Biotechnology Journal
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2023
·Vol. 19
·期 1
·2300273
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IF 4.7
Wenqi Hu,Yidan Zhu,Qu Tang,Xiaolei Ji,Lei Wang,Weijun Ou,Guo Li,Li Wu,Hui Cong,Yuling Qin
The chip-based digital polymerase chain reaction (PCR) is an indispensable technique for amplifying and quantifying nucleic acids, which has been widely employed in molecular diagnostics at both fundamental and clinical levels. However, the previous designs have yet to achieve widespread application due to limitations in complex chip fabrication, pretreatment procedures, special surface properties, and low throughput. This study presents a facile digital microfluidic chip driven by centrifugal force for digital PCR analysis. Interestingly, regardless of the hydrophilicity or hydrophobicity of the inner chip surface, an efficient digitization process can be achieved. PCR reagents introduced into the inlet can be allocated to 9600 microchambers and subsequently isolated by the immiscible phase (silicone oil). The centrifugal priming approach offers a facile means to achieve high-throughput analysis. The design was further employed for the quantification of nucleic acids using digital PCR. The calculated result exhibited a strong correlation with the measured value at the concentrations from 1 copy/μL to 1000 copies/μL ( R 2 = 0.99). Additionally, the chip also allowed digital multiplexed analysis, thereby indicating its potential for multi-target detection applications.
研究领域:
分子诊断
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生物技术
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微流控技术
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核酸分析
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DOI
PubMed
高影响因子
Biomed Research International
·
2020
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IF 2.276
Jiyu Chen,Zhenzhen Bao,Yanli Huang,Zhenglong Wang,Yucheng Zhao
Quantitative real-time PCR (qPCR) has become a widely used approach to analyze the expression level of selected genes. However, owing to variations in cell types and drug treatments, a suitable reference gene should be selected according to special experimental design. In this study, we investigated the expression level of ten candidate reference genes in hepatoma carcinoma cell (HepG2) and human hepatocyte cell line (L02) treated with ethanol (EtOH), hydrogen peroxide (H2O2), acetaminophen (APAP), and carbon tetrachloride (CCl4), respectively. To analyze raw cycle threshold values (Cp values) from qPCR run, three reference gene validation programs, including Bestkeeper, geNorm, and NormFinder, were used to evaluate the stability of ten candidate reference genes. The results showed that TATA-box binding protein (TBP) and tubulin beta 2a (TUBB2a) presented the highest stability for normalization under different treatments and were regarded as the most suitable reference genes of HepG2 and L02. In addition, this study not only identified the most stable reference genes of each treatment, but also suggested that β-actin (ACTB), glyceraldehade-3-phosphate dehydrogenase (GAPDH), tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta (YWHAZ), and beta-2 microglobulin (B2M) were the least stable reference genes in HepG2 and L02. This work was the first report to systematically explore the stability of reference genes in injured models of HepG2 and L02.
研究领域:
分子生物学
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遗传学
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DOI
PubMed
高影响因子
MICROCHEMICAL JOURNAL
·
2022
·
IF 5.304
Jia Wei,Yaoqi Wang,Jiaxue Gao,Zhenshengnan Li,Renzhu Pang,Tianchao Zhai,Yuan Ma,Zhenxin Wang,Xianying Meng
Qualitative and quantitative analysis of carcinogenic mutations in circulating tumor DNA (ctDNA) plays a unique role in the early screening and precise diagnosis of cancer. Herein, we introduced an electrochemical-enrichment assisted amplification refractory mutation system-quantitative real-time PCR (EC-ARMS-qPCR) assay to detect the BRAF V600E carcinogenic mutation of thyroid cancer in plasma samples. Cell-free DNA (cfDNA) were firstly enriched by an electric field-driven adsorption and desorption process, and then ctDNA in cfDNA were analyzed by a designed allele-specific qPCR assay. The EC-ARMS-qPCR assay provides good sensitivity and specificity for the identification of single nucleotide polymorphisms (SNPs) of BRAF V600E mutant ctDNA in a complex system, which can detect as low as 2 BRAF V600E mutant copies in Tris buffer and 25 BRAF V600E mutant copies in Tris buffer containing 50% plasma with a allele frequency of 0.01% (i.e., mutant-to-wild-type ratio of 1: 9999). The assay was assessed in plasma samples of patients with papillary thyroid cancer (PTC) and benign thyroid nodule (TN). The EC-ARMS-qPCR assay detected 40.74% and 10.00% BRAF V600E mutation ctDNA in plasma samples of 54 PTC patients and 20 TN patients, respectively. The overall concordance between BRAF V600E mutant ctDNA by EC-ARMS-qPCR assay and BRAF V600E mutant of matched pathological tissue genes by fine needle aspiration biopsy (FNAB) is 73.08%, suggesting that the EC-ARMS-qPCR assay is appropriate for detecting carcinogenic mutations in liquid biopsy samples.
研究领域:
肿瘤学
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分子生物学
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医学遗传学
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临床诊断
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产品关联
DOI
高影响因子
FOOD CHEMISTRY
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2023
·
IF 8.8
Bingxue Hu,Hongbin Pu,Da-Wen Sun
In this study, a high-performance, stable and homogeneous [email protected] /CMC/qPCR flexible film surface-enhanced Raman scattering (SERS) substrate was constructed by synergistically stabilizing and protecting bimetallic core–shell [email protected] nanorods ( [email protected] ) with carboxymethylcellulose (CMC) and fluorescent-quantitative-polymerase-chain-reaction (qPCR) film. The network structure of CMC immobilized and aligned [email protected] through coordination of carboxyl groups with surface Ag atoms to provide intensive and stable ‘hot spots’, and the qPCR bilayer film performed as carrier and barrier to protect [email protected] from oxidation, humidity and optical damage and improved the robustness and stability. The [email protected] /CMC/qPCR film was used for in-situ extraction and SERS detection of thiabendazole on nectarine (0.24 ppm) and lemon (0.27 ppm) with low detection of limits. Furthermore, it retained 98.6% SERS performance after storage for 90 days under ambient conditions, revealing the great potential in promoting the commercialization of the SERS technique for sensitive contaminants sensing with simple fabrication procedures, homogeneity, reproducibility and long-term stability.
研究领域:
材料科学
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分析化学
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食品安全
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纳米技术
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产品关联
DOI
PubMed
高影响因子
ANALYTICAL CHEMISTRY
·
2024
·
IF 6.7
Jingyu Cui,Jiafang Piao,Houyu Han,Weipan Peng,Mengyao Lin,Dianming Zhou,Cheng Zhu,Xiaoqun Gong
Circulating microRNAs (miRNAs) have recently emerged as noninvasive disease biomarkers. Quantitative detection of circulating miRNAs could offer significant information for clinical diagnosis due to its significance in the development of biological processes. In response to the current challenges of circulating miRNA detection, we introduce a sensitive, selective, and versatile circulating miRNA detection strategy using terminal deoxynucleotidyl transferase (TdT)-catalyzed RNA-primed DNA polymerization (TCRDP) coupled with semiarbitrary qPCR (SAPCR). Semiarbitrary qPCR was first developed here to detect long fragment targets with only a short-known sequence or to detect a short fragment target after extension with terminal transferase. Besides, the subsequent results show that TdT has a preference for RNA, particularly for extending RNAs with purine-rich and unstructured ends. Consequently, utilizing this assay, we have successfully applied it to the quantitative analysis of circulating miR-122 in animal models, a sensitive and informative biomarker for drug-induced liver injury, and as low as 200 zmol of the target is detected with desirable specificity and sensitivity, indicating that the TCRDP-SAPCR can offer a promising platform for nucleic acids analysis.
研究领域:
分子生物学
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临床诊断
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生物技术
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纳米技术
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产品关联
DOI
PubMed
高影响因子
Agronomy-Basel
·
2025
·Vol. 15
·期 8
·1813
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IF 3.4
Yufei Zhao,Xu Yang,Qiang Hu,Jie Zhang,Sumei Wan,Wen Chen
Asia minor bluegrass (Polypogon fugax), a widespread Poaceae weed, exhibits broad tolerance to abiotic stresses. Validated reference genes (RGs) for reliable RT-qPCR normalization in this ecologically and agriculturally significant species remain unidentified. This study identified eight candidate RGs using transcriptome data from seedling tissues. We assessed the expression stability of these eight RGs across various abiotic stresses and developmental stages using Delta Ct, BestKeeper, geNorm, and NormFinder algorithms. A comprehensive stability ranking was generated using RefFinder, with validation performed using the target genesCOR413andP5CS. Results identifiedEIF4AandTUBas the optimal RG combination for normalizing gene expression during heat stress, cold stress, and growth stages.EIF4AandACTwere most stable under drought stress,EIF4Aand28Sunder salt stress, andEIF4AandEF-1 under cadmium (Cd) stress. Furthermore,EIF4AandUBQdemonstrated optimal stability under herbicide stress. Additionally, application of validated RGs revealed higher acetyl-CoA carboxylase gene (ACCase) expression in one herbicide-resistant population, suggesting target-site gene overexpression contributes to resistance. This work presents the first systematic evaluation of RGs inP. fugax. The identified stable RGs provide essential tools for future gene expression studies on growth and abiotic stress responses in this species, facilitating deeper insights into the molecular basis of its weediness and adaptability.
研究领域:
植物学
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分子生物学
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植物遗传学
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杂草科学
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胁迫生理学
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产品关联
DOI
高影响因子
VACUUM
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2024
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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
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医学检测
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材料科学
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DOI
高影响因子
ANALYTICAL AND BIOANALYTICAL CHEMISTRY
·
2026
·
IF 4.1
Li Jiahao,Zhang Chuhan,Ou Zongdong,Zhao Yiran,Ji Zhirun,Feng Yufan,Chen Ting-Hsuan,Yang Zhugen,Wu Minghui,Mao Ruiqi,Zhang Chunyang,Chu Rui,Yang Xinyi,Liu Tianmi,Shan Yanke,Liu Fei
Rapid and reliable diagnostic methods are crucial for controlling animal disease outbreaks and reducing mortality, especially when effective drugs or vaccines are unavailable. While real-time quantitative polymerase chain reaction (qPCR) remains the gold standard for nucleic acid detection due to its superior sensitivity and specificity, conventional workflows are inherently cumbersome. Traditional extraction and amplification processes typically require 1.5 to 3 h, with further delays incurred by transporting samples to centralized laboratories. To overcome these limitations, this study introduces a point-of-care testing platform for on-site pathogen diagnostics. This system synergistically combines a centrifuge-free, glass fiber (GF) paper-based nucleic acid extraction card, room-temperature-stable lyophilized qPCR reagents, and a portable qPCR instrument. By eliminating the dependency on bulky benchtop equipment and cold-chain logistics, this platform streamlines lysis, extraction, and amplification, delivering sample-to-answer results within 45 min. The optimized system demonstrated excellent analytical sensitivity, achieving limits of detection (LoDs) of 10 copies/μL for the N gene of porcine epidemic diarrhea virus (PEDV) and 50 copies/μL for the PirA gene of Vibrio parahaemolyticus AHPND (VP AHPND ). Furthermore, clinical validation using complex field samples yielded a 100% diagnostic concordance rate with standard laboratory equipment. This study highlights the potential of the proposed system for early diagnosis and monitoring of animal diseases, providing a promising solution for rapid on-site screening in resource-constrained settings. Graphical abstract
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DOI
高影响因子
MICROCHEMICAL JOURNAL
·
2025
·
IF 5.1
Weijie Yuan,Yuzhu Wang,Runze Zhang,Yansong Li,HongLin Ren,Bo Zhang,Ruoran Shi,Chengwei Li,Jiaqi Hou,Shaohui Hu,Xueyu Hu,Shiying Lu,Xiaoxu Wang,Pan Hu
The present study reports a dual-functional platform that couples vancomycin-functionalized magnetic beads (VMBs) with real-time quantitative polymerase chain reaction (qPCR) to achieve rapid, culture-free discrimination and quantification of live and dead Listeria monocytogenes in complex food matrices. The exploitation of vancomycin's high-affinity binding to the D-Ala–D-Ala motif of intact peptidoglycan enables the selective capture and removal of viable cells, thereby leaving only DNA from non-viable bacteria for SYBR Green qPCR analysis. Optimisation of bead coupling and adsorption kinetics, in conjunction with a pre-calibrated live/dead correction algorithm, yields an LOD of 10 2 CFU/mL over a 10 1 –10 7 CFU/mL linear range and completes within 12 h—a process that is significantly faster than conventional culture methods. The validation of the method in beef, salad, cheese, and salmon matrices has yielded recoveries ranging from 96.4 to 99.2 %, with RSDs consistently below 3.6 %. Stability tests have further confirmed that dead-cell DNA remains amplifiable for a minimum of one week, and non-specific capture remains below 5 %. In contrast to isothermal or immunomagnetic approaches, the VMB-qPCR system offers a unique capability to provide unambiguous live/dead resolution without the use of photoactive dyes or extended enrichment, thereby providing a robust, high-throughput tool for food safety surveillance and clinical pathogen monitoring.
研究领域:
食品微生物学
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分子诊断
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生物传感器
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病原体检测
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生物技术
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分析化学
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产品关联
DOI
高影响因子
ACS Applied Materials & Interfaces
·
2023
·
IF 9.5
Xiuli Wang,Weiwei Fei,Zhifan Zhou,Mengjing Zhu,Yinghao Chang,Qilin Guo,Jia Guo,Changchun Wang
Magnetic-assisted DNA testing technology has attracted much attention in genetics, clinical diagnostics, environmental microbiology, and molecular biology. However, achieving satisfying DNA adsorption and desorption efficiency in real samples is still a big challenge. In this paper, a new kind of high-quality magnetic composite microsphere of MM@PGMA–PA-Ti4+ was designed and prepared for DNA extraction and detection based on the strong interaction of Ti4+ and phosphate groups. By taking the advantages of high magnetic susceptibility and high Ti4+ content, the MM@PGMA–PA-Ti4+ microspheres possessed remarkable extraction capacity for mimic biological samples (salmon sperm specimens) with saturated loadings up to 533.0 mg/g. When the DNA feeding amount was 100 μg and the MM@PGMA–PA-Ti4+ dosage was 1 mg, the adsorption and desorption efficiencies were 80 and 90%, respectively. The kinetic and equilibrium extraction data were found to fit well with the pseudo-second-order model and Freundlich isotherm model. Furthermore, the MM@PGMA–PA-Ti4+ microspheres were successfully employed for DNA extraction from mouse epithelial-like fibroblasts. The extraction ability (84 ± 4 μg/mg) and DNA purity were superior to the comparative commercial spin kits, as evaluated by electrophoresis assays and qPCR analysis. The experimental results suggest that the MM@PGMA–PA-Ti4+ microspheres possess great potential as an adsorbent for DNA purification from complex biological samples.
研究领域:
分子生物学
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临床诊断学
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环境微生物学
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产品关联
DOI
PubMed
高影响因子
ACS Sensors
·
2021
·
IF 7.711
Yongzhong Jiang,Menglu Hu,An-An Liu,Yi Lin,Linlin Liu,Bo Yu,Xiaoming Zhou,Dai-Wen Pang
The outbreak of COVID-19 caused a worldwide public health crisis. Large-scale population screening is an effective means to control the spread of COVID-19. Reverse transcription–polymerase chain reaction (RT-qPCR) and serology assays are the most available techniques for SARS-CoV-2 detection; however, they suffer from either less sensitivity and accuracy or low instrument accessibility for screening. To balance the sensitivity, specificity, and test availability, here, we developed enhanced colorimetry, which is termed as a magnetic pull-down-assisted colorimetric method based on the CRISPR/Cas12a system (M-CDC), for SARS-CoV-2 detection. By this method, SARS-CoV-2 RNA from synthetic sequences and cultured viruses can be detected by the naked eye based on gold nanoparticle (AuNP) probes, with a detection limit of 50 RNA copies per reaction. With CRISPR/Cas12a-assisted detection, SARS-CoV-2 can be specifically distinguished from other closely related viruses. M-CDC was further used to analyze 41 clinical samples, whose performance was 95.12%, consistent with that of an approved Clinical RT-qPCR Diagnosis kit. The developed M-CDC method is not dependent on sophisticated instruments, which makes it potentially valuable to be applied for SARS-CoV-2 screening under poor conditions.
研究领域:
分子生物学
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病毒学
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诊断医学
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产品关联
DOI
PubMed
高影响因子
ANALYTICAL CHEMISTRY
·
2022
·
IF 8.008
Jia-Hui Dong,Chen-Yu Xue,Xin-Ying Zhong,Ying-Lin Zhou,Xin-Xiang Zhang
Oxidative DNA damage is tightly linked to the development of multiple age-related diseases. The prominent oxidation product is 8-oxo-7,8-dihydroguanine (OG), which has been proved to be an important epigenetic-like biomarker. Quantification of the locus-specific OG frequency includes quantitative and locating information, which is of great significance for exploring the functional roles of OG in disease induction and gene regulation. Herein, an ultrasensitive quantification of OG at single-base resolution was established using real-time fluorescence quantitative polymerase chain reaction as an amplification tool. Based on the coding property of Bsu DNA polymerase that incorporates adenine on the opposite site of OG and the selectivity of the ligase for perfectly matched sequences, the difference between OG and G on the sequence could be enlarged. Well-performed Taq DNA ligase was selected out, and as low as 46.2 zmol of target DNA with an OG site and an OG frequency of 5% could be detected. G contents on a specific site were also detectable based on the similar principle, thus the OG frequency of this locus could be accurately determined by a standard addition method. This strategy was successfully applied to the evaluation of locus-specific OG in both model DNA and genomic DNA from human cervical carcinoma cell lines under multiple oxidative stress, showing the potential for functional research and dynamic monitoring of critical OG sites.
研究领域:
生物化学
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分子生物学
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基因组学
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疾病研究
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产品关联
DOI
PubMed
高影响因子
危险品化学品经营许可证（带存储）