Biotechnology Journal
·
2023
·Vol. 19
·期 1
·2300273
·
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
Agronomy-Basel
·
2025
·Vol. 15
·期 8
·1813
·
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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DOI
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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DOI
PubMed
高影响因子
JOURNAL OF ETHNOPHARMACOLOGY
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2023
·
IF 5.4
Li Zhu,Xiao-jia Li,Chao Gu,Yuan Gao,Chun-sheng Zhang,Lu-yu Wang,Nai-hong Chen,Gang Li
Ethnopharmacological relevance The main clinical manifestations of eczema include itching, erythema, swelling and pain. Currently, allergies and TH1/TH2 cytokine imbalances are significant causes of eczema. TCM believes that eczema is mainly caused by incongruity between dry and wet. Wenguanmu ointment is a classic Mongolian medicine, which mainly composed of Xanthoceras sorbifolia Bunge , Coptis chinensis Franch and Bezoar. These ingredients can clear heat and dampness, dispel wind and dehumidification, anti-inflammatoryad analgesic. In this study, it was found that Wenguanmu ointment can treat eczema with anti-inflammatory, analgesic and antipruritic. Aim of the study In this study, the content of main components in Wenguanmu ointment was tested. Moreover, the therapeutic effect and mechanism of Wenguanmu ointment on eczema model mice were studied. Materials and methods Kunming mice (25 ± 2 g) were randomly divided into 6 groups: Control group; Model group; Vehicle group; Wenguanmu ointment group; Compound dexamethasone acetate cream group; Chushizhiyang ointment group. The eczema mouse model was established by DNCB. HPLC and TLC tests were used to determine the content of the main components in Wenguanmu ointment. HE staining was used to assess skin damage in mice. In order to detect the anti-inflammatory effect of Wenguanmu ointment on eczema, The levels of IgE, TNF-α, IFN-γ, COX-2 and IL-4 in serum was measured by ELISA. Genecards and Online Mendelian Inheritance in Man databases were used to analyze potential target gene predictions, and it was speculated that Wenguanmu ointment was associated with NF−κB signaling pathway and chemokine signaling pathway. To detect this inference, RT-qPCR and western blotting were used to detect protein and mRNA levels of CKLF-1, IκB-α, and NF-κB. Results Wenguanmu ointment can repress the symptoms of eczema caused by 2, 4-dinitrochlorobenzene, and inhibit the level of serum immunoglobulin E. Simultaneously it restrain the elevation of miscellaneous pro-inflammatory cytokines and chemokines, as well as reducing the expression of CKLF-1 and NF-κB protein in the nucleus, and increasing the protein expression of IκB to improve eczema. Conclusions The ameliorating effect of Wenguanmu ointment on eczema lesions can play a importment role by inhibiting the CKLF-1/NF-κB pathway.
研究领域:
民族药理学
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药理学
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免疫学
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分子生物学
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中医药
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DOI
PubMed
高影响因子
BIOPROCESS AND BIOSYSTEMS ENGINEERING
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2020
·Vol. 43
·期 5
·831-838
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IF 2.419
Liu Bin,Wang Xufeng,Zhao Jiahao,Qin Liehao,Shi Lei,Zhou Tao,Liu Shuang,Wang Chunling
Zygosaccharomyces rouxii referred as Yeast S is an important microorganism widely applied in traditional fermented food to accelerate flavor formation. Z. rouxii 3-2 referred as Yeast S 3-2 constructed previously is a new strain having higher salt tolerance than wild type. In this study, salt stress response of synthesis key flavor metabolites 3-methylthiopropanol, 2-phenylethyl alcohol, and isoamyl acetate in Yeast S and S 3-2 were investigated based on SPME–GC–MS and RT-qPCR. Analysis of GC–MS data showed that high salinity led to increase the contents of 3-methylthiopropanol and 2-phenylethyl alcohol both in Yeast S and Yeast S 3-2, while inhibited the synthesis of isoamyl acetate, and the induced effect on Yeast S 3-2 was more pronounced than Yeast S. Furthermore, the RT-qPCR results revealed that the salinity could enhance the genes expression of ARO10 and PDC1 , and stronger effect on Yeast S 3-2 than S. However, the gene expression of AFT1 was reduced in high-salt culture conditions, which was consistent with the results of GC–MS data. The results presented in this study can provide theoretical support of Z. rouxii 3-2 application during food fermentation.
研究领域:
微生物学
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食品科学
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分子生物学
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DOI
PubMed
高影响因子
BIOSENSORS & BIOELECTRONICS
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2022
·
IF 12.545
Hai-Yan Jia,Hong-Li Zhao,Ting Wang,Pin-Ru Chen,Bin-Cheng Yin,Bang-Ce Ye
MicroRNAs (miRNAs) play an essential role in cancer diagnosis and prognosis. Developing a new method for sensitive detection of miRNA is constantly in demand. CRISPR/Cas12a system can nonspecifically cleave single-stranded DNA after specific recognition of target DNA, showing tremendous potential in molecular diagnostics. However, CRISPR-based detection methods require synthesizing different crRNAs for detecting different targets, which limit their widespread application. Herein, we design a versatile and sensitive miRNA detection platform based on CRISPR/Cas12a system combined with a hybridization chain reaction (HCR) circuit. In this design, the HCR circuit as the signal transducer converts each miRNA into multiple DNA duplexes, which act as the activators to activate the trans -cleavage activity of Cas12a for further signal amplification . More importantly, this platform can sensitively detect different miRNAs without changing the spacer sequence of crRNA due to the fixed activators formed by HCR. In addition, the consistency between the proposed platform and RT-qPCR in miRNA detection extracted from different cell lines validated its practicability, demonstrating the potential in clinical diagnosis of cancers and monitoring therapy.
研究领域:
分子生物学
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生物技术
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癌症研究
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诊断技术
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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
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分子生物学
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DOI
PubMed
高影响因子
ANALYTICAL CHEMISTRY
·
2024
·
IF 6.7
Benyu He,Lingfeng Wang,Xinyu Jin,Xing Zhang,Rui Sha,Yong Liang,Yawei Wang,Wenjing Xie,Jianbo Shi,Hanyong Peng
The detection of virus RNA in wastewater has been established as a valuable method for monitoring Coronavirus disease 2019. Carbon nanomaterials hold potential application in separating virus RNA owing to their effective adsorption and extraction capabilities. However, carbon nanomaterials have limited separability under homogeneous aqueous conditions. Due to the stabilities in their nanostructure, it is a challenge to efficiently immobilize them onto magnetic beads for separation. Here, we develop a porous agarose layered magnetic graphene oxide (GO) nanocomposite that is prepared by agglutinating ferroferric oxide (Fe3O4) beads and GO with agarose into a cohesive whole. With an average porous size of approximately 500 nm, the porous structure enables the unhindered entry of virus RNA, facilitating its interaction with the surface of GO. Upon the application of a magnetic field, the nucleic acid can be separated from the solution within a few minutes, achieving adsorption efficiency and recovery rate exceeding 90% under optimized conditions. The adsorbed nucleic acid can then be preserved against complex sample matrix for 3 days, and quantitatively released for subsequent quantitative reverse transcription polymerase chain reaction (RT-qPCR) detection. The developed method was successfully utilized to analyze wastewater samples obtained from a wastewater treatment plant, detecting as few as 10 copies of RNA molecules per sample. The developed aMGO-RT-qPCR provides an efficient approach for monitoring viruses and will contribute to wastewater-based surveillance of community infections.
研究领域:
环境科学
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纳米技术
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病毒学
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分析化学
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产品关联
DOI
PubMed
高影响因子
CHEMOSPHERE
·
2022
·
IF 8.943
Si-Qi Zhang,Ping Li,Shu-Wen He,Shao-Ying Xing,Zhi-Han Cao,Xue-Li Zhao,Cuici Sun,Zhi-Hua Li
This paper evaluates the coexistence risks of triphenyltin (TPT) and norfloxacin (NOR) to aquatic organisms in the aquatic environment. Carp ( Cyprinus carpio ) was used as the test organism, the control and exposure groups (1 μg/L TPT), 1 mg/L (NOR), 1 μg/LTPT+1 mg/LNOR (TPT_NOR)) were set up according to the environmental concentration in the severely polluted area for 42 days. The single/combined toxic effects of TPT and NOR on aquatic organisms were evaluated by analyzing carp brain transcriptome sequencing, gut microbiota structure, and detection of biochemical indicators and RT-qPCR. Our results show that TPT and NOR induce lipid metabolism disorder in carp brain tissue, affecting the metabolism of cytochrome P450 to exogenous substances, and NOR also induces immunosuppression in carp. Long-term exposure to TPT combined with NOR amplifies the monotoxicity of TPT or NOR on lipid metabolism and immunosuppression in carp, induces immune dysfunction in brain tissue and changes in gut microbiota structure. However, TPT_NOR has no obvious neurotoxicity on the brain, but it can inhibit the level of intestinal MDA. This highlights that co-exposure of TPT and NOR amplifies metabolic disorders and immunosuppressive functions in carp.
研究领域:
环境毒理学
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水生毒理学
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免疫毒理学
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代谢调控
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DOI
PubMed
高影响因子
Frontiers in Cardiovascular Medicine
·
2024
·
IF 2.8
Yang Mengyue,Bi Wen,Zhang Zhijie
Background: The relationship between human immunodeficiency virus (HIV) infection and pulmonary arterial hypertension (PAH) has garnered significant scrutiny. Individuals with HIV infection have a higher risk of developing PAH. However, the specific mechanism of HIV-associated PAH remains unclear. Our study aims at investigating the shared biomarkers in HIV infection and PAH and predicting the potential therapeutic target for HIV-associated PAH.Methods: Data for HIV infection and PAH were downloaded from Gene Expression Omnibus (GEO) database. Differentially expressed genes (DEGs) analysis was performed to detect shared genes in HIV infection and PAH. Enrichment analysis was conducted to identify the function of common DEGs. Protein-protein interaction (PPI) analysis was used to detect key genes. These crucial genes were subsequently verified by RT-qPCR. Finally, candidate drugs were identified by using the Drug Signatures Database (DSigDB).Results: Nineteen common DEGs were identified in HIV infection and PAH. Enrichment analysis exhibited that the functions of these genes were mainly enriched in inflammatory responses, mainly including cellular immunity and interaction between viral proteins and cytokines. By constructing PPI networks, we identified the key gene CC-type chemokine ligand 5 (CCL5), and we verified that CCL5 was highly expressed in hypoxia induced human pulmonary artery endothelial cells (hPAECs) and human pulmonary artery smooth muscle cells (hPASMCs). In addition, we predicted 10 potential drugs targeting CCL5 by Autodock Vina.Conclusion: This study revealed that CCL5 might be a common biomarker of HIV infection and PAH and provided a new therapeutic target for HIV-associated PAH. However, further clinical validation is still indispensable.
研究领域:
免疫学
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分子生物学
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药理学
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生物信息学
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DOI
高影响因子
Marine Drugs
·
2024
·Vol. 22
·期 10
·439
·
IF 4.9
Huiwen Zhang,Chen Sun,Qing Xia,Peihai Li,Kechun Liu,Yun Zhang
Existing antithrombotic drugs have side effects such as bleeding, and there is an urgent need to discover antithrombotic drugs with better efficacy and fewer side effects. In this study, a zebrafish thrombosis model was used to evaluate the antithrombotic activity and mechanism of Brevianamide F, a deep-sea natural product, with transcriptome sequencing analysis, RT-qPCR analysis, and molecular docking. The results revealed that Brevianamide F significantly attenuated the degree of platelet aggregation in the thrombus model zebrafish, leading to an increase in the number of circulating platelets, an augmentation in the return of blood to the heart, an elevated heart rate, and a significant restoration of caudal blood flow velocity. Transcriptome sequencing and RT-qPCR validation revealed that Brevianamide F may exert antithrombotic effects through the modulation of the MAPK signaling pathway and the coagulation cascade reaction. Molecular docking analysis further confirmed this result. This study provides a reference for the development of therapeutic drugs for thrombosis.
研究领域:
药理学
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分子生物学
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血栓研究
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产品关联
DOI
PubMed
高影响因子
SENSORS AND ACTUATORS B-CHEMICAL
·
2019
·
IF 6.393
Hui Zhao,Feng Liu,Yuxun Lu,Lian Jin,Shuang Tan,Yaping Zhang,Can-Peng Li
Alternative polyadenylation (APA) is a novel mechanism in the expressional regulation of most human oncogenes. This process also is involved in cancer progression and prognosis. The potential of APA events as novel prognostic biomarkers has been suggested. CCND2 gene, which is overexpressed in various cancer tissues, tends to utilize the proximal APA site and produces a more prominent mRNA isoform with shorter 3’ untranslated region in the cancer cells. In this study, an ultrasensitive detection for CCND2 APA was successfully achieved using electrochemical biosensors constructed by gold nanoparticles-Fe 3 O 4 nanocomposite and p-sulfonated calix[8]arene functionalized reduced graphene oxide as nanocarriers. The detection ranges of CCND2-S and CCND2-L by the constructed biosensors were 10 −18 –10 −11 and 10 −17 –10 −11 M, respectively. The limit of detection (LOD) of CCND2-S was 0.176 aM (the RNA concentration at the single-cell level). To the best of our knowledge, this value was the lowest LOD among the reported non-coding RNA detection. In real samples, the results of the electrochemical detection using the constructed biosensors showed that the lung cancer cell H292 expressed considerably higher CCND2-S than CCND2-L. This result was in agreement with those obtained by RT-qPCR method. The electrochemical method had showed simpler operation, accurate quantitation, faster detection, and cheaper supplies compared with traditional methods. Therefore, this technology not only provides an ultrasensitive and feasible method for APA detection, but can also be applied in the prognosis and diagnosis of cancer progression.
研究领域:
分子生物学
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癌症研究
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生物医学工程
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基因组学
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产品关联
DOI
高影响因子
危险品化学品经营许可证（带存储）