JOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE
·
2021
·Vol. 101
·期 11
·4490-4497
·
IF 3.639
Ming Li,Xia Hong,Xuchun Qiu,Chuqin Yang,Yuhao Mao,Yan Li,Zhenjiang Liu,Daolin Du
BACKGROUND The ultrasensitive monitoring strategy of zearalenone (ZEN) is essential and desirable for food safety and human health. In the present study, a coupling of gold nanoparticles-DNA barcode and direct competitive immunoassay-based real-time polymerase chain reaction signal amplification (RT-IPCR) for ZEN close to the sensitivity of PCR-like levels is described and evaluated. RESULTS The RT-IPCR benefited from the use of a DNA barcode and RT-PCR detection strategy, thus resulting in ultrasensitive and simple detection for ZEN. Under the optimal RT-IPCR, the linear range of detection was from 0.5 to 1000 pg mL −1 and the limit of detection was 0.5 pg mL −1 , which was 400-fold lower than the enzyme-linked immunosorbent assay. The detection procedure was simplified and the detection time was shortened. The specificity, accuracy and precision of the RT-IPCR confirmed a high performance. ZEN-positive contamination levels were from 0.056 to 152.12 ng g −1 by the RT-IPCR, which was demonstrated to be highly reliable by liquid chromatography-tandem mass spectrometry. CONCLUSION The proposed RT-IPCR could be used as an alternative for detecting ZEN with satisfactory ultrasensitivity, simplicity, low cost and high-throughput. The present study could provide a strategy for the ultrasensitive detection of the small molecule with a simple and practical approach, which has significant appeal and application prospects.
研究领域:
食品安全
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分子生物学
·
免疫学
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产品关联
DOI
PubMed
高影响因子
Animals
·
2023
·Vol. 13
·期 20
·3177
·
IF 3
Hongbo Zhang,Nan Zhang,Jianhua Li,Panpan Zhao,Xin Li,Xiaocen Wang,Xu Zhang,Bao Yuan,Fei Gao,Pengtao Gong,Xichen Zhang
Simple SummaryTritrichomonas muris(T. muris) mainly parasitizes the ceca of different rodent species worldwide. To date, there are no ideal methods for the detection ofT. murisinfections in laboratory mice. This study developed a nested PCR reaction using a novel specific primer based on the conserved regions of the SSU rRNA gene ofT. murisfor a molecular epidemiological survey (investigation) ofT. murisinfections in laboratory mice. The results showed that the nested PCR system has higher sensitivity, reliability, and specificity. The nested PCR system showed an infection rate ofT. murisof 18.96% (58/306), which was higher than the infection rate of 14.05% (43/306) that was detected via smear microscopy in fecal samples from five mouse strains. The sensitivity and specificity of nested PCR in detectingT. muriswas found to be 100%, and it demonstrated a 26% increase in diagnostic sensitivity compared to the smear microscopy method. The present study provides a new method for the molecular epidemiological investigation ofT. murisinfections in laboratory mice.AbstractA variety of rodent ceca are parasitized byTritrichomonas muris(T. muris), a flagellated protozoan. To date, there are no ideal methods for the detection ofT. murisinfections in laboratory mice; thus, new molecular methodologies for its specific detection need to be developed. In this study, using staining and SEM, it was observed thatT. murishas a pear-shaped body and contains three anterior flagella. A nested PCR system with novel specific primers was designed based on the conserved regions of the SSU rRNA gene ofT. muris. The nested PCR system forT. murisshowed good specificity and high sensitivity for at least 100T. muristrophozoites/mL and 0.1 ng/μL of fecal genomic DNA, which means that 176 trophozoites per gram of mouse feces could be detected. When using this nested PCR system, the detection rate was 18.96% (58/306), which was higher than the detection rate of 14.05% (43/306) detected via smear microscopy in fecal samples from five mouse strains. The sensitivity and specificity of nested PCR in detectingT. muriswas found to be 100%, and it demonstrated a 26% increase in diagnostic sensitivity compared to the smear microscopy method in the present study. In conclusion, the nested PCR developed with novel primers based on the SSU rRNA gene ofT. murishas good accuracy, specificity, and sensitivity for the detection ofT. murisinfections in laboratory mice.Keywords:Tritrichomonas muris;SSU rRNA;primer;nested PCR;laboratory mice
研究领域:
分子生物学
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寄生虫学
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兽医学
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DOI
PubMed
高影响因子
Small
·
2025
·
IF 12.1
Yu Zhang,Jin Li,Yixuan Wu,Yunqing Wang,Xiaoyan Wang,Changjun Lv,Lingxin Chen
Pulmonary fibrosis (PF) is a severe respiratory disease, but early diagnosis of PF is challenging. Here, a non-invasive approach is developed to rapidly detect bronchoalveolar lavage fluid (BALF)-derived exosomal microRNAs (miRNAs) for PF progression based on hybrid nanoheater-driven digital polymerase chain reaction (nanodPCR). The hybrid nanoheaters, composed of plasmonic nanostructures and attached black hole quenchers (BHQs), generate a synergistic photothermal effect from the plasmonic field amplification. Gelatin microcarriers doped with such nanoheaters enable ultra-rapid thermal cycling (9.5 °C s −1 heating rate) under near-infrared laser irradiation, which is sufficient for ultrafast thermal cycling. This rapid thermal cycling capability, coupled with fluorescence detection, allows precise quantification of exosomal miRNAs through nanodPCR, thereby facilitating longitudinal monitoring of PF progression with an ultrashort turnover time (8.8 min), nearly seven-fold faster than conventional PCR protocols (≈60 min). Compared with normal mice, expression of miR-142-3p and miR-21-5p in PF mice at different stages significantly increases over time, while let-7d-5p expression decreases. Furthermore, clinical validation in 40 PF patients through this nanodPCR platform identifies disease-stage-specific miRNA profiles from healthy controls. Therefore, this work not only presents a robust photothermal-driven PCR platform but also demonstrates the potential of BALF-derived exosomal miRNA as non-invasive biomarkers for PF progression.
研究领域:
生物医学工程
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分子诊断
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纳米技术
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呼吸系统疾病
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microRNA生物标志物
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即时检测
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产品关联
DOI
PubMed
高影响因子
CHEMICAL ENGINEERING JOURNAL
·
2026
·
IF 12.5
Jian Duan,Kun Wang,Xiaoqiang Shu,Lianhao Zhao,Changhui Chen,Shifang Fei,Min Xi,Xiang Chen,Diankai Wang,Kaihuan Zhang,Sijia Xie,Bo Liu,Chang Chen,Zheng Deng
Rapid and timely nucleic acid detection through polymerase chain reaction (PCR) is an urgent demand in epidemic prevention and control. However, conventional Peltier-based PCR instruments are too slow to meet this requirement. Herein, an ultra-fast photothermal real-time quantitative PCR (qPCR) system that integrates a metal-organic framework (MOF)-based photothermal membrane with a silicon-based microfluidic chip is constructed for rapid SARS-CoV-2 detection. After optimization of the synthesis temperature of zirconium-ferrocenyl (Zr-Fc) MOFs and the thickness of Zr-Fc MOF/SWCNTs (ZFC) membranes, the resultant ZFC-22 membrane exhibits excellent and robust photothermal performance with an ultra-fast heating rate of 60.3 °C/s, enabling a rapid heat generation rate. Subsequently, the ZFC-22 membrane is integrated into a custom-designed silicon-based microfluidic chip that not only enhances heat conduction but also effectively confines heat in the PCR microchannel via thermal-isolation grooves, thereby reducing heat loss. Finally, a photothermal real-time qPCR system consisting of photothermal modules, microfluidic chips, control modules, and fluorescence imaging modules is constructed, which completes 40 nucleic acid amplification cycles within 10 min with an excellent amplification efficiency (98.6%), a low limit of detection (10 copies/μL), and an extremely low average optical power (1 W/well).
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产品关联
DOI
高影响因子
SENSORS
·
2026
·Vol. 26
·期 7
·2126
·
IF 4
Zhenqing Li,Yueqing Wang,Jing Yang,Bo Yang,Yuan Zeng,Shinichi Sekine,Yoshinori Yamaguchi
Polymerase chain reaction (PCR) is widely regarded as the gold standard for nucleic acid analysis; however, conventional thermal cycling limits its applicability in rapid and compact analytical systems. Here, we report an oscillating-flow microfluidic PCR method that enables rapid and flexible amplification by repeatedly shuttling the reaction mixture between two fixed-temperature zones. Unlike continuous-flow PCR, the proposed approach decouples PCR cycle number from microchannel geometry, allowing programmable cycling while reducing chip footprint. To enhance analytical reliability, polymer-assisted surface passivation using polyvinylpyrrolidone was employed to suppress nonspecific adsorption in polydimethylsiloxane (PDMS) microchannels, significantly improving amplification efficiency. Using Porphyromonas gingivalis and Treponema denticola as representative periodontal pathogens, 35-cycle amplification was completed within 20 min with reliable product yield. The proposed method advances oscillating-flow PCR toward a robust analytical strategy for rapid pathogen detection and related microfluidic nucleic acid analysis.
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产品关联
DOI
PubMed
高影响因子
Frontiers in Plant Science
·
2023
·
IF 5.6
Guohui Li,Muhammad Aamir Manzoor,Guoyu Wang,Cunwu Chen,Cheng Song
Introduction KNOX plays a pivotal role in governing plant growth, development, and responses to diverse abiotic and biotic stresses. However, information on the relationship between the KNOX gene family and expression levels under different treatments in Dendrobium is still limited. Methods To address this problem, we first used bioinformatics methods and revealed the presence of 19 KNOX genes distributed among 13 chromosomes in the Dendrobium huoshanense genome. Through an analysis of phylogenetic relationships, these genes were classified into three distinct clades: class I, class II, and class M. Our investigation included promoter analysis, revealing various cis -acting elements associated with hormones, growth and development, and abiotic stress responses. Additionally, qRT-PCR experiments were conducted to assess the expression patterns of DhKNOX genes under different treatments, including ABA, MeJA, SA, and drought. Results The results demonstrated differential expression of DhKNOX genes in response to these treatments, thereby highlighting their potential roles in stress adaptation. Discussion Overall, our results contribute important insights for further investigations into the functional characterization of the Dendrobium KNOX gene family, shedding light on their roles in plant development and stress responses.
研究领域:
植物学
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分子生物学
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基因组学
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产品关联
DOI
PubMed
高影响因子
JOURNAL OF PHARMACEUTICAL AND BIOMEDICAL ANALYSIS
·
2023
·
IF 3.4
Yuxiang Zhou,Yanyun Wang,Mengyuan Song,Lanrui Jiang,Chaoran Sun,Shuangshuang Wang,Hewen Yao,Zefei Wang,Xindi Wang,Chunhui Liu,Haibo Luo,Feng Song
The droplet digital Polymerase Chain Reaction (ddPCR) has garnered recognition for its distinctive attribute of absolute quantification. And it has found practical utility in age prediction through DNA methylation profiles. However, a prevalent limitation in current ddPCR methodologies is the restricted capacity to detect only two targets concurrently in most instruments, leading to high costs, sample wastage, and labor-intensive procedures. To address the limitations, a novel high-throughput ddPCR system allowing for the simultaneous detection of eight targets was developed. Through the implementation of a new 8-plex ddPCR assay, coupled with comprehensive linear regression analyses involving primers and probes ratios, diverse inputs of single CpG sites with distinct primers and probes, and varying plex assay configurations, stable DNA methylation values for four CpGs and stable measurement precisions for distinct multiplex systems were consistently observed. These findings pave the way for advancing the field of chemistry science by enabling more efficient and cost-effective methods. Furthermore, the comparative validation of ddPCR and SNaPshot demonstrated a remarkable concordance in results, and the system also displayed well in the field of various aspects, including species specificity, DNA input, and aged samples. In this study, the recommended input of bisulfite-converted DNA was determined to be 10–50 ng due to the double-positive droplets. Notably, the Pearson correlation coefficient squared values of four CpGs were 0.4878 (ASPA), 0.4832 (IGSF1), 0.6881 (COL1A1), and 0.6475 (MEIS1-AS3). And the testing set exhibited a mean absolute error of 4.5923 years, indicating the robustness and accuracy of the age-predictive model.
研究领域:
分子生物学
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法医学
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化学科学
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DOI
PubMed
高影响因子
Scientific Reports
·
2014
·Vol. 4
·期 1
·1-8
·
IF 5.078
Zhou Dinggang,Guo Jinlong,Xu Liping,Gao Shiwu,Lin Qingliang,Wu Qibin,Wu Luguang,Que Youxiong
To meet the demand for detection of foreign genes in genetically modified (GM) sugarcane necessary for regulation of gene technology, an efficient method with high specificity and rapidity was developed for the cry1Ac gene, based on loop-mediated isothermal amplification (LAMP). A set of four primers was designed using the sequence of cry1Ac along with optimized reaction conditions: 5.25 mM of Mg 2+ , 4:1 ratio of inner primer to outer primer, 2.0 U of Bst DNA polymerase in a reaction volume of 25.0 μL. Three post-LAMP detection methods (precipitation, calcein (0.60 mM) with Mn 2+ (0.05 mM) complex and SYBR Green I visualization), were shown to be effective. The sensitivity of the LAMP method was tenfold higher than that of conventional PCR when using templates of the recombinant cry1Ac plasmid or genomic DNA from cry1Ac transgenic sugarcane plants. More importantly, this system allowed detection of the foreign gene on-site when screening GM sugarcane without complex and expensive instruments, using the naked eye. This method can not only provide technological support for detection of cry1Ac , but can also further facilitate the use of this detection technique for other transgenes in GM sugarcane.
研究领域:
分子生物学
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植物生物技术
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基因工程
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产品关联
DOI
PubMed
高影响因子
SENSORS AND ACTUATORS B-CHEMICAL
·
2017
·
IF 5.401
Xiangyu Meng,Fulai Li,Fan Li,Yonghua Xiong,Hengyi Xu
In this study, vancomycin (Van) functionalized PEGylated-magnetic nanoparticles (Van-PMs) combined with polymerase chain reaction (PCR) showed great potential for highly efficient detection of L. monocytogenes at low infectious dose. Herein, Van-PMs played as universal molecular probes for targeting bacterial cells, while PEG was used to help Van close to D-alanyl- d -alanine (D-Ala- d -Ala) moieties on surface of L. monocytogenes . Under optimum conditions, the developed Van-PMs complexes exhibited extremely high capture efficiency (CE) in a 10 mL bacterium suspension with the value was higher than 90% and 83% in the phosphate-buffered saline (PBS) solution and lettuce samples, respectively. Meanwhile, Gram-negative bacteria slightly influenced CE even under concentrations as high as 10 6 CFU/mL. The assay of Van-PMs-PCR was applied to detect L. monocytogenes with detection limits of 30 CFU/mL and 30 CFU/g in PBS solution and lettuce samples, respectively. The whole experiment was completed within 4 h. This assay demonstrated the potential of this modified method in practical application involving the detection of L. monocytogenes or other Gram-positive pathogenic bacteria.
研究领域:
微生物学
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纳米技术
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分子生物学
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食品安全
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DOI
高影响因子
COLLOIDS AND SURFACES B-BIOINTERFACES
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2016
·
IF 3.902
Yalong Bai,Yan Cui,George C. Paoli,Chunlei Shi,Dapeng Wang,Min Zhou,Lida Zhang,Xianming Shi
Magnetic separation has great advantages over traditional bio-separation methods and has become popular in the development of methods for the detection of bacterial pathogens, viruses, and transgenic crops. Functionalization of magnetic nanoparticles is a key factor for efficient capture of the target analytes. In this paper, we report the synthesis of amino-rich silica-coated magnetic nanoparticles using a one-pot method. This type of magnetic nanoparticle has a rough surface and a higher density of amino groups than the nanoparticles prepared by a post-modification method. Furthermore, the results of hydrochloric acid treatment indicated that the magnetic nanoparticles were stably coated. The developed amino-rich silica-coated magnetic nanoparticles were used to directly adsorb DNA. After magnetic separation and blocking, the magnetic nanoparticles and DNA complexes were used directly for the polymerase chain reaction (PCR), without onerous and time-consuming purification and elution steps. The results of real-time quantitative PCR showed that the nanoparticles with higher amino group density resulted in improved DNA capture efficiency. The results suggest that amino-rich silica-coated magnetic nanoparticles are of great potential for efficient bio-separation of DNA prior to detection by PCR.
研究领域:
纳米技术
·
生物化学
·
分子生物学
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产品关联
DOI
PubMed
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
·
生物技术
·
分析化学
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产品关联
DOI
高影响因子
TALANTA
·
2021
·
IF 6.057
Min-Xi Li,Yao Chen,Zeng-Ping Chen,Ru-Qin Yu
Herein, a label free and sensitive miRNA detection method with enhanced practical applicability was developed based on the locked nucleic acid (LNA) assisted repeated fishing amplification strategy. The working mechanism of the proposed method is as follows: 1) a DNA probe (i.e, L-DNA) with LNA bases is immobilized onto the surface of a gold foil. The L-DNA hybridizes with the 3′ terminus of the first strands of complementary deoxyribonucleic acid (cDNA) of the target miRNA in the test samples; 2) The protruding 5′ terminus of the cDNA serves as a ‘fishhook’ to repeatedly fish the products of a hybridization chain reaction (HCR) out from a ‘reaction tube’; 3) The HCR products can be unloaded from the gold foil into a ‘product tube’ through temperature-controlled dehybridization; 4) The concentration of the target miRNA is determined based on the fluorescence intensity generated by the addition of SYBR-Green I (SG) into the ‘product tube’. The proposed platform was applied to the detection of miRNA-122 in cell lysate samples and obtained quantitative results with accuracy comparable to the quantitative reverse transcription PCR method (qRT-PCR). It is worth pointing out that the proposed platform achieved a limit of detection value of 2.9 fM for miRNA-122 by a simple but effective LNA-assisted repeated fishing amplification strategy instead of complicated enzyme-based amplification techniques. It is reasonable to expect that the proposed method provides a competitive alternative for designing practically applicable, cost-effective and label-free miRNA detection methods.
研究领域:
生物化学
·
分子生物学
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纳米技术
·
分析化学
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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）