Natural Products and Bioprospecting
·
2024
·Vol. 14
·期 1
·1-16
·
IF 4.8
Yang Ya-Ya,Jing Shun-Li,Shao Jia-Li,Chen Ji-Xuan,Zhang Wei-Feng,Wan Si-Yuan,Shen Yu-Ping,Yang Huan,Yu Wei
Functionalized mesoporous materials have become a promising carrier for enzyme immobilization. In this study, Santa Barbara Amorphous 15 (SBA-15) was modified by N-aminoethyl- γ -aminopropyl trimethoxy (R). R-SBA-15 was employed to purify and immobilize recombinant β -glucosidase from Terrabacter ginsenosidimutans (BgpA) in one step for the first time. Optimum pH of the constructed R-SBA-15@BgpA were 7.0, and it has 20 ℃ higher optimal temperature than free enzyme. Relative activity of R-SBA-15@BgpA still retained > 70% at 42 ℃ after 8-h incubation. The investigation on organic reagent resistance revealed that the immobilized enzyme can maintain strong stability in 15% DMSO. In leaching test and evaluation of storage stability, only trace amount of protein was detected in buffer of the immobilized enzyme after storage at 4 ℃ for 33 days, and the immobilized BgpA still maintained > 50% relative activity. It also demonstrated good reusability, with 76.1% relative activity remaining after fourteen successive enzymatic hydrolyses of epimedin A to sagittatoside A. The newly proposed strategy is an effective approach for the purification and immobilization of BgpA concurrently. In addition, R-SBA-15@BgpA was demonstrated to have high efficiency and stability in this application, suggesting its great feasibility and potential to produce bioactive compounds such as secondary glycosides or aglycones from natural products. Graphical Abstract
研究领域:
生物化学
·
酶工程
·
材料科学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
Royal Society Open Science
·
2020
·Vol. 7
·期 9
·
IF 2.646
Siqi Liu,Xiaolin Xu,Yanshun Kang,Yingtian Xiao,Huan Liu
Ligninolytic enzymes, including laccase (Lac), manganese peroxidase (MnP) and lignin peroxidase (LiP), have attracted much attention in the degradation of contaminants. Genes of Lac (1827 bp), MnP (1134 bp) and LiP (1119 bp) were cloned from Aspergillus sp. TS-A, and the recombinant Lac (69 kDa), MnP (45 kDa) and LiP (35 kDa) were secretory expressed in Pichia pastoris GS115, with enzyme activities of 34, 135.12 and 103.13 U l−1, respectively. Dyes of different structures were treated via the recombinant ligninolytic enzymes under the optimal degradation conditions, and the result showed that the decolourization rate of Lac on Congo red (CR) in 5 s was 45.5%. Fourier-transform infrared spectroscopy, gas chromatography–mass spectrometry analysis and toxicity tests further proved that the ligninolytic enzymes could destroy the dyes, both those with one or more azo bonds, and the degradation products were non-toxic. Moreover, the combined ligninolytic enzymes could degrade CR more completely compared with the individual enzyme. Remarkably, besides azo dyes, ligninolytic enzymes could also degrade triphenylmethane and anthracene dyes. This suggests that ligninolytic enzymes from Aspergillus sp. TS-A have the potential for application in the treatment of contaminants.
研究领域:
环境科学
·
生物技术
·
微生物学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
ANALYTICAL CHEMISTRY
·
2023
·
IF 7.4
Yin Zhao,Dongjuan Chen,Zhichen Xu,Tao Li,Jiang Zhu,Rui Hu,Guoyong Xu,Ying Li,Yunhuang Yang,Maili Liu
Fast, simplified, and multiplexed detection of human papillomaviruses (HPVs) is of great importance for both clinical management and population screening. However, current HPV detection methods often require sophisticated instruments and laborious procedures to detect multiple targets. In this work, we developed a simple microfluidic dual-droplet device (M-D3) for the simultaneous detection of HPV16 and HPV18 by combining the CRISPR-Cas12a system and multiplexed recombinase polymerase amplification (RPA) assay. A new approach of combining pressure/vacuum was proposed for efficient droplet generation with minimal sample consumption. Two groups of droplets that separately encapsulate the relevant Cas12a/crRNA and the fluorescent green or red reporters are parallelly generated, followed by automatic imaging to discriminate the HPV subtypes based on the specific fluorescence of the droplets. The M-D3 platform performs with high sensitivity (∼0.02 nM for unamplified plasmids) and specificity in detecting HPV16 and HPV18 DNA. By combining the RPA and Cas12a assay, M-D3 allows on-chip detection of HPV16 and HPV18 DNA simultaneously within 30 min, reaching a detection limit of 10–18 M (∼1 copy/reaction). Moreover, the outstanding performance of M-D3 was validated in testing 20 clinical patient samples with HPV infection risk, showing a sensitivity of 92.3% and a specificity of 100%. By integrating the dual-droplet generator, CRISPR-Cas12a, and multiplexed RPA, the M-D3 platform provides an efficient way to discriminate the two most harmful HPV subtypes and holds great potential in the applications of multiplexed nucleic acid testing.
研究领域:
分子生物学
·
医学检测
·
生物技术
✓ 已验证
产品关联
DOI
PubMed
高影响因子
INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
·
2025
·
IF 8.5
Fan’ge Kong,Shichao Peng,Yuchong Zhang,He Zhang,Jiasi Wang,Di Wang
Cladobotryum mycophilum , a pathogen responsible for cobweb disease, caused significant and irreversible losses in the mushroom industry. Effective monitoring and early prevention rely on the development of advanced diagnosis methods. This study introduced a novel hydrogel-based C. mycophilum detection method that integrates recombinase-aided amplification (RAA) with the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) system (CRISPR/Cas12a), referred as RCCH. The RAA reaction occurs within cross-linked PEG hydrogel, which is subsequently overlaid with a CRISPR/Cas12a-functionalized hydrogel. The porous network of the PEG hydrogel traps essential enzymes, facilitating spatial co-localization of target DNA and the CRISPR/Cas12a-crRNA complex. Upon activation of Cas12a's trans -cleavage activity, clear and countable fluorescent spots are generated for visual detection. RCCH demonstrates a limit of detection as low as 1 fg/μL, and exceptional selectivity against common fungi Trichoderma viride and T. harzianum and the host mushroom Lentinula edodes . The entire process is completed in under 40 min, indicating RCCH's potential as a rapid, accurate, and practical detection method for monitoring mushroom diseases. This innovative approach offers significant support for enhancing safety in the mushroom industry.
研究领域:
微生物学
·
分子生物学
·
食品科学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
Infectious Medicine
·
2022
·
IF 0
Jinrong Wang,Guowei Song,Yue Ming,Jing Pan,Ruiqing Zhang,Guohao Fan,Xinxin Shen,Xuejun Ma,Lixin Li
Background Nucleic acid amplification enhancers suitable for use in a recombinase-aided amplification (RAA) assay were studied for the first time, and amplification of a long-fragment (509 bp) was initially explored. Methods Using recombinant plasmids and clinical samples, RAA fluorescence and basic methods were used to evaluate the efficacy. The fluorescence method was evaluated by threshold time and fluorescence value, and the basic method was characterized by 2% agarose gel electrophoresis. Results Taking a previously established RAA assay for HPV18 as an example, we demonstrated that the addition of 0.2 M, 0.4 M, and 0.6 M betaine and 10% pullulan could enhance the RAA. The new RAA assays with betaine and pullulan were named B-RAA and P-RAA, respectively. Using the B-RAA and P-RAA fluorescence methods, the threshold time values could be shortened by 1.72–2.32 minutes and 2.60 minutes, respectively, and the fluorescence values could be enhanced by 8847.25–9094.37 mv and 5250 mv, respectively. Using the basic method, the sensitivity could be increased 10-fold. We successfully amplified a long-fragment of 509 bp using a P-RAA assay with a sensitivity of 10 2 copies/µL (compared with 10 3 copies/µL in the RAA assay). Conclusions Thus, we concluded that betaine and pullulan are effective additives to enhance the sensitivity of RAA assays.
研究领域:
分子生物学
·
生物化学
·
临床医学
✓ 已验证
DOI
BIOPROCESS AND BIOSYSTEMS ENGINEERING
·
2020
·Vol. 44
·期 4
·727-735
·
IF 2.419
Wang Hongrao,Han Shuran,Wang Jiayi,Yu Shuyu,Li Xiaoyan,Lu Lei
Laccases are versatile oxidases that are capable of decolorizing various synthetic dyes. Recombinant Bacillus amyloliquefaciens laccase was immobilized as magnetic cross-linked enzyme aggregates (M-CLEAs) for application in dye decolorization. Several parameters influencing the activity recovery were evaluated during the synthesis of M-CLEAs. With ammonium sulfate as precipitant, maximum activity was recovered by cross-linking with 0.16% glutaraldehyde for 1 h. The prepared M-CLEAs exhibited improved activity under alkaline conditions. It remained 74% activity after incubation at 60 °C for 5 h. Enhanced tolerance towards NaCl was also observed for the M-CLEAs, with 68% activity remaining in the presence of 1 M NaCl. The immobilized laccase could rapidly decolorize more than 93% of reactive black 5 and indigo carmine in 1 h, while its catalytic efficiency towards reactive blue 19 was relatively low. After four cycles of consecutive reuse, the M-CLEAs could decolorize 92% of indigo carmine. The easy recovery and reusability of M-CLEAs facilitate the potential application of bacterial laccase in dye decolorization.
研究领域:
生物化学
·
环境科学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
FOOD CHEMISTRY
·
2025
·
IF 9.8
Jiaqi Feng,Mengyuan Xiong,Yongsheng Qiao,Ping Wang,Xianfeng Lin,Lixin Kang,Nuo Duan,Zhouping Wang,Shijia Wu
A portable dual-mode RPA-based LFA sensor was developed for Salmonella detection in lettuce and milk samples. The fimY gene of Salmonella was chosen as specific conserved sequences, their corresponding primers based on RPA conditions were designed and optimized, and respectively labeled with Biotin and FAM groups at 5′ end, then applied for efficient fimY gene fragments enrichment via RPA. Sandwich construction of streptavidin-RPA products-AuNPs@FAM antibody was generated on T-line, showing positive results within 10 min, colorimetric-fluorescent signals were captured by a smartphone and converted into gray intensities and fluorescence intensities, further enabling the quantification of Salmonella with detection limits of 10 cfu/mL and 1 cfu/mL for colorimetric and fluorescent modes, respectively, over a linear range of 10 1 –10 6 cfu/mL. The proposed strategy provided a valuable reference for Salmonella one-site detection, which would be utilized as a universal method for other food-borne pathogens detection by matching specific conserved sequences and primer designs.
研究领域:
食品科学
·
分子生物学
·
检验医学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
Parasites & Vectors
·
2024
·Vol. 17
·期 1
·1-10
·
IF 3
Huang Yue,Li Jinli,Pei Shifeng,You Heng,Liu Huimin,Guo Yaqiong,Xu Rui,Li Na,Feng Yaoyu,Xiao Lihua
Background The dimerizable Cre recombinase system (DiCre) exhibits increased leaky activity in Cryptosporidium, leading to unintended gene editing in the absence of induction. Therefore, optimization of the current DiCre technique is necessary for functional studies of essential Cryptosporidium genes.Methods Based on the results of transcriptomic analysis of Cryptosporidium parvum stages, seven promoters with different transcriptional capabilities were screened to drive the expression of Cre fragments (FKBP-Cre59 and FRB-Cre60). Transient transfection was performed to assess the effect of promoter strength on leakage activity. In vitro and in vivo experiments were performed to evaluate the leaky activity and cleavage efficiency of the optimized DiCre system by polymerase chain reaction (PCR), nanoluciferase, and fluorescence analyses.Results The use of promoters with lower transcriptional activity, such as pcgd6_4110 and pcgd3_260, as opposed to strong promoters such as pActin, pα-Tubulin, and pEnolase, reduced the leakage rate of the system from 35–75% to nearly undetectable levels, as verified by transient transfection. Subsequent in vitro and in vivo experiments using stable lines further demonstrated that the optimized DiCre system had no detectable leaky activity. The system achieved 71% cleavage efficiency in vitro. In mice, a single dose of the inducer resulted in a 10% conditional gene knockout and fluorescent protein expression in oocysts. These fluorescently tagged transgenic oocysts could be enriched by flow sorting for further infection studies.Conclusion sA DiCre conditional gene knockout system for Cryptosporidium with good cleavage efficiency and reduced leaky activity has been successfully established.Graphical Abstract
研究领域:
寄生虫学
·
分子生物学
·
基因组编辑
✓ 已验证
产品关联
DOI
PubMed
高影响因子
Analytical Methods
·
2023
·Vol. 15
·期 17
·2105-2113
·
IF 3.1
Furong Zhao,Pei Wang,Haoxuan Wang,Sirui Liu,Muhammad Sohail,Xing Zhang,Bingzhi Li,He Huang
The spread of the monkeypox virus (MPXV) from Central and West Africa to previously non-endemic regions has caused a global panic. In this context, the rapid, specific, and ultrasensitive detection of MPXV is crucial to contain its spread, though such technology has seldom been reported. Herein, we proposed an MPXV assay combining recombinase-aided amplification (RAA) and CRISPR/Cas12a. This assay targeted the highly conserved MPXV F3L gene and demonstrates a low detection limit (LOD) of 101 copies per μL. By leveraging the high specificity nature of RAA and CRISPR/Cas12a, we rationally optimized probes and conditions to achieve high selectivity that differentiates MPXV from other orthopox viruses and current high-profile viruses. To facilitate on-site screening of potential MPXV carriers, a kit integrating lateral flow strips was developed, enabling naked-eye MPXV detection with a LOD of 104 copies per μL. Our RAA-Cas12a-MPXV assay was able to detect MPXV without the need for sophisticated operation and expensive equipment. We believe that this assay can be rapidly deployed in emerging viral outbreaks for on-site surveillance of MPXV.
研究领域:
病毒学
·
分子诊断
·
生物技术
✓ 已验证
DOI
PubMed
高影响因子
ANALYTICA CHIMICA ACTA
·
2023
·
IF 6.2
Ling Deng,Xiaoyi He,Ke Liu,Yuanli Li,Han Xia,Hang Qian,Xiaoxue Lu,Xuhu Mao,Yang Xiang
Burkholderia pseudomallei is the causative agent of melioidosis, a potentially life-threatening infectious disease, and poses public health risks in endemic areas. Due to the high mortality, intrinsic antibiotic resistance , and atypical manifestations, establishing a rapid, accurate, and sensitive identification of B. pseudomallei enables earlier diagnosis, proper treatments, and better outcomes of melioidosis. Herein, we present a O ne- P ot C RISPR-integrated assay for I nstant and V isual D etection (termed OPC-IVD) of B. pseudomallei . The integration of recombinase polymerase amplification and CRISPR-Cas12a recognition-activated trans -cleavage, achieved a true all-in-one single-tube reaction system, initiating the amplification and cleavage simultaneously, which realized a facile sample-to-answer assay. This approach could be performed with simplified DNA extraction and completed around 30 min by holding the reaction tube in the hand. The detection limit of our OPC-IVD was determined to be 2.19 copy/uL of plasmid DNA, 12.5 CFU/mL of B. pseudomallei , and 61.5 CFU/mL of bacteria in spiked blood samples, respectively. Furthermore, the introduction of internal amplification control effectively reduced the occurrence of false negatives, which was incorporated in the reaction system, and amplified simultaneously with the target and read by naked eyes. The assay exhibited 100% accuracy when evaluated in clinical isolates and samples. The streamlined workflow of our OPC-IVD of B. pseudomallei enables a field-deployable, instrument-free, and ultra-fast approach that can be utilized by non-expert personnel in the field of molecular diagnosis of melioidosis especially in under-resourced setting.
研究领域:
医学诊断
·
分子生物学
·
传染病学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
MICROCHEMICAL JOURNAL
·
2024
·
IF 4.9
Yang Song,Yiwen Xu,Ruirui Wang,Jianping Wang,Xiaoping Yu,Xuping Shentu
Pathogenic bacterial contamination of food is a major challenge in food safety and public health management. To monitor pathogenic bacteria in food, a rapid, sensitive, and specific pathogen detection method is needed. CRISPR-Cas12a has been developed as a sensitive method for detecting pathogenic bacteria, but its output signal is fluorescent, which need equipment to detect. Here, the nanoparticle probe was designed to detect the activity of Cas12a and were able to visualize the assay results by observing the color change by naked eyes. The target sequences of pathogenic bacteria were amplified by recombinase polymerase, then recognized and bound by the crRNA and Cas12a complex. The activated Cas12a cleaved the linker-ssDNA connecting the nanoparticles, causing the nanoparticles to transition from aggregation to dispersion, and the color showed as purple. The sensitivity analysis showed that the limits of detection (LOD) reached 1 × 10°CFU/mL, 1 × 10 4 CFU/mL, 1 × 10 2 CFU/mL, 1 × 10 3 CFU/mL, 1 × 10 1 CFU/mL for Escherichia coli , Salmonella typhimurium , Vibrio parahaemolyticus , Staphylococcus aureus , and Listeria monocytogenes , which were all specific for five bacteria in the milk samples. Therefore, this method is a dependable, fast, sensitive and specific method, which make the CRIPSR-Cas12a detection method more useful in bacterial pathogenic detection.
研究领域:
食品科学
·
分子生物学
·
生物技术
✓ 已验证
产品关联
DOI
高影响因子
JOURNAL OF FOOD COMPOSITION AND ANALYSIS
·
2024
·
IF 4
Haibin Liu,Tingting Cao,Jing Wang,Yi Yuan,Haijie Li,Kuo He,Hongshuo Chen,Lei Wang
Meat adulteration is a growing concern for consumers and researchers, necessitating the development of a simple and robust method for identifying animal sources in meat products. In this study, a double-tailed recombinase polymerase amplification (RPA) integrated with surface-enhanced Raman scattering (SERS) based two-color lateral flow nucleic acid hybridization strip (LFNAHS) was established for simultaneous detection of pork and horse meat. Magenta and cyan gold nanoparticles functionalized with specific probes and Raman dyestuff were used to color the LFNAHS based on RPA amplification products. The assay successfully detected colored test lines for horse and pork adulteration, with quantitative analysis achieved through Raman intensity measurements. The method showed a high specificity with no cross-reactivity and a detection limit of 0.01 % (wt%). Authenticity testing of 40 donkey products yielded results consistent with PCR-agarose gel electrophoresis. This assay can be extended to identify other meat sources by replacing the specific primer sets. This versatile method can be adapted for identifying other meat sources, making it a valuable tool for accurate, sensitive, and quantitative meat authentication in resource-limited settings.
研究领域:
食品安全
·
分子生物学
·
分析化学
✓ 已验证
DOI
高影响因子
危险品化学品经营许可证（带存储）