly in the recent years, and numerous medical researchers are focusing on it. Such techniques could not only achieve prenatal diagnosis accurately, but also prevent tangential illness in fetuses and thus, reduce the incidence of diseases. Moreover, it is non-invasive prenatal gene diagnosis that prevents potential threaten and danger to both mothers and fetuses. Therefore, it is welcomed by clinical gynecologist and obstetrian, researchers of medical genetics, and especially, pregnancies. This review article touches briefly on the advanced development of using cell-free DNA, RNA in maternal plasma and urine for non-invasive prenatal gene diagnosis.展开更多
In this paper, the DNA-templated Ag/Pt bimetallic nanoclusters were successfully synthesized using an optimized synthetic scheme. The obtained DNA-Ag/Pt NCs have an ultrasmall particle size and excellent distribution....In this paper, the DNA-templated Ag/Pt bimetallic nanoclusters were successfully synthesized using an optimized synthetic scheme. The obtained DNA-Ag/Pt NCs have an ultrasmall particle size and excellent distribution. The DNA-Ag/Pt NCs show intrinsic peroxidase-mimicking activity and can effectively catalyze the H2O2-mediated oxidation of a substrate, 3,30,5,50-tetramethylbenzidine（TMB）, to produce a blue colored product. Based on this specific property, we employed the aptamer of VEGF to design a label-free electrochemical biosensor for VEGF detection. Under the optimized experimental conditions, a linear range from 6.0 pmol/L to 20 pmol/L was obtained with a detection limit of 4.6 pmol/L. The proposed biosensor demonstrated its high specificity for VEGF and could directly detect the VEGF concentration in human serum samples of breast cancer patients with satisfactory results. This novel electrochemical aptasensor was simple and convenient to use and was cost-effective and label-free in design, and would hold potential applications in medical diagnosis and treatment.展开更多
A dual-amplification system is reported to apply in DNA sensing via the assembly of DNA and protein. In this process, the biotinylatedcapature DNA bounded with streptavidin（SA） through the biotinstreptavidin reactio...A dual-amplification system is reported to apply in DNA sensing via the assembly of DNA and protein. In this process, the biotinylatedcapature DNA bounded with streptavidin（SA） through the biotinstreptavidin reaction, and then the assembly of DNA and protein was triggered by the linker DNA after the target hybridized with biotinylatedcapature DNA. Sequentially, the 3,3＇,5,5＇-tetramethylbenzidine（TMB）was oxidized by H_2O_2 under the catalysis of horseradish peroxidase. Based on the variation of the color and the UV–vis absorbance intensities, qualitative and quantitative DNA analyses were realized. This proposed method could detect the target DNA as low as 1.75 pmol/L and discriminate perfectly matched target DNA from the mismatch DNA. What＇s more, it can be expanded to detect other molecules with a reasonable design of the corresponding DNA sequences.展开更多
The mixtures of two polymers, poly (N,N-dimethylacrylamide) (PDMA) and polyvinylpyrrolidone (PVP) were synthesized and used as the separation medium for double-stranded and single-stranded DNA fragments by capillary e...The mixtures of two polymers, poly (N,N-dimethylacrylamide) (PDMA) and polyvinylpyrrolidone (PVP) were synthesized and used as the separation medium for double-stranded and single-stranded DNA fragments by capillary electrophoresis with UV detector. On optimal conditions, 2%w/v PDMA + 2%w/v PVP can be used to separate the doublet 123/124bp in pBR322/Hae III Markers.展开更多
基金the Experimental Technology Research Project of Zhejiang University(SYB202138)National Natural Science Foundation of China(32000195).
文摘Soil DNA extraction,such as microbial community analysis and gene drift detection,is an important basis for multiple analyses in different fields.Nevertheless,the soil DNA extraction methods for field detection are still lacking.This study established a rapid soil DNA extraction(RSDE)method that can be used in field detection.In this method,we first utilized the optimized lysate to isolate DNA from soil and then used a filtration membrane and a DNA adsorption membrane to purify the DNA via the column method.Moreover,we used the pressure from the syringe instead of the conventional centrifugal force of the centrifuge to assist the sample filtration,resulting in very low requirements for this method,with an extraction time of less than 20 min.Furthermore,we demonstrated that the RSDE method was applicable for DNA extraction from different types of soils,with the demand for soil samples as low as 0.1 g and that the amount of obtained DNA was,to some extent,greater than that obtained by a commercial kit.Further analysis revealed that this extracted genomic DNA can be used directly for polymerase chain reaction(PCR)analysis,including ordinary PCR,real-time fluorescent quantitative PCR,and recombinase polymerase amplification(RPA)-CRISPR/Cas12a visual assays.In addition,we demonstrated that this method can be used to extract DNA from residual plant roots in addition to soil microbes,which lays a foundation for the comprehensive analysis of soil plants and microorganisms.In summary,the RSDE method proposed in this study may have wide application prospects.
文摘Liver fibrosis is an important pathological precondition for hepatocellular carcinoma.The degree of hepatic fibrosis is positively correlated with liver cancer.Liver fibrosis is a series of pathological and physiological process related to liver cell necrosis and degeneration after chronic liver injury,which finally leads to extracellular matrix and collagen deposition.The early detection and precise staging of fibrosis and cirrhosis are very important for early diagnosis and timely initiation of appropriate therapeutic regimens.The risk of severe liver fibrosis finally progressing to liver carcinoma is＆gt;50%.It is known that biopsy is the gold standard for the diagnosis and staging of liver fibrosis.However,this method has some limitations,such as the potential for pain,sampling variability,and low patient acceptance.Furthermore,the necessity of obtaining a tissue diagnosis of liver fibrosis still remains controversial.An increasing number of reliable non-invasive approaches are now available that are widely applied in clinical practice,mostly in cases of viral hepatitis,resulting in a significantly decreased need for liver biopsy.In fact,the noninvasive detection and evaluation of liver cirrhosis now has good accuracy due to current serum markers,ultrasound imaging,and magnetic resonance imaging quantification techniques.A prominent advantage of the non-invasive detection and assessment of liver fibrosis is that liver fibrosis can be monitored repeatedly and easily in the same patient.Serum biomarkers have the advantages of high applicability（〉95%）and good reproducibility.However,their results can be influenced by different patient conditions because none of these markers are liver-specific.The most promising techniques appear to be transient elastography and magnetic resonance elastography because they provide reliable results for the detection of fibrosis in the advanced stages,and future developments promise to increase the reliability and accuracy of the staging of hepatic fibrosis.This article aims to describe the recent progress in the development of non-invasive assessment methods for the staging of liver fibrosis,with a special emphasize on computer-aided quantitative and deep learning methods.
基金supported by National Key R&D Program of China(No.2021YFA0910100)National Natural Science Foundation of China(No.82374544,82204828,82422078)+2 种基金Healthy Zhejiang One Million People Cohort(No.K-20230085)Program of Zhejiang Provincial TCM Sci-tech Plan(No.GZY-ZJ-KJ-230003,No.GZY-ZJ-KJ23048)Natural Science Foundation of Zhejiang Province(No.LHDMY22H160008)。
文摘Objective:Plasma cell-free DNA(cfDNA)methylation has shown potential in the detection and prognostic testing of multiple cancers.Here,we comprehensively investigate the performance of cfDNA methylation for gastric cancer(GC)detection and prognosis.Methods:GC-specific differentially methylated regions(DMRs)were identified by sequencing 56 GC tissues and 59 normal adjacent tissues(NATs).We then performed targeted bisulfite sequencing of cfDNA from 294 GC and 446 non-gastric cancer(NGC)plasma samples,identifying 179 DMRs that overlapped with those in tissue samples.The efficacy of plasma cfDNA methylation markers for GC detection and prognosis was evaluated.Results:Based on the 179 DMRs overlapping with those in tissue samples,the random forest(RF)model using28 DMRs achieved an area under the curve(AUC)of 0.998 in the training cohort,whereas further refinement to the top 6 DMRs resulted in an AUC of 0.985.Consistent results were obtained in the validation cohort(28 DMR AUC:0.985;6 DMR AUC:0.988).Support vector machine(SVM)and logistic regression(LR)models also demonstrated robust performance.Additionally,an 11-DMR signature was developed for prognostic prediction,successfully identifying high-risk GC patients with significantly shorter overall survival.Conclusions:Our study highlights the potential utility of cfDNA methylation markers for both the detection and prognostication of GC.
文摘Detecting lung cancer early is crucial for improving survival rates,yet it remains a significant challenge due to many cases being diagnosed at advanced stages.This review aims to provide advances in epigenetics which have highlighted DNA methylation patterns as promising biomarkers for early detection,prognosis,and treatment response in lung cancer.Techniques like bisulfite conversion followed by PCR,digital droplet polymerase chain reaction,and next-generation sequencing are commonly used for detecting these methylation patterns,which occur early in the cancer development process and can be detected in non-invasive samples like blood and sputum.Key genes such as SHOX2 and RASSF1A have demonstrated high sensitivity and specificity in clinical studies,making them crucial for diagnostic purposes.However,several challenges remain to be overcome before these biomarkers can be widely adopted for use in clinical practice.Standardizing the assays and validating their effectiveness are critical steps.Additionally,integrating methylation biomarkers with existing diagnostic tools could significantly enhance the accuracy of lung cancer detection,providing a more comprehensive diagnostic approach.Although progress has been made in understanding and utilizing DNA methylation patterns for lung cancer detection,more research and extensive clinical trials are necessary to fully harness their potential.These efforts will help establish the robustness of methylation patterns as biomarkers and therapeutic targets,ultimately leading to better prevention,diagnosis,and treatment strategies for lung cancer.In conclusion,DNA methylation states represent a promising avenue for advancing early detection,accurate diagnosis,and management of lung cancer.
基金supported by the National Natural Science Foundation of China(Nos.21874060 and 22174058,U21A20282)the Science and Technology program of Gansu Province(No.22JR5RA476)。
文摘DNA repair enzymes are important in the repair of DNA lesions for maintaining the genome stability,and their abnormal expression induced various human cancers.Simultaneous detection of these DNA enzymes could provide convincing evidence based on the comparison of the activity of multiple enzymes than on that of single enzyme.Although fluorescence approach has been applied for the simultaneous detection both of DNA repair enzymes,the spectral overlap and multiwavelength excitation severely restrict the number of available fluorophores.Thus,it is difficult to simultaneously detect three enzymes in a single analysis by fluorescence detection.Herein,we developed a method for the simultaneous determination of three DNA repair enzymes including human flap DNA endonuclease 1(FEN1),human alkyladenine DNA glycosylase(hAAG)and uracil DNA glycosylase(UDG)based on the combination of template-free amplification system with capillary electrophoresis-laser induced fluorescence(CE-LIF)detection.The amplification system was adopted to transfer and amplify the enzymatic products into different length DNA fragments which could be separated effectively by CE-LIF without the complicated modification of the capillary inner wall or labeling different tails on signal probes for separation.The method demonstrated a detection limit of 0.07 U/mL(0.08-160 U/mL)for FEN1,2.40 U/mL(2.5-250U/mL)for hAAG and 2.1×10^(-4)U/mL(0.0004-2.5 U/mL)for UDG,the relative standard deviations(RSDs)of peak time and peak area for different analytes were as follows:2.50%-4,37%and 3.24%-7.18%(inter-day);1.37%-2.71%and 1.43%-3.02%(intra-day),4.28%-6.08%and 4.16%-7.57%(column to column),respectively.And it can identify the inhibitor-like drugs,evaluate enzymatic kinetics and achieve the detection of three enzymes in cell extracts,providing a simple and powerful platform for simultaneous detection of more DNA repair enzymes.
基金supported by the Beijing Nova Program of Science and Technology(grant number:20230484397)the National Natural Science Foundation of China(grant number:82273726).
文摘1.Introduction With an estimate of 19,976,499 newly diagnosed cases and 9,743,832 deaths occurred in 2022 worldwide,cancer continues to impose a significant health and economic burden worldwide.1 The development of cancer is a complex interplay between genetic and environmental factors.2 In addition to genetic modifications,there is a growing body of evidence suggesting that epigenetic changes,which influence gene expression without modifying the DNA sequence,are playing an increasingly significant role in the development of cancer.DNA methylation,a key epigenetic mechanism,has been notably implicated in the early stages of cancer development,positioning it as a potential biomarker for cancer risk assessment.3 Studies have identified a diverse array of DNA methylation biomarkers for the early detection and diagnosis of cancer,utilizing DNA extracted from tissues,blood,stool,urine,and bowel lavage fluid.4 Research of DNA methylation has focused on two primary sources:peripheral blood mononuclear cell or white blood cell(WBC)DNA methylation,5 linked to cancer susceptibility and tumor-derived cell-free DNA(cfDNA)methylation,6 which has gained significant attention in recent years as a promising biomarker for cancer screening and diagnosis.
基金supported by the National Natural Science Foundation of China (No. 20707034, 20877091,20890112, 20921063)the National Basic Research Program (973) of China (No. 09CB421605,2010CB933500, 2011CB936001)
文摘An E. coli SOS-EGFP biosensor which expresses enhanced green fluorescent protein as a reporter protein under the control of recA gene promoter in SOS response was constructed for detection of DNA damage and evaluation of DNA damaging chemicals. The chemicals that may cause substantial DNA damage will trigger SOS response in the constructed bacterial biosensor, and then the reporter egfp gene under the control of recA promoter is stimulated to express as a fluorescent protein, allowing fast and sensitive fluorescence detection. Interestingly, this biosensor can be simultaneously applied for evaluation of genotoxicity and cytotoxicity. The SOS-EGFP bacterial biosensor provides a sensitive, specific and simple method for detecting known and potential DNA damaging chemicals.
基金Supported by the Changzhou High-Level Medical Talents Training Project,No.2016ZCLJ002
文摘Colorectal cancer(CRC)remains a major contributor to the number of cancerrelated deaths that occur annually worldwide.With the development of molecular biology methods,an increasing number of molecular biomarkers have been identified and investigated.CRC is believed to result from an accumulation of epigenetic changes,and detecting aberrant DNA methylation patterns is useful for both the early diagnosis and prognosis of CRC.Numerous studies are focusing on the development of DNA methylation detection methods or DNA methylation panels.Thus,this review will discuss the commonly used techniques and technologies to evaluate DNA methylation,their merits and deficiencies as well as the prospects for new methods.
基金the Major Scientific and Technological Application Program of Chengdu(No.2019-YF09-0081-SN)the Major Scientific and Technological Special Program of Sichuan Province(No.2018SZDZX0027)the Fundamental Research Funds for Central Universities(No.2018SCUH0075)。
文摘For efficient and quantitative DNA detection,fluorescence staining is the most often explored approach,which relies on non-covalent binding of dyes with double stranded DNA(dsDNA).Ethidium bromide(EB)is the most classic DNA stain,but suffers from its high carcinogenicity.A series of less toxic alternatives were developed,many of which contain the core structure of the benzothiazole ring.However,the relationship between the structure and the DNA detection performance was not illustrated.Herein,five benzothiazole dyes,namely thiazole orange,SYBR Green I,Pico Green,SYBR Safe,and thioflavine-T,were compared for DNA detection through direct fluorescence and gel electrophoresis,with particular focus on the structure-performance relationship.It turned out that SYBR Green I is currently the best choice for DNA detection.The results in this work may be useful for future DNA-staining dye developments.
基金funded by the National Natural Science Foundation of China(Nos.21103059,51136002 and 51076079)the China Key Technologies R&D Program(No.2012BAJ02B03)
文摘In the present study, a disposable electrochemical DNA nano-biosensor is proposed for the rapid detection of genotoxic compounds and bio-analysis of water pollution. The DNA nano-biosensor is prepared by immobilizing DNA on Au nanoparticles and a self-assembled monolayer of cysteamine modified Au electrode. The assembly processes of cysteamine, Au nanoparticles and DNA were characterized by cyclic voltammetry （CV）. The Au nanoparticles enhanced DNA immobilization resulting in an increased guanine signal. The interaction of the analyte with the immobilized DNA was measured through the variation of the electrochemical signal of guanine by square wave voltammetry （SWV）. The biosensor was able to detect the known genotoxic compounds： 2-anthramine, acridine orange and 2- naphthylamine with detection limits of 2, 3 and 50 nmol/L, respectively. The biosensor was also used to test actual water samples to evaluate the contamination level. Additionally, the comparison of results from the classical genotoxiciw bioassay has confirmed the applicability of the method for real samoles.
基金Project supported by the Key Program of the National Natural Science Foundation of China (Grant No. 61036004)the Shenzhen Science & Technology Foundation, China (Grant No. CXB201005250031A)+1 种基金the Fundamental Research Project of Shenzhen Science & Technology Foundation, China (Grant No. JC201005280670A)the International Collaboration Project of Shenzhen Science & Technology Foundation, China (Grant No. ZYA2010006030006A)
文摘In this paper we report on a study of the CMOS image sensor detection of DNA based on self-assembled nano- metallic particles, which are selectively deposited on the surface of the passive image sensor. The nano-metallic particles effectively block the optical radiation in the visible spectrum of ordinary light source. When such a technical method is applied to DNA detection, the requirement for a special UV light source in the most popular fluorescence is eliminated. The DNA detection methodology is tested on a CMOS sensor chip fabricated using a standard 0.5 gm CMOS process. It is demonstrated that the approach is highly selective to detecting even a signal-base mismatched DNA target with an extremely-low-concentration DNA sample down to 10 pM under an ordinary light source.
基金Supported by the National Natural Science Foundation of China under Grant Nos 51301119 and 11204201the Natural Science Foundation for Young Scientists of Shanxi Province under Grant No 2013021010-1
文摘The adsorption of DNA bases on a magnetic probe composed of Fe atoms and graphene is studied by using first- principles calculations. The stability of geometry, the electronic structure and magnetic property are investigated. The results indicate that four DNA bases, i.e., adenine, thymine, cytosine and guanine, can all be adsorbed on the probe solidly. However, the magnetic moments of the composite structure can be observed only when adenine adsorbs on the probe. In the cases of the adsorption of the other three bases, the magnetic moments of the composite structure are zero. Based on the significant change of magnetic moment of the composite structure, adenine can be distinguished conveniently from thymine, cytosine and guanine. This work may provide a new way to detect DNA bases.
文摘Colorectal cancer(CRC) is one of the most prevalent malignancies in the world. CRC-associated morbidity and mortality is continuously increasing, in part due to a lack of early detection. The existing screening tools such as colonoscopy, are invasive and yet high cost, affecting the willingness of patients to participate in screening programs. In recent years, evidence is accumulating that the interaction of aberrant genetic and epigenetic modifications is the cornerstone for the CRC development and progression by alternating the function of tumor suppressor genes, DNA repair genes and oncogenes of colonic cells. Apart from the understanding of the underlying mechanism(s) of carcinogenesis, the aforementioned interaction has also allowed identification of clinical biomarkers, especially epigenetic, for the early detection and prognosis of cancer patients. One of the ways to detect these epigenetic biomarkers is the cell-free circulating DNA(circ DNA), a blood-based cancer diagnostic test, mainly focusing in the molecular alterations found in tumor cells, such as DNA mutations and DNA methylation.In this brief review, we epitomize the current knowledge on the research in circ DNA biomarkers-mainly focusing on DNA methylation-as potential blood-based tests for early detection of colorectal cancer and the challenges for validation and globally implementation of this emergent technology.
基金Supported by the National High Technology Research and Development Program of China(863 Program)(No.2012AA092001)the Science and Technology Project of Zhejiang Province(No.2013C03045-1)+2 种基金the Zhejiang Marine Biotechnology Innovation Team(No.2010R50029-12)the Natural Science Foundation of Ningbo City of China(No.2013A610168)the KC Wong Magna Fund in Ningbo University
文摘Rapid,high-throughput and reliable methods are urgently required to accurately detect and monitor harmful algae,which are responsible for algal blooms,such as red and green tides. In this study,we successfully developed a multiplex PCR-based DNA microarray method capable of detecting nine harmful algal species simultaneously,namely A lexandrium tamarense,Gyrodinium instriatum,Heterosigma akashiwo,Karenia mikimotoi,Prorocentrum donghaiense,Prorocentrum minimum,Ulva compressa,Ulva ohnoi and Ulva prolifera. This method achieved a limit of detection(LOD) of 0.5 ng of genomic DNA(orders of magnitude of the deci-nanogram range) in the tested algae cultures. Altogether,230 field samples from ship ballast waters and seaport waters were used to evaluate the DNA microarray. The clinical sensitivity and specificity of the DNA microarray assay in detecting field samples were 96.4% and 90.9%,respectively,relative to conventional morphological methods. This indicated that this high-throughput,automatic,and specific method is well suited for the detection of algae in water samples.
基金Supported by National Natural Science Foundation of China(Nos.21077321,21105028,21075128)K.C.Wong Education Foundation,Hong Kong,China
文摘Deoxyribonucleic acid (DNA) damage arising from radiations widely occurred along with the development of nuclear weapons and clinically wide application of computed tomography (CT) scan and nuclear medicine. All ionizing radiations (X-rays, γ-rays, alpha particles, etc.) and ultraviolet (UV) radiation lead to the DNA damage. Polymerase chain reaction (PCR) is one of the most wildly used techniques for detecting DNA damage as the amplification stops at the site of the damage. Improvements to enhance the efficiency of PCR are always required and remain a great challenge. Here we establish a multiplex PCR assay system (MPAS) that is served as a robust and efficient method for direct detection of target DNA sequences in genomic DNA. The establishment of the system is performed by adding a combination of PCR enhancers to standard PCR buffer. The performance of MPAS was demonstrated by carrying out the direct PCR amplification on 1.2 mm human blood punch using commercially available primer sets which include multiple primer pairs. The optimized PCR system resulted in high quality genotyping results without any inhibitory effect indicated and led to a full-profile success rate of 98.13%. Our studies demonstrate that the MPAS provides an efficient and robust method for obtaining sensitive, reliable and reproducible PCR results from human blood samples.
文摘BACKGROUND Patients diagnosed with non-small-cell lung cancer with activated epidermal growth factor receptor mutations are more likely to develop leptomeningeal(LM)metastasis than other types of lung cancers and have a poor prognosis.Early diagnosis and effective treatment of leptomeningeal carcinoma can improve the prognosis.CASE SUMMARY A 55-year-old female with a progressive headache and vomiting for one month was admitted to Peking University First Hospital.She was diagnosed with lung adenocarcinoma with osseous metastasis 10 months prior to admittance.epidermal growth factor receptor(EGFR)mutation was detected by genomic examination,so she was first treated with gefitinib for 10 months before acquiring resistance.Cell-free cerebrospinal fluid(CSF)circulating tumor DNA detection by next-generation sequencing was conducted and indicated the EGFR-Thr790Met mutation,while biopsy and cytology from the patient’s CSF and the first enhanced cranial magnetic resonance imaging(MRI)showed no positive findings.A month later,the enhanced MRI showed linear leptomeningeal enhancement,and the cytology and biochemical examination in CSF remained negative.Therefore,osimertinib(80 mg/d)was initiated as a second-line treatment,resulting in a good response within a month.CONCLUSION This report suggests clinical benefit of osimertinib in LM patients with positive detection of the EGFR-Thr790Met mutation in CSF and proposes that the positive findings of CSF circulating tumor DNA as a liquid biopsy technology based on the detection of cancer-associated gene mutations may appear earlier than the imaging and CSF findings and may thus be helpful for therapy.Moreover,the routine screening of chest CT with the novel coronavirus may provide unexpected benefits。
文摘Non-invasive prenatal gene diagnosis has been developed rapidly in the recent years, and numerous medical researchers are focusing on it. Such techniques could not only achieve prenatal diagnosis accurately, but also prevent tangential illness in fetuses and thus, reduce the incidence of diseases. Moreover, it is non-invasive prenatal gene diagnosis that prevents potential threaten and danger to both mothers and fetuses. Therefore, it is welcomed by clinical gynecologist and obstetrian, researchers of medical genetics, and especially, pregnancies. This review article touches briefly on the advanced development of using cell-free DNA, RNA in maternal plasma and urine for non-invasive prenatal gene diagnosis.
基金support of the National Natural Science Foundation of China (Nos. 21375017, 21105012 and 21205015)the National Science Foundation for Distinguished Young Scholars of Fujian Province (No. 2013J06003)+3 种基金the Key Project of Fujian Science and Technology (No. 2013Y0045)the Program for New Century Excellent Talents of Colleges and Universities in Fujian Province (Nos. JA13130 and JA13088)the Program for Fujian University Outstanding Youth Scientific Research (Nos. JA11105 and JA10295)the Foundation of Fuzhou Science and Technology Bureau (No. 2013-S-122-4)
文摘In this paper, the DNA-templated Ag/Pt bimetallic nanoclusters were successfully synthesized using an optimized synthetic scheme. The obtained DNA-Ag/Pt NCs have an ultrasmall particle size and excellent distribution. The DNA-Ag/Pt NCs show intrinsic peroxidase-mimicking activity and can effectively catalyze the H2O2-mediated oxidation of a substrate, 3,30,5,50-tetramethylbenzidine（TMB）, to produce a blue colored product. Based on this specific property, we employed the aptamer of VEGF to design a label-free electrochemical biosensor for VEGF detection. Under the optimized experimental conditions, a linear range from 6.0 pmol/L to 20 pmol/L was obtained with a detection limit of 4.6 pmol/L. The proposed biosensor demonstrated its high specificity for VEGF and could directly detect the VEGF concentration in human serum samples of breast cancer patients with satisfactory results. This novel electrochemical aptasensor was simple and convenient to use and was cost-effective and label-free in design, and would hold potential applications in medical diagnosis and treatment.
基金supported by the National Science Foundation of China (No. 21205089)
文摘A dual-amplification system is reported to apply in DNA sensing via the assembly of DNA and protein. In this process, the biotinylatedcapature DNA bounded with streptavidin（SA） through the biotinstreptavidin reaction, and then the assembly of DNA and protein was triggered by the linker DNA after the target hybridized with biotinylatedcapature DNA. Sequentially, the 3,3＇,5,5＇-tetramethylbenzidine（TMB）was oxidized by H_2O_2 under the catalysis of horseradish peroxidase. Based on the variation of the color and the UV–vis absorbance intensities, qualitative and quantitative DNA analyses were realized. This proposed method could detect the target DNA as low as 1.75 pmol/L and discriminate perfectly matched target DNA from the mismatch DNA. What＇s more, it can be expanded to detect other molecules with a reasonable design of the corresponding DNA sequences.
基金supported by the Innovation Fund(KSCX1-06)of Chinese Academy of Sciences.
文摘The mixtures of two polymers, poly (N,N-dimethylacrylamide) (PDMA) and polyvinylpyrrolidone (PVP) were synthesized and used as the separation medium for double-stranded and single-stranded DNA fragments by capillary electrophoresis with UV detector. On optimal conditions, 2%w/v PDMA + 2%w/v PVP can be used to separate the doublet 123/124bp in pBR322/Hae III Markers.
