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
·
分析化学
✓ 已验证
产品关联
DOI
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
✓ 已验证
DOI
高影响因子
BIOSENSORS & BIOELECTRONICS
·
2025
·
IF 10.5
Junjie Zeng,Yanping Li,Qinghua He,Ji Xia,Jiebiao Guo,Zhui Tu
Phage display-mediated immuno-polymerase chain reaction (PD-IPCR) combines the strengths of phage display technology and immuno-PCR. The link between genotype and phenotype has been established by displaying antibody fragments on the surface of phage particles, allowing signal transmission. In this study, we utilized aflatoxin B 1 (AFB 1 ) as a model system to investigate the efficacy of PD-IPCR. Signal amplification was achieved using both real-time fluorescence quantitative PCR (qPCR) and a custom-made droplet digital PCR (dPCR) system. Under optimized conditions, the assay using dPCR achieved a limit of detection of 0.015 ng/mL, a 6-fold improvement over qPCR. The validation of our method using major agricultural produce, such as corn, flour, and soy sauce, shows excellent recoveries ranging from 85.77 % to 108.28 %, with a total assay time of less than 2 h. Furthermore, we compared our results with a high-performance liquid chromatography (HPLC) method for AFB 1 content in corn and flour, revealing consistent outcomes (r = 0.9847). These results demonstrate the potential of nanobody-based PD-IPCR as a reliable tool for detecting mycotoxins.
研究领域:
食品安全
·
分子生物学
✓ 已验证
产品关联
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
✓ 已验证
DOI
PubMed
高影响因子
Analytical Methods
·
2024
·Vol. 16
·期 8
·1281-1287
·
IF 2.7
Keyu Li,Peng Xiao,Ningning Yuan,Shujuan Yan,Pei Zhao,Guowei Zuo
MiRNAs are critical regulators of target gene expression in many biological processes and are considered promising biomarkers for diseases. In this study, we developed a simple, specific, and sensitive miRNA detection method based on proximity ligation reaction, which is easy to operate. The method uses a pair of target-specific DNA probes immobilized on the same gold nanoparticles (AuNPs), which hybridize to the target miRNA. Hybridization brings the probes close together, allowing the formation of a continuous DNA sequence that can be amplified by Quantitative Real-time PCR (qPCR). This method eliminates the need for complex reverse transcription design and achieves high specificity for discriminating single base mismatches between miRNAs through a simple procedure. This method can sensitively measure three different miRNAs with a detection limit of 20 aM, providing high versatility and sensitivity, even distinguishing single-base variations among members of the miR-200 family with high selectivity. Due to its high selectivity and sensitivity, this method has important implications for the investigation of miRNA biological functions and related biomedical research.
研究领域:
分子生物学
·
纳米生物技术
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医学检测
✓ 已验证
产品关联
DOI
PubMed
高影响因子
CELLULAR AND MOLECULAR BIOLOGY
·
2023
·Vol. 69
·期 9
·
IF 1.6
Aili Xuan,Mei Yang,Qun Xia,Qian Sun
Asthma is a respiratory inflammatory disease, and nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4) is involved in the progression of respiratory diseases. However, the role of NOX4 in asthma remains unclear. In the present study, we aimed to explore the effects of NOX4 on airway remodeling and inflammation. NOX4 expression was measured using immunocytochemistry (IHC), western blot, and real-time PCR (qPCR). Lung tissues were stained using the H&E assay. ELISA was used to examine the levels of airway remodeling-related indicators, and qPCR was used to detect airway inflammatory factors. The results indicated that NOX4 is highly expressed in lung tissues, bronchoalveolar lavage fluid (BALF), and serum of OVA-treated mice. Inhibition of NOX4 alleviated OVA-induced airway remodeling and inflammation. Similarly, TGF-β1 was also upregulated in BALF and serum OVA-induced mice. Inhibition of TGF-β1 signaling also improved airway remodeling and inflammation induced by OVA. Moreover, the downregulation of NOX4 inactivated the TGF-β1-Smad2/3 pathway, and TGF-β1 decreased Smad2/3 expression. Moreover, inhibition of the TGF-β1 was enhanced, while TGF-β1 reversed the effects on airway remodeling and inflammation induced by NOX4 inhibition. Taken together, the downregulation of NOX4 improves airway remodeling and inflammation via inactivation of the TGF-β1-Smad2/3 pathway in asthma mice, suggesting that NOX4 may be a therapeutic target for asthma.
研究领域:
呼吸系统疾病
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分子生物学
·
免疫学
✓ 已验证
DOI
PubMed
高影响因子
Plant Stress
·
2024
·
IF 6.8
Haiyang Chi,Shasha Bai,Chenxiao Chen,Yuying Bao,Xiaoxin Qu,Shitao Sun,Jiangpeng Pan,Xiushi Yang,Chunsheng Hou,Yanchun Deng
Squalene plays a crucial role in plant growth, development, and stress tolerance. To select the best reference genes (RGs) for expression profile analysis of genes involved in squalene biosynthesis in olives under stress tolerance, the expression stability of 22 candidate RGs across four tissues (root, stem, leaf, and fruit) and five representative cultivars under drought stress was assessed using six methods. Our study showed that, ubiquitin-conjugating enzyme1 ( UBC1 ) and 60S ribosomal protein L18-2-like ( 60S ) were the most stable RGs across five different cultivars and various tissues. Additionally, elongation factor 1-alpha-like ( EF1-α2) and UBC1 were the most stable RG in olives under drought stress. UBC1 was the appropriate RG for further study, qPCR analysis showed that five certain genes involved in the squalene biosynthesis exhibited significant differential expression under varying conditions. Specially, the expression level of squalene synthase (SQS) in the leaves was highest, and HPLC analysis showed that squalene content was the highest in leaves. Likewise, the expression levels of SQS and farnesyl diphosphate synthase ( FPPS ) in the leaves of the cultivar Arbosana and Chelmsford Lal were significantly higher than those of the other cultivars, respectively, and HPLC analysis also showed that squalene content was highest in the two cultivars. Interestingly, the amount of squalene increased dramatically in olive under drought stress, as lupeol synthase ( LUPS ) was significantly up-regulated. To date, this study firstly provided the comprehensive analysis of RGs related to squalene biosynthesis in various cultivars and tissues of olives under drought stress.
研究领域:
植物科学
·
分子生物学
·
胁迫生理学
✓ 已验证
产品关联
DOI
高影响因子
BMC PLANT BIOLOGY
·
2023
·Vol. 23
·期 1
·1-17
·
IF 5.3
Sun Meiyu,Zhu Li,Zhang Yanan,Liu Ningning,Zhang Jinzheng,Li Hui,Bai Hongtong,Shi Lei
Background
Thyme derived essential oil and its components have numerous applications in pharmaceutical, food, and cosmetic industries, owing to their antibacterial, antifungal, and antiviral properties. To obtain thyme essential oil with different terpene composition, we developed new germplasm resources using the conventional hybridization approach. Results Phenotypic characteristics, including essential oil yield and composition, glandular trichome density, plant type, and fertility, of three wild Chinese and seven European thyme species were evaluated. Male-sterile and male-fertile thyme species were crossed in different combinations, and two F 1 populations derived from Thymus longicaulis (Tl) × T. vulgaris ‘Fragrantissimus’ (Tvf) and T. vulgaris ‘Elsbeth’ (Tve) × T. quinquecostatus (Tq) crosses were selected, with essential oil yield and terpene content as the main breeding goals. Simultaneously, simple sequence repeat (SSR) primers were developed based on the whole-genome sequence of T. quinquecostatus to authenticate the F 1 hybrids. A total of 300 primer pairs were selected, and polymerase chain reaction (PCR) was carried out on the parents of the two hybrid populations (Tl, Tvf, Tve, and Tq). Based on the chemotype of the parents and their F 1 progenies, we examined the expression of genes encoding two γ-terpinene synthases, one α-terpineol synthase, and maybe one geraniol synthase in all genotypes by quantitative real-time PCR (qRT-PCR). Conclusion We used hybridization to create new germplasm resources of thyme, developed SSR markers based on the whole-genome sequence of T. quinquecostatus , and screened the expression of monoterpene synthase genes in thyme. The results of this study provide a strong foundation for the creation of new germplasm resources, construction of the genetic linkage maps, and identification of quantitative trait loci (QTLs), and help gain insight into the mechanism of monoterpenoids biosynthesis in thyme.
研究领域:
植物科学
·
分子生物学
·
遗传学
·
药学
·
农业遗传学
✓ 已验证
DOI
PubMed
高影响因子
PEST MANAGEMENT SCIENCE
·
2020
·Vol. 76
·期 12
·4036-4045
·
IF 3.75
Yifan Li,Hong Sun,Zhen Tian,Xinxin Su,Yue Li,Xuan Ye,Yifei Zhou,Shengli Zheng,Jiyuan Liu,Yalin Zhang
BACKGROUND Insect glutathione S-transferases (GSTs) play a crucial role in insecticide detoxification. However, there remains a distinct lack of information regarding the role of GSTs in the detoxification of Tolfenpyrad (TFP) in insects. RESULTS Real-time quantitative PCR showed significant upregulation of PxGSTs after exposure to TFP for 6 h. An in vitro inhibition assay showed that TFP could inhibit PxGSTδ, PxGSTε and PxGSTσ, and the most pronounced inhibitory effect was on PxGSTσ. Metabolism assays displayed that PxGSTσ was superior to other test PxGSTs in metabolizing TFP. The molecular docking of TFP and PxGSTσ revealed that the H-bond provided by the sidechains of Tyr107 and Tyr162 were key to the detoxification of TFP by PxGSTσ. Further tests using mutant PxGSTσ proteins at the sites of Tyr107 (PxGSTσY107A) and Tyr162 (PxGSTσY162A) corroborated that the individual replacement of Tyr107 and Tyr162 could greatly weaken the binding and metabolic abilities to TFP. CONCLUSION Metabolic interactions between the Plutella xylostella (L.) GSTs (PxGSTs) and TFP were deciphered. This study illustrates the molecular metabolism mechanism of PxGSTσ towards TFP and provides theoretical underpinnings for the design and optimization of novel TFP-like insecticides. © 2020 Society of Chemical Industry
研究领域:
昆虫学
·
分子生物学
·
农药科学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
JOURNAL OF NANOPARTICLE RESEARCH
·
2014
·Vol. 16
·期 11
·1-13
·
IF 2.278
Liu Wenjuan,Yao Jun,Cai Minmin,Chai Hankuai,Zhang Chi,Sun Jingjing,Chandankere Radhika,Masakorala Kanaji
A new nanopesticide, carboxymethyl-β-cyclodextrin-Fe 3 O 4 magnetic nanoparticles-Diuron (CM-β-CD-MNPs-Diuron), was synthesized from an inclusion complex of CM-β-CD-MNPs as host and diuron as guest molecules. The transmission electron microscopy revealed it had an average diameter of 25 nm which is more or less the same as that of MNPs (average diameter ~23 nm). The CM-β-CD grafting was confirmed by infrared spectroscopy, and the amount of CM-β-CD grafted on the surface of MNPs was determined to be 144.1 mg/g by thermogravimetry. The feasibility of using CM-β-CD-MNPs as a nanocarrier for loading diuron was verified by investigating the formation of inclusion complex. The complexation of CM-β-CD-MNPs with diuron followed the Langmuir adsorption isotherm. In this work, the potential toxic effect of CM-β-CD-MNPs-Diuron on soil microbial was evaluated by microcalorimetry, urease enzyme and real-time quantitative PCR (qPCR). The thermokinetic parameters were observed to decrease with increase in the loading of CM-β-CD-MNPs-Diuron in soil. The urease activity data showed that there was a significant effect ( p < 0.05) of CM-β-CD-MNPs-Diuron on the enzyme activity. The microcalorimetric analysis was in agreement with qPCR, confirming the toxic effect of this nanopesticide on microorganism in soil.
研究领域:
环境科学
·
微生物学
·
纳米技术
✓ 已验证
产品关联
DOI
高影响因子
BMC PLANT BIOLOGY
·
2019
·Vol. 19
·期 1
·1-16
·
IF 3.67
Cao Yunpeng,Meng Dandan,Han Yahui,Chen Tianzhe,Jiao Chunyan,Chen Yu,Jin Qing,Cai Yongping
Background Studies have demonstrated that BBX (B-BOX) genes play crucial roles in regulatory networks controlling plant growth, developmental processes and stress response. Nevertheless, comprehensive study of BBX genes in orchids (Orchidaceae) is not well studied. The newly released genome sequences of Dendrobium officinale and Phalaenopsis equestris have allowed a systematic analysis of these important BBX genes in orchids. Results Here we identified 19 ( DoBBX01 – 19 ) and 16 ( PeBBX01–16 ) BBX genes from D. officinale and P. equestris , respectively, and clustered into five clades (I-V) according to phylogenetic analysis. Thirteen orthologous, two DoBBXs paralogous and two PeBBXs paralogous gene pairs were validated. This gene family mainly underwent purifying selection, but five domains experienced positive selection during evolution. Noteworthy, the expression patterns of root, root_tips, stem, leaf, speal, column, lip, and flower_buds revealed that they might contribution to the formation of these tissues. According to the cis-regulatory elements analysis of BBX genes, qRT-PCR experiments were carried out using D. officinale PLBs (protocorm-like bodies) and displayed that these BBX genes were differentially regulated under AgNO 3 , MeJA (Methyl Jasmonate), ABA (abscisic acid) and SA (salicylic acid) treatments. Conclusions Our analysis exposed that DoBBX genes play significant roles in plant growth and development, and response to different environmental stress conditions of D. officinale , which provide aid in the selection of appropriate candidate genes for further functional characterization of BBX genes in plants.
研究领域:
植物生物学
·
基因组学
·
分子遗传学
·
生物信息学
✓ 已验证
DOI
PubMed
高影响因子
TALANTA
·
2020
·
IF 5.339
Wancun Zhang,Kangbo Liu,Pin Zhang,Weyland Cheng,Yaodong Zhang,Linfei Li,Zhidan Yu,Mengmeng Chen,Lin Chen,Lifeng Li,Xianwei Zhang
Rapid, simple, specific and sensitive approaches for single nucleotide polymorphisms (SNPs) detection are essential for clinical diagnosis. In this study, all-in-one approaches, consisting of the whole detection process including ligase detection reaction (LDR) and real time quantitative polymerase chain reaction performed in one PCR tube by a one-step operation on a real-time PCR system using molecular beacon (MB) as turn-on probe, were developed for rapid, simple, specific and sensitive quantifcation of SNPs. High specificity of the all-in-one approach was achieved by using the LDR, which employs a thermostable and single-base discerning Hifi Taq DNA ligase to ligate adjacently hybridized LDR-specific probes. In addition, a highly specific probe, MB, was used to detect the products of all-in-one approach, which doubly enhances the specificity of the all-in-one approach. The linear dynamic range and high sensitivity of mutant DNA (MutDNA) and wild-type DNA (WtDNA) all-in-one approaches for the detection of MutDNA and WtDNA were studied in vitro , with a broad linear dynamic range of 0.1 fM to 1 pM and detection limits of 65.3 aM and 31.2 aM, respectively. In addition, the MutDNA and WtDNA all-in-one approaches were able to accurately detect allele frequency changes as low as 0.1%. In particular, the epidermal growth factor receptor T790M MutDNA frequency in the tissue of five patients with non-small cell lung cancer detected by all-in-one approaches were in agreement with clinical detection results, indicating the excellent practicability of the developed approaches for the quantification of SNPs in real samples. In summary, the developed all-in-one approaches exhibited promising potential for further applications in clinical diagnosis.
研究领域:
分子生物学
·
医学诊断
·
遗传学
✓ 已验证
DOI
PubMed
高影响因子
危险品化学品经营许可证（带存储）