Analytical Methods
·
2024
·Vol. 16
·期 7
·1003-1011
·
IF 2.7
Yongwei Fang,Chuang Wang,Yuli Wang,Jiandong Hu,Miao Lai,Pengfei Liu,Hong Zhang
Plant viral diseases can seriously affect the yield and quality of crops. In this work, a convenient and highly sensitive biosensor for the visual detection of plant viral disease is proposed by the PCR-induced generation of DNAzyme. In the absence of nucleic acid for a target plant virus, the primers prohibited the production of G-quadruplex by forming a hairpin structure. However, PCR amplification occurred and generated a number of specific PCR products with free G-quadruplex sequences at both ends in the presence of the target cDNA. A catalytically active G-quadruplex DNAzyme was formed with the help of K+ and hemin, resulting in the formation of colored products visible to the naked eye and a strong absorbance by the addition of ABTS2− and H2O2. The absorbance and the logarithm of target cDNA concentrations showed a good linear relationship in the range of 10 fM–1.0 nM, with a linear regression equation of A = 0.1402 lg c + 0.3761 (c: fM) and a detection limit of 0.19 fM. This method was successfully applied to the analysis of emerging tobacco mosaic virus (TMV) infections in tobacco leaf samples collected in the field due to its flexibility and convenience, indicating a potential application for the early detection of plant viral disease.
研究领域:
植物病理学
·
分子生物学
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产品关联
DOI
PubMed
高影响因子
VACUUM
·
2024
·
IF 3.8
Xun Zhou,Xin Yi,Zhixian Liang,Jie Zhang,Guixiang Li,Yuanliang Tang,Kun Zhou,Shaohui Zhang
In this work, the photothermal effect of aminomodified MWCNTs/TiO 2 /SiO 2 /PDMS nanocomposites was utilized as a photothermal substrate to replace the metal heating block of traditional qPCR combining of the advantages of high photothermal effect of composite materials and low sample volume of static microfluidic PCR. Meanwhile, Multiple PCR rapid thermal cycles under 808 nm laser irradiation was accomplished through a non-contact energy conversion method and the use of chamber-immobilized PCR chips as specific reaction vessels. Notably, the chip chamber architecture requires merely about 8 μL of reagents compared to traditional PCR tubes. While reducing the amount of PCR reagents used, the higher specific surface area results in a larger contact surface (SSA) between the heat source and the solution. Thereafter, the CCD image sensor is then used as a fluorescence detector to monitor the amplification of the target DNA during the PCR reaction, thus enabling simultaneous fluorescence detection. In addition, the threshold cycle (Ct) value of qPCR and standard PCR curves for SARS-CoV-2 virus have been analysed in depth. Importantly, this study has important implications for the development of low-cost materials, miniaturized and portable Point-of-Care Testing (POCT) chips/systems.
研究领域:
生物技术
·
医学检测
·
材料科学
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DOI
高影响因子
MICROCHEMICAL JOURNAL
·
2026
·
IF 5.2
Shengjie Gao,Xuanxuan Liu,Lili Yao,Hui Zhang,Zhiwen Guan,Chuanlong Men,Yu Mao,Lu Wang,Lei Zheng
Developed a high-annealing-temperature PCR-assisted GO-SELEX screening strategy. • Enriched GC-rich sequences with strong G-quadruplex folding potential. • Identified a Pb 2+ -specific G4 aptamer Pb-GC-1 with high affinity and selectivity. • Constructed a Pb 2+ -responsive G4 allosteric colorimetric biosensor. • Achieved 10 nM detection limit and high recovery in real food sample testing. Heavy metal pollution, particularly from lead, poses a severe threat to global food safety. We present a novel G-quadruplex (G4) aptamers screening strategy based on high-annealing-temperature PCR-assisted graphene oxide-SELEX (HAT-GO-SELEX) for efficient development of highly specific and sensitive probes targeting Pb 2+ . By systematically elevating the PCR annealing temperature during selection, we enriched sequences with high GC content and structural stability. Analysis of the top 100 candidate sequences from high-throughput sequencing revealed significant enrichment of G-tracts, with 20% consensus G4 formation potential across multiple prediction algorithms. A Pb 2+ -specific G4 aptamer, Pb-GC-1, exhibited high affinity ( K d = 167 nM) and strong specificity. A colorimetric biosensor based on Pb 2+ -induced G4 allosteric switching enabled sensitive detection in real food samples, achieving a detection limit of 10 nM and recoveries of 96.7–102.0%. This work presents an efficient strategy for screening functional G4-based aptamers, advancing the development of rapid detection platforms for heavy metal contaminants. Download: Download high-res image (136KB) Download: Download full-size image
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高影响因子
Foods
·
2026
·Vol. 15
·期 10
·1790
·
IF 6
Xiaorong Xu,Jie Fang,Lingyan Mao,Yingying Wu,Hai Cheng,Jinru Lin,Liyu Shi,Jiali Xing,Xiaohu Luo
DNA must be efficiently extracted from samples to accurately test the authenticity of food, particularly from processed matrices in which DNA integrity may be compromised. We systematically evaluated the efficiency of extracting DNA from dairy and blood products by four methods, namely SDS-CTAB, SDS-isopropanol precipitation, guanidine isothiocyanate magnetic beads, and a commercial kit. The guanidine isothiocyanate-magnetic bead method yields high quantities and purity of DNA; for example, the yield obtained from chicken blood samples was 318.34 ± 4.77 ng/µL, with an A260/A280 ratio ranging from 1.8 to 2.0. The processing time of this method was compared with the DNA Extraction Kit shorter by 40% and unlike methods such as the SDS-CTAB protocol, does not require the use of toxic reagents such as phenol or chloroform, meeting green chemistry requirements. Among the dairy and blood samples tested, it enables the extraction of DNA in quantities comparable to those obtained using commercial kits; moreover, the DNA yield achieved is 20–30% higher than that of these kits. Furthermore, this method is free from the limitations associated with protein contamination and amplification instability often encountered in protocols such as the CTAB-SDS and SDS-isopropanol methods. The magnetic bead approach was adaptable for complex matrices and demonstrated strong tolerance to coexisting contaminants, thereby improving extraction performance in challenging food samples. The magnetic bead surface functionalization and buffer systems could be improved to further increase their versatility. This method enables reliable DNA extraction and advanced technical support for DNA analysis.
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DOI
PubMed
高影响因子
MOLECULAR BREEDING
·
2012
·Vol. 31
·期 3
·729-735
·
IF 2.852
Shirao Tsukasa,Ueno Kei-ichiro,Abe Tomoko,Matsuyama Tomoki
Four chrysanthemum cultivars were generated through (carbon) ion-beam irradiation of the original ‘Jimba’ ( Chrysanthemum morifolium Ramat.). The new cultivars had acquired a number of superior cultivation traits, while remaining identical to the commercially available ‘Jimba’ in appearance. In this study, polymerase chain reaction (PCR) assays were used to detect the mutated region of each strain, thereby allowing clear identification at the molecular level. PCR assays were performed with 446 primer sets, including random amplified polymorphic DNA (RAPD) primer sets (10-mer RAPD), arbitrarily primed (AP)-PCR primers based on retrotransposon-like sequences and modified RAPD primers (15-mer RAPD). 15-mer RAPD primers generated a 1.49-fold increased band number at high annealing temperatures compared with the original 10-mer RAPD primers and could thus be effective for detection of polymorphic patterns. Our results provide information on the mutated regions of these ion-beam-irradiated chrysanthemum cultivars. Thus, specific DNA markers could be used to improve identification of new cultivars of chrysanthemum as well as other clonal cultivars of horticultural and agricultural crops.
研究领域:
植物学
·
遗传学
·
分子生物学
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DOI
高影响因子
ANALYTICA CHIMICA ACTA
·
2017
·
IF 4.95
Chenguang Wang,Nan Cheng,Longjiao Zhu,Yuancong Xu,Kunlun Huang,Pengyu Zhu,Shuifang Zhu,Wei Fu,Wentao Xu
A colorimetric biosensor for DNA screening was designed based on the conformational changes of the horseradish peroxidase (HRP)-mimicking DNAzyme. The scheme of DNA biosensing was designed based on the base pairing of DNAzyme sequence to inhibit the formation of HRP-mimicking hemin/G-quadruplex structures in the process of amplification. DNA could be amplified via the universal primer multiplex polymerase chain reaction (UP- M -PCR) and innovatively detected as color disappear in the reaction visible to the naked eye. The input of key factors and the output of optical characteristics in the reaction inspired the development of an OR logic gate operation for DNA detection. This biosensor overcomes self-inhibition and amplification disparity with the help of UP- M -PCR, thereby exhibiting high specificity and high-throughput without the requirement of gel analysis work. This biosensing system also presented 1% sensitivity and approximately 180 copy numbers in triplicate. The biosensor was used to screen elements from genetically modified organisms (GMOs) and covered more than 90% of all globally authorized events in the world. The designed colorimetric biosensor is a rapid, portable and versatile tool for nucleic acids detection and diagnosis in the field.
研究领域:
生物化学
·
分子生物学
·
分析化学
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DOI
PubMed
高影响因子
PROCESS BIOCHEMISTRY
·
2019
·
IF 2.883
Beibei Guo,Yuanhao Chen,Lu Lv,Hafiz Adeel Ahmad,Shou-Qing Ni,Liying Ren,Zhaojie Cui,Xu Fang,Zhuangming Qiao,Shaowu Ding
In this paper, three bio-reactors were designed to determine the effects of different zero-valent iron (ZVI) powder dosages on anammox. Initially, a high ZVI dosage inhibited the anammox process, while anammox adopted the habitat after long-term culture. The continuous experiment indicated that R2 (100 mg ZVI/L) had no notable effect on shortening the anammox start-up time, while R3 (1000 mg ZVI/L) reduced the start-up time from 96 to 87 days. The stable nitrogen isotope test suggested that the anammox activity of R3 was higher than that of R1 (without ZVI powder) during the stabilisation stage. The anammox growth rate increased by 20% in R3, which was verified by the real-time quantitative PCR results. In comparison to the control, the anammox quantity increased by approximately 44.5% and 54% at 100 and 1000 mg/L on day 120, respectively, which is likely due to lower dissolved oxygen and oxide-reduce potential created by the ZVI powder. The addition of ZVI may release iron ions, reduce the DO and ORP, and regulate the habitat pH conditions, which are beneficial for promoting anammox bacteria. Moreover, ZVI may directly partake in the anammox reaction pathway and improve the total nitrogen removal.
研究领域:
环境工程
·
微生物学
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DOI
高影响因子
ANALYST
·
2012
·Vol. 138
·期 1
·197-203
·
IF 4.23
Sai Bi,Yangyang Cui,Li Li
An ultrasensitive and highly selective method for polymerase chain reaction-free (PCR-free) messenger RNA (mRNA) expression profiling is developed through a novel cross-rolling circle amplification (C-RCA) process based on DNA-rotaxane nanostructures. Two species of DNA pseudorotaxane (DPR) superstructures (DPR-I and DPR-II) are assembled by threading a linear DNA rod through a double-stranded DNA (dsDNA) ring containing two single-stranded gaps. In this assay, cDNA that is specific for β-actin (ACTB) mRNA is taken as a model analyte. Upon the introduction of the target cDNA, the cDNA and the biotin-modified primer are hybridized to the single-stranded regions of the DNA rod and the gap-ring, respectively. As a result, the DPR-I dethreads into free DNA macrocycle and a dumbbell-shaped DNA nanostructure. In the presence of DNA polymerase/dNTPs, two release-DNA on the DPR-I are replaced by polymerase with strand-displacement activity, which can act as the input of the DPR-II to trigger the dethreading of DPR-II and the RCA reaction, releasing another two specified release-DNA strands those in turn serve as the “mimic cDNA” for DPR-I. The C-RCA reaction then proceeds autonomously. To overcome the high background induced by hemin itself, the biotinylated rolling circle products are captured by streptavidin-coated MNPs, achieving a detection limit as low as 0.1 zmol cDNA. The assay also exhibits an excellent selectivity due to its unique DNA nanostructure fabricated through base pairing hybridization. The ACTB mRNA expression in mammary cancer cells (MCF-7) is successfully detected.
研究领域:
分子生物学
·
生物化学
·
纳米医学
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DOI
PubMed
高影响因子
Biomaterials Science
·
2017
·Vol. 5
·期 7
·1279-1292
·
IF 4.21
Yawen Liu,Zhaozhu Zheng,He Gong,Meng Liu,Shaozhe Guo,Gang Li,Xiaoqin Wang,David L. Kaplan
The structure of DNA is susceptible to alterations at high temperature and on changing pH, irradiation and exposure to DNase. Options to protect and preserve DNA during storage are important for applications in genetic diagnosis, identity authentication, drug development and bioresearch. In the present study, the stability of total DNA purified from human dermal fibroblast cells, as well as that of plasmid DNA, was studied in silk protein materials. The DNA/silk mixtures were stabilized on filter paper (silk/DNA + filter) or filter paper pre-coated with silk and treated with methanol (silk/DNA + PT-filter) as a route to practical utility. After air-drying and water extraction, 50–70% of the DNA and silk could be retrieved and showed a single band on electrophoretic gels. 6% silk/DNA + PT-filter samples provided improved stability in comparison with 3% silk/DNA + filter samples and DNA + filter samples for DNA preservation, with ∼40% of the band intensity remaining at 37 °C after 40 days and ∼10% after exposure to UV light for 10 hours. Quantitative analysis using the PicoGreen assay confirmed the results. The use of Tris/borate/EDTA (TBE) buffer enhanced the preservation and/or extraction of the DNA. The DNA extracted after storage maintained integrity and function based on serving as a functional template for PCR amplification of the gene for zinc finger protein 750 (ZNF750) and for transgene expression of red fluorescence protein (dsRed) in HEK293 cells. The high molecular weight and high content of a crystalline beta-sheet structure formed on the coated surfaces likely accounted for the preservation effects observed for the silk/DNA + PT-filter samples. Although similar preservation effects were also obtained for lyophilized silk/DNA samples, the rapid and simple processing available with the silk–DNA–filter membrane system makes it appealing for future applications.
研究领域:
遗传学
·
生物技术
·
材料科学
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产品关联
DOI
PubMed
高影响因子
ANALYTICAL CHEMISTRY
·
2022
·
IF 8.008
Chunyang Wei,Chengzhuang Yu,Shanshan Li,Jiyu Meng,Tiejun Li,Jingmeng Cheng,Feng Pan,Junwei Li
Digital polymerase chain reaction (PCR) plays important roles in the detection and quantification of nucleic acid targets, while there still remain challenges including high cost, complex operation, and low integration of the instrumental system. Here, in this work, a novel microfluidic chip based on co-flow step emulsification is proposed for droplet digital PCR (ddPCR), which can achieve droplet generation, droplet array self-assembly, PCR amplification, and fluorescence detection on a single device. With the combination of single-layer lithography and punching operation, a step microstructure was constructed and it served as the key element to develop a Laplace pressure gradient at the Rayleigh–Plateau instability interface so as to achieve droplet generation. It is demonstrated that the fabrication of step microstructure is low cost, easy-to-operate, and reliable. In addition, the single droplet volume can be adjusted flexibly due to the co-flow design; thus, the ddPCR chip can get an ultrahigh upper limit of quantification to deal with DNA templates with high concentrations. Furthermore, the volume fraction of the resulting droplets in this ddPCR chip can be up to 72% and it results in closely spaced droplet arrays, makes the best of CCD camera for fluorescence detections, and is beneficial for the minimization of a ddPCR system. The quantitative capability of the ddPCR chip was evaluated by measuring template DNA at concentrations from 20 to 50 000 copies/μL. Owing to the characteristics of low cost, easy operation, excellent quantitative capability, and minimization, the proposed ddPCR chip meets the requirements of DNA molecule quantification and is expected to be applied in the point-of-care testing field.
研究领域:
生物技术
·
分子生物学
·
医学检验
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产品关联
DOI
PubMed
高影响因子
INTERNATIONAL JOURNAL OF LEGAL MEDICINE
·
2021
·Vol. 135
·期 2
·431-440
·
IF 2.686
Jiang Bowei,He Wei,Jin Chuan,Liu Ying,Wen Dan,Wang Chudong,ZEYE Moutanou Modeste Judes,Li Jienan,Zha Lagabaiyila
Genotyping of short tandem repeat (STR) markers is the basic method of forensic science. Enhanced technologies are needed to meet the requirements of databasing and casework samples. The STRscan-17LC kit is a 6 Dye STR kit which amplifies 16 STR loci: D3S1358, TPOX, D16S539, vWA, D2S1338, CSF1PO, D19S433, D7S820, FGA, D8S1179, D5S818, D13S317, D18S51, TH01, D12S391, and D21S11 and the sex-determinant locus amelogenin. This kit is designed for better tolerance to PCR inhibitors and analysis of mildly degraded samples with all fragments smaller than 330 bases. In this study, the STRscan-17LC kit is validated according to the SWGDAM (Scientific Working Group on DNA Analysis Methods) guidelines, including PCR-based studies, sensitivity, precision and accuracy, inhibitors, species specificity, DNA mixture studies, population, and concordance studies. The validation results suggest that the STRscan-17LC kit is a useful tool for forensic application.
研究领域:
法医学
·
分子生物学
·
遗传学
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DOI
PubMed
高影响因子
Frontiers in Microbiology
·
2019
·
IF 4.259
He Shengnan,An Tai,A Runa,Liu Shouan
The tea plant [Camellia sinensis (L.) O. Kuntze] is one of the most important leaf crops, and it is widely used for the production of non-alcoholic beverages worldwide. Tea also has a long history of medicinal use. Colletotrichum camelliae Massee is one of the dominant fungal pathogens that infects tea leaves and causes severe tea anthracnose disease. To analyze the molecular biology of C. camelliae, the quantification of pathogen gene expression by the RT-qPCR method is necessary. Reliable RT-qPCR results require the use of stable reference genes for data normalization. However, suitable reference genes have not been reported in C. camelliae thus far. In this study, 12 candidate genes (i.e., CcSPAC6B12.04c, CcWDR83, Cchp11, Ccnew1, CcHplo, CcRNF5, CcHpcob, CcfaeB-2, CcYER010C, CcRNM1, CcUP18, and CcACT) were isolated from C. camelliae and assessed as potential reference genes. The expression stability of these genes in C. camelliae during spore germination and mycelial growth and interaction with host plants was first evaluated using several statistical algorithms, such as geNorm, NormFinder, and Bestkeeper. A web-based analysis program, Refinder, was then used to find the most suitable reference genes. Our results indicated that Cenew1, CcHplo, and CcSPAC6B12.04c were the most stable reference genes in C. camelliae under all conditions. Our work provided the most suitable reference genes for future studies performed to quantify the target gene expression levels of C. camelliae.
研究领域:
植物病理学
·
分子生物学
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DOI
PubMed
高影响因子
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