Escherichia coli O157:H7 and Listeria monocytogenes (E. coli O157:H7 and LM), as common foodborne pathogens can cause a variety of diseases following infection. Early and accurate detection plays a pivotal role in the food safety and human health. In the study, a visualized detection platform with paper-based lateral flow assay (LFA) based on recombinase polymerase amplification (RPA), the clustered regularly interspaced short palindromic repeats (CRISPR) system, and self-developed CdSe/ZnS core-shell quantum dot (QDs) based test strips was constructed for the detection of E. coli O157:H7 and LM, called RPA-Cas12a-LFA. For target DNA, the visual limits of detection (vLODs) for E. coli O157:H7 and LM were 29.0 fg/mu L and 25.1 fg/mu L. For target bacteria, the vLODs for E. coli O157:H7 and LM were 223 CFU/mL and 19.5 CFU/mL. The limits of quantitative (LOQs) were 2.93 fg/mu L and 2.62 fg/mu L for DNA of E. coli O157:H7 and LM, and were 2.26 CFU/mL and 2.03 CFU/mL for E. coli O157:H7 and LM, respectively. Moreover, the approach demonstrated excellent specificity and showed no cross-reactivity with other non-target bacteria. The proposed RPA-CRISPR-LFA was successfully applied to the direct detection of real food samples, which indicates that it has great potential for field application.

Lettuce (Lactuca sativa L.) is an economically important leafy vegetable worldwide. Improving lettuce yield is vital to stabilize vegetable supply and satisfy market demand in China. Plant growth-promoting rhizobacteria (PGPR) represent a sustainable and environmentally friendly approach to facilitate crop growth. To uncover cultivar-specific growth responses of lettuce to PGPR and reveal the underlying molecular regulatory mechanisms, two Bacillus velezensis strains (JB0319 and SAAS-6–3) were tested on two newly bred lettuce cultivars, Huqian and Binfen-3.Our results revealed obvious cultivar-dependent growth phenotypes. Inoculation with either strain significantly improved the biomass of Huqian, with SAAS-6–3 showing superior growth-promoting efficiency compared with JB0319. However, neither strain induced significant growth stimulation in Binfen-3. Physiological measurements demonstrated that bacterial inoculation increased soluble protein concentration in Huqian leaves. Endogenous hormone quantification further confirmed elevated levels of gibberellin (GA₃) and auxin (IAA) upon inoculation.To dissect the molecular mechanism, integrated transcriptomic and metabolomic analyses were carried out on Huqian. We identified 672 differentially expressed genes (DEGs), mainly enriched in plant hormone signal transduction, zeatin biosynthesis, phenylpropanoid and anthocyanin biosynthesis pathways. The detected differentially accumulated metabolites (DAMs) were predominantly associated with ABC transporters, amino acid metabolism, carbohydrate metabolism and the tricarboxylic acid (TCA) cycle. Weighted gene co-expression network analysis (WGCNA) further identified two core modules (MEblue and MEyellow) closely linked to SAAS-6–3 treatment, forming a coordinated “material supply - signal regulation” cascade supporting lettuce growth.Collectively, strain SAAS-6–3 stimulates Huqian growth by activating IAA and GA₃ biosynthetic pathways. The increased endogenous phytohormones synergistically modulate central carbon and nitrogen metabolism as well as transport processes, eventually enhancing biomass accumulation. This work clarifies the cultivar-specific effects of B. velezensis and provides theoretical support for precision application of PGPR agents in sustainable lettuce cultivation.

A visual and dual-targets detection platform for Staphylococcus aureus and Vibrio parahaemolyticus was established based on recombinant polymerase amplification (RPA), CRISPR/Cas12a and lateral flow test strip. The test strip was constructed using multicolored microspheres labeling antibody as tracer, and the results of detection of two kinds of bacteria could be displayed by different colored test lines on the test strip through the antigen-antibody specific recognition function, which realized results visualization and avoid cross-reaction. Due to the target-specific amplification of RPA and the sequence-specific recognition of Cas12a, the detection strategy could still show good sensitivity, specificity and accuracy in complex matrices. The visual limit of detection was as low as 50 CFU/mL, and the entire detection process could be completed within 42 min. In conclusion, a rapid and efficient detection platform for Staphylococcus aureus and Vibrio parahaemolyticus was developed, and this method is expected to provide a solution for the on-site detection of other foodborne pathogens in market and environmental food by simply changing the labeled antibody.

Psychrotrophic bacteria are major contributors to spoilage in refrigerated foods, posing significant challenges for food safety and quality management. Consequently, the development of rapid, on-site detection methods in specific food matrices is essential for controlling the proliferation of these microorganisms and reducing economic losses. This study aimed to develop a portable dual-color test strip platform based on recombinase polymerase amplification (RPA) and CRISPR/Cas12a technology for the simultaneous detection of Pseudomonas fluorescens (ATCC 17397) and Bacillus cereus(ATCC 14579). The platform integrates RPA, CRISPR/Cas12a-specific recognition and cleavage, and dual-color latex microsphere-labeled lateral flow immunoassay. The system was evaluated using purified bacterial DNA to assess sensitivity and specificity. The results demonstrated high sensitivity, with a detection limit of 10 copies/mu L for both target bacteria, no cross-reactivity against five common foodborne pathogens, and a complete assay time of within 45 min. This portable platform offers a sensitive, specific, and user-friendly solution for monitoring psychrotrophic bacteria in food matrices, facilitating timely intervention and contributing to improved food safety management.

Bacilus cereus and Pseudomonas fluorescens are major foodborne psychrotrophic bacteria posing global health and economic risks. B. cereus has a 23.8% food prevalence worldwide. P. fluorescens is a leading cause of spoilage in refrigerated products. Their rapid detection is crucial for food safety. However, existing detection methods often rely on open-tube operations, risking aerosol contamination. In this study, we developed two independent one-tube RPA-CRISPR/Cas12a visual detection assays for B. cereus and P. fluorescens. Using a physical separation design, the recombinase polymerase amplification (RPA) and CRISPR/Cas12a detection were pre-assembled in a single reaction tube. After incubation, a brief centrifugation combined the components for enclosed detection. This step is compatible with portable mini-centrifuges. The assays can be completed within 40 min at 37 °C, with results visualized directly under blue light. Both assays demonstrated good specificity against six common non-target pathogens. The visual detection limits were 5.1 × 101 copies/μL for B. cereus and 2.1 × 101 copies/μL for P. fluorescens. Each assay was applied to 14 types of real-world food samples (naturally contaminated and uncontaminated, confirmed by PCR), achieving 100% concordance with conventional PCR. The one-tube assays are tailored for psychrotrophic bacteria in refrigerated foods. They minimize aerosol contamination risk and provide a reliable solution for on-site cold-chain food safety monitoring.

A signal enhanced lateral flow immunochromatographic test strip (ICS) using CdSe/ZnS core-shell quantum dots (QDs) labeled antigen and antibody for the rapid and sensitive detection of CP4-EPSPS protein in genetically modified crops was successfully developed. In the ICS, signal amplification was realized based on aminated CdSe/ZnS QDs labeled CP4-EPSPS antigen protein and carboxylated QDs labeled anti-CP4-EPSPS monoclonal antibody 2, respectively, which were forming two fluorescent probes. The CP4-EPSPS test antibody 1 and the goat anti-mouse IgG were coated on test area and control area of the nitrocellulose membrane to form the test line and control line. The visual detection limits of the test strip were 0.01 % for soybean RRS, 0.05 % for maize NK603, 0.001 % for labeled soybean RRS, and 0.005 % for labeled maize NK603. Compared with carboxylated QDs based ICS and gold nanoparticles based ICS, the signal enhanced ICS succeeded in increasing sensitivity by 10 similar to 20 times with the same detection time and operation steps. Consequently, it could be a potential tool for sensitive, on-site screening genetically modified crops.

Genetically modified (GM) crops have been widely cultivated worldwide. The development of rapid, ultrasensitive platforms suitable for point-of-care testing (POCT) is critical for the regulation. In this study, an ultrasensitive and specific metal-enhanced fluorescence (MEF)-induced lateral flow biosensor was developed, which was based on strongly plasmonic gold nanorods (AuNRs) and highly fluorescent carboxylated CdSe/ZnS quantum dots (AuNRs@QDs nanohybrids). The CP4-EPSPS protein from GM crops can be quantitatively and accurately determined within 5 min by using these nanohybrids as probe in the lateral flow biosensor. By utilizing the MEF mechanism of QDs on AuNRs, this biosensor exhibits lower detection limits of 0.005 % and 0.01 % for soybean RRS and maize NK603, respectively. It is 2 and 2.5-fold higher than conventional fluorescent QDs based lateral flow biosensor and 10 times higher than that of commonly used colloidal gold-based test strip. Moreover, CP4-EPSPS protein extracted from GM crops without complicated sample pretreatment makes the lateral flow biosensor based on AuNRs@QDs promising for POCT.

Development of a highly sensitive visualization platform for multiplex genetic detection could significantly improve efficiency and reliability of on-site detection of foodborne pathogens. In this study, coupling recombinase polymerase amplification (RPA) with lateral flow immunoassay (LFIA) readout system was proposed for Staphylococcus aureus and Vibrio parahaemolyticus detection. Taking the advantage of the isothermal amplification of RPA, dual primers modified with different labeling groups were designed to realize target signal amplification. LFIA coated with anti-digoxigenin antibody and streptavidin as test line 1 and 2 were designed to detect the two RPA products. The proposed method (dual RPA-LFIA) could realize visual detection using LFIA through rapid RPA amplification within 20 min, exhibiting a lowest detection limit of 4.6 x 102 CFU/mL for Staphylococcus aureus and Vibrio parahaemolyticus. The dual RPA-LFIA is characterized by simultaneous detection of dual targets in one RPA reaction and colorimetric readout through LFIA, thus ensuring high sensitivity and efficiency, and showing great potential to address the on-site detection of foodborne pathogens in the future.

The poor mechanical properties of bio-based films are the main drawbacks that limit their application. To solve this problem, the fish gelatin (FG) mixed with corn starch (CS) were prepared by melt extrusion, and pure FG films as control to study the effects of freeze-thaw cycles. The post-treatment boosted tensile strength from 11.89 to 17.77 MPa of FG/CS, improved thermal stability and optimized water vapor barrier. The addition of 20 % (w/w) CS reduced swelling rate to 262 %, and all films degraded in 3 days. In the chemical characterization, fourier transform infrared spectroscopy and protein solubility tests confirmed new hydrogen bonds between FG molecular chains and molten starch. X-ray diffraction showed the treated FG/CS film had the highest diffraction peak at 15-25° (2θ) and strongest crystallinity. In summary, the innovative use of freeze-thaw cycles as a modification method provides a high performance alternative to petroleum-based plastics while reducing costs.

At present, herbicide-resistant crops (containing CP4-EPSPS protein) constitute a significant proportion of GM crops, and many countries have implemented strict genetically modified (GM) labeling regulations, so the detection of GM crops is necessary. Herein, a portable detection method integrating aggregation-induced emission nanoparticles (AIENPs) with lateral flow immunoassay (LFIA) was developed for the detection of CP4-EPSPS protein in herbicide-resistant GM crops. Antibodies conjugated with AIENPs were used as probes to generate fluorescent signal amplification. Qualitative detection of herbicide-resistant GM crops could be completed within 10 min, and the results are visible to the naked eye under UV light. The fluorescent signal intensity of the test line was measured by using ImageJ software to obtain quantitative analysis results. The visual limits of detection were 0.005 % for soybean RRS and 0.05 % for maize NK603, and the limits of quantitation were 0.0015 % for soybean RRS and 0.023 % for maize NK603, respectively. These are more sensitive than most existing methods. Importantly, the AIE-LFIA proved the robustness and reliability when applied in real samples. Consequently, it holds promise for the detection of other GM crops in food and agricultural samples.

In recent years, foodborne diseases have become a public health problem of global importance, and with the rapid development of globalization, frequent personnel exchanges and fast flow of materials have led to a significant increase in the incidence of foodborne diseases. The most common foodborne pathogen in foodborne infections is Escherichia coli O157:H7. Escherichia coli O157:H7 was used as the research object, combined with recombinase polymerase amplification (RPA) technology, CRISPR/Cas12a and single-tube equipment to construct an RPA-CRISPR/Cas12a single-tube detection platform. The platform enables ultra-sensitive and portable visualization of E. coli O157:H7 in less than 40 min. This platform requires only a portable detection bag (a constant-temperature metal bath and a handheld blue light lamp) to complete the entire detection process. It achieves sensitivity as low as 12 fg/mu L and offers advantages such as high specificity, ultra-sensitivity, and visual detection. During the inspection process, the results can be visualized without opening the lid, which greatly reduces the risk of contamination. Therefore, the one-tube detection method developed in this study can provide a good platform for on-site rapid detection of foodborne pathogens and fully ensure social food safety.

Traditional transgenic detection methods require high test conditions and struggle to be both sensitive and efficient. In this study, a one-tube dual recombinase polymerase amplification (RPA) reaction system for CP4-EPSPS and Cry1Ab/Ac was proposed and combined with a lateral flow immunochromatographic assay, named ''Dual-RPA-LFD'', to visualize the dual detection of genetically modified (GM) crops. In which, the herbicide tolerance gene CP4-EPSPS and the insect resistance gene Cry1Ab/Ac were selected as targets taking into account the current status of the most widespread application of insect resistance and herbicide tolerance traits and their stacked traits. Gradient diluted plasmids, transgenic standards, and actual samples were used as templates to conduct sensitivity, specificity, and practicality assays, respectively. The constructed method achieved the visual detection of plasmid at levels as low as 100 copies, demonstrating its high sensitivity. In addition, good applicability to transgenic samples was observed, with no cross-interference between two test lines and no influence from other genes. In conclusion, this strategy achieved the expected purpose of simultaneous detection of the two popular targets in GM crops within 20 min at 37 ℃ in a rapid, equipment-free field manner, providing a new alternative for rapid screening for transgenic assays in the field.

Objectives Vibrio parahaemolyticus is the primary species that causes vibriosis. In this study, a point-of-care detection method was developed for V. parahaemolyticus.Materials and Methods The detection platform targeted the thermolabile haemolysin (tlh) gene of V. parahaemolyticus based on recombinant polymerase amplification (RPA) and clustered regularly spaced short palindromic repeat (CRISPR/Cas) systems. The platform was combined with an immunochromatographic test strip (ICS) that enables low-cost, simple, visual detection of V. parahaemolyticus.Results The detection limit was 2.5x102 fg/mu L for plasmids and 1.4x102 CFU/mL for V. parahaemolyticus. In addition, V. parahaemolyticus in salmon sashimi could be detected at a concentration of 154 CFU/g without enrichment, and the entire detection time was around 30 min. After enrichment for 6 h, 2 CFU/g V. parahaemolyticus could be detected.Conclusions Consequently, the proposed RPA-CRISPR/Cas12a-ICS platform could detect V. parahaemolyticus in seafood intuitively, quickly, and sensitively, leading to high practical application value.

Plant growth-promoting microorganisms (PGPMs), such as Pantoea sp. YSD J2, promote plant development and stress resistance, while their role in flavonoids accumulation still needs to be further understood. To investigate the complex flavonoid biosynthesis pathway of Cyperus esculentus L. var. sativus (tigernut), we compared Pantoea sp. YSD J2 inoculation (YSD J2) and water inoculation (CK) groups. YSD J2 significantly elevated the content of indole-3-acetic acid (IAA) and orientin. Furthermore, when analyzing flavonoid metabolome, YSD J2 caused increased levels of uralenol, petunidin-3-O-glucoside-5-O-arabinoside, luteolin-7-O-glucuronide-(2 → 1)-glucuronide, kaempferol-3-O-neohesperidoside, cyanidin-3-O-(2″-O-glucosyl)glucoside, kaempferol-3-O-glucuronide-7-O-glucoside, quercetin-3-O-glucoside, luteolin-7-O-glucuronide-(2 → 1)-(2″-sinapoyl)glucuronide, and quercetin-4′-O-glucoside, which further enhanced antioxidant activity. We then performed RNA-seq and LC-MS/MS, aiming to validate key genes and related flavonoid metabolites under YSD J2 inoculation, and rebuild the gene-metabolites regulatory subnetworks. Furthermore, the expression patterns of the trans cinnamate 4-monooxygenase (CYP73A), flavonol-3-O-L-rhamnoside-7-O-glucosyltransferase (UGT73C6), shikimate O-hydroxycinnamoyltransferase (HCT), chalcone isomerase (CHI), flavonol synthase (FLS), and anthocyanidin synthase (ANS) genes were confirmed by qRT-PCR. Additionally, 4 transcription factors (TF) (especially bHLH34, Cluster-37505.3) under YSD J2 inoculation are also engaged in regulating flavonoid accumulation. Moreover, the current work sheds new light on studying the regulatory effect of Pantoea sp. YSD J2 on tigernut development and flavonoid biosynthesis.

To avoid the unreasonable use of chemical fertilizer, an environmentally friendly means of improving soil fertility is required. This study explored the role of the plant growth-promoting rhizosphere bacteria (PGPR) strain Bacillus velezensis SAAS-63 in improving nutrient stress in lettuce. Compared with no inoculation, B. velezensis SAAS-63 inoculants exhibited significantly increased fresh weight, root length, and shoot height under nutrient deficiency, as well as improved antioxidant activities and proline contents. The exogenous addition of B. velezensis SAAS-63 also significantly increased the accumulation of macroelements and micronutrients in lettuce. To elucidate the resistance mechanisms induced by B. velezensis SAAS-63 under nutrient stress, high-throughput sequencing and multi-omics analysis were performed. Inoculation with B. velezensis SAAS-63 altered the microbial community of the rhizosphere and increased the relative abundances of Streptomyces, Actinoallomurus, Verrucomicrobia, and Chloroflexi. It is worth noting that the inoculant SAAS-63 can affect plant rhizosphere metabolism. The inoculant changed the metabolic flow of phenylpropanoid metabolic pathway under nutrient deficiency and promoted phenylalanine to participate more in the synthesis of lignin precursors and coumarin substances by inhibiting the synthesis of flavone and isoflavone, thus improving plant resistance. This study showed that the addition of inoculant SAAS-63 could help plants recruit microorganisms to decompose and utilize trehalose and re-established the carbon metabolism of the plant rhizosphere. Additionally, microbes were found to be closely related to the accumulation of metabolites based on correlation analysis. The results indicated that the addition of PGPRs has an important role in regulating soil rhizosphere microbes and metabolism, providing valuable information for understanding how PGPRs affect complex biological processes and enhance plant adaptation to nutrient deficiency. • Inoculation with SAAS-63 significantly promoted plant growth under nutrient-deficient conditions • Inoculation with SAAS-63 affected rhizosphere microbial diversity and community structure • Inoculation with SAAS-63 affected plant rhizosphere metabolism and induced plants to synthesize substances that resist stress

Escherichia coli O157:H7 (E. coli O157:H7) is a foodborne pathogenic microorganism that is commonly found in the environment and poses a significant threat to human health, public safety, and economic stability worldwide. Thus, early detection is essential for E. coli O157:H7 control. In recent years, a series of E. coli O157:H7 detection methods have been developed, but the sensitivity and portability of the methods still need improvement. Therefore, in this study, a rapid and efficient testing platform based on the CRISPR/Cas12a cleavage reaction was constructed. Through the integration of recombinant polymerase amplification and lateral flow chromatography, we established a dual-interpretation-mode detection platform based on CRISPR/Cas12a-derived fluorescence and lateral flow chromatography for the detection of E. coli O157:H7. For the fluorescence detection method, the limits of detection (LODs) of genomic DNA and E. coli O157:H7 were 1.8 fg/µL and 2.4 CFU/mL, respectively, within 40 min. Conversely, for the lateral flow detection method, LODs of 1.8 fg/µL and 2.4 × 102 CFU/mL were achieved for genomic DNA and E. coli O157:H7, respectively, within 45 min. This detection strategy offered higher sensitivity and lower equipment requirements than industry standards. In conclusion, the established platform showed excellent specificity and strong universality. Modifying the target gene and its primers can broaden the platform’s applicability to detect various other foodborne pathogens.

The salty oligopeptides from Stropharia rugosoannulata have been proven to be potential ACE inhibitors. To investigate the ACE receptor binding properties and interaction mechanisms of salty oligopeptides, the molecular interaction, dynamics simulation, and antihypertensive evaluation cross-validation strategy were employed to reveal the oligopeptides' binding reactions and modes with the ACE receptor. Single oligopeptide (ESPERPFL, KSWDDFFTR) had exothermic and specific binding reactions with the ACE receptor, driven by hydrogen bonds and van der Waals forces. The coexistence of the multiple oligopeptide molecules did not produce the apparent ACE receptor competition binding reactions. The molecular dynamics simulation verified that the two oligopeptides disturbed the ACE receptor's different residue regions. Both oligopeptides could form stable complexes with the ACE receptor. Based on the classification of 50 oligopeptides' binding modes, ESPERPFL and KSWDDFFTR belonged to different classes, and their receptor binding modes and sites complemented, resulting in a potential synergistic effect on ACE inhibition. The antihypertensive effect of KSWDDFFTR and its distribution in the body were evaluated using SHR rats orally and ICR mice by tail vein injection, and KSWDDFFTR had antihypertensive effects within 8 h. The study provides a theoretical basis for understanding salty oligopeptides' ACE receptor binding mechanism and their antihypertensive effects.

Foodborne pathogens have caused many food safety problems, which have long been serious threats to human health, therefore, a rapid and on-site method for detecting foodborne pathogenic microorganisms is urgently needed. In this study, a visual and dual-targets detection platform for Staphylococcus aureus and Vibrio parahaemolyticus was established here based on recombinant polymerase amplification, CRISPR/Cas12a and lateral flow test strip. The test strip was constructed using colored microspheres labeling antibody, and the detection results of two kinds of bacteria could be displayed by different colored lines on the test strip through the antigen-antibody specific recognition function, which realized results visualization and avoid cross-reaction. Due to the specific amplification of RPA and the precise recognition of Cas12a, the detection strategy could still show good accuracy in proteins from complex matrices or other genomic DNA environments. The visual limit of detection was as low as 50 CFU/mL, and entire detection process could be completed within 42 min. In conclusion, a rapid and efficient detection platform for Staphylococcus aureus and Vibrio parahaemolyticus was developed, and this process is expected to provide a solution for the on-site detection of other foodborne pathogens in market and environmental food.

Advanced multiplexed testing techniques should be designed and developed to ensure an accurate and reliable evaluation for unknown samples. In this study, an efficient platform coupled with the “Blue-Red-Purple” strategy based on recombinant polymerase amplification (RPA), CRISPR/Cas12a and lateral flow strip was established, which could realize the dual-target detection of CP4-EPSPS and Cry1Ab/Ac in genetically modified crops. The lateral flow immunoassay was developed using different colored microspheres to label the antibodies to realize the visualization of results and avoid cross-reactions. The proposed method exhibits high specificity, sensitivity and stability. The visual detection limits of standard plasmids and real samples reached 10 copies/μL and 0.5 %, respectively, which could be stored at 4 °C for 12 months with high detection ability. Moreover, the entire detection process could be completed within 50 min without any complex instruments or professional operators. These findings indicated that a sensitive, specific, rapid and accurate method was established for on-site detection of GM crops.

Background Escherichia coli O157:H7 ( E. coli O157:H7) is a foodborne pathogenic microorganism that is commonly found in the environment and poses a significant threat to human health, public safety, and economic stability worldwide. Thus, early detection is essential for E. coli O157:H7 control. In recent years, a series of E. coli O157:H7 detection methods have been developed, but the sensitivity and portability of the methods still need improvement. Methods and Results In this study, a rapid and efficient testing platform based on CRISPR/Cas12a cleavage reaction was constructed. Through the integration of recombinant polymerase amplification and lateral flow chromatography, we established a dual-interpretation-mode detection platform based on CRISPR/Cas12a-derived fluorescence and lateral flow chromatography for the detection of E. coli O157:H7. For the fluorescence detection method, the limits of detection (LODs) of genomic DNA and E. coli O157:H7 were 1.8 fg/µL and 2.4 CFU/mL, respectively, within 40 min. Conversely, for the lateral flow detection method, LODs of 1.8 fg/µL and 2.4 × 10 2 CFU/mL were achieved for genomic DNA and E. coli O157:H7, respectively, within 45 min. Conclusions This detection strategy offered higher sensitivity and lower equipment requirements than industry standards, and showed excellent specificity and strong universality. Modifying the target gene and its primers can broaden the platform’s applicability to detect various other foodborne pathogens.