Multiple Perspectives of Study on the Potential of Bacillus amyloliquefaciens JB20221020 for Alleviating Nutrient Stress in Lettuce.

Soil nutrient deficiency has become a key factor limiting crop growth. Plant growth-promoting rhizobacteria (PGPR) are vital in resisting abiotic stress. In this study, we investigated the effects of inoculation with Bacillus amyloliquefaciens JB20221020 on the physiology, biochemistry, rhizosphere microorganisms, and metabolism of lettuce under nutrient stress. Pot experiments showed that inoculation with B. amyloliquefaciens JB20221020 significantly promoted lettuce growth under nutrient deficiency. At the same time, the activities of the antioxidant enzymes superoxide dismutase, peroxidase, and catalase and the content of proline increased, and the content of Malondialdehyde decreased in the lettuce inoculated with B. amyloliquefaciens JB20221020. Inoculation with B. amyloliquefaciens JB20221020 altered the microbial community of the rhizosphere and increased the relative abundances of Myxococcales, Deltaproteobacteria, Proteobacteria, Devosia, and Verrucomicrobia. Inoculation also altered the rhizosphere metabolism under nutrient deficiency. The folate metabolism pathway was significantly enriched in the Kyoto Encyclopedia of Genes and Genomes enrichment analysis. This study explored the interaction between plants and microorganisms under nutrient deficiency, further explained the critical role of rhizosphere microorganisms in the process of plant nutrient stress, and provided a theoretical basis for the use of microorganisms to improve plant resistance.

A Dual-mode platform for the rapid detection of Escherichia coli O157:H7 based on CRISPR/Cas12a and RPA.

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.

Using multi-omics to explore the effect of Bacillus velezensis SAAS-63 on resisting nutrient stress in lettuce.

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. KEY POINTS: • 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.

Isothermal Amplification and CRISPR/Cas12a-System-Based Assay for Rapid, Sensitive and Visual Detection of Staphylococcus aureus.

Staphylococcus aureus exists widely in the natural environment and is one of the main food-borne pathogenic microorganisms causing human bacteremia. For safe food management, a rapid, high-specificity, sensitive method for the detection of S. aureus should be developed. In this study, a platform for detecting S. aureus (nuc gene) based on isothermal amplification (loop-mediated isothermal amplification-LAMP, recombinase polymerase amplification-RPA) and the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas12a) proteins system (LAMP, RPA-CRISPR/Cas12a) was proposed. In this study, the LAMP, RPA-CRISPR/Cas12a detection platform and immunochromatographic test strip (ICS) were combined to achieve a low-cost, simple and visualized detection of S. aureus. The limit of visual detection was 57.8 fg/µL of nuc DNA and 6.7 × 102 CFU/mL of bacteria. Moreover, the platform could be combined with fluorescence detection, namely LAMP, RPA-CRISPR/Cas12a-flu, to establish a rapid and highly sensitive method for the detection of S. aureus. The limit of fluorescence detection was 5.78 fg/µL of genomic DNA and 67 CFU/mL of S. aureus. In addition, this detection platform can detect S. aureus in dairy products, and the detection time was ~40 min. Consequently, the isothermal amplification CRISPR/Cas12a platform is a useful tool for the rapid and sensitive detection of S. aureus in food.

A rapid and high-throughput system for the detection of transgenic products based on LAMP-CRISPR-Cas12a.

With the increasing acreage of genetically modified crops worldwide, rapid and efficient detection technologies have become very important for the regulation and screening of GM organisms. We constructed a method based on loop-mediated isothermal amplification (LAMP), CRISPR-Cas12a and lateral flow assay (LAMP-CRISPR-Cas12a-LFA). It is an intuitive, sensitive and specific fluorescence detection and test strip system to detect CP4-EPSPS and Cry1Ab/Ac genes in field screening. The LAMP-CRISPR-Cas12a-LFA method has a limit of detection (LOD) of 100 copies based on lateral flow test strips after optimization of the conditions with screened specific primers, and the entire detection process can be completed within 1 h at 61 °C. The system was used to evaluate field test samples and showed high reproducibility after testing products containing CP4-EPSPS and Cry1Ab/Ac genes, and both were detectable. The LAMP-CRISPR-Cas12a-LFA method established in this paper functions as a rapid field detection method. It requires only one portable thermostatic instrument, which renders it compatible with the rapid detection of field samples and useable at experimental workstations, in law enforcement field work, and in local inspection and quarantine departments.

Two electrochemiluminescence immunosensors for the sensitive and quantitative detection of the CP4-EPSPS protein in genetically modified crops.

Two different models of electrochemiluminescence (ECL) immunosensors for the sensitive and quantitative detection of the CP4-EPSPS protein in genetically modified (GM) crops were proposed in this study. One was a signal-reduced ECL immunosensor based on nitrogen-doped graphene, graphitic carbon nitride and polyamide-amine (GN-PAMAM-g-C3N4) composites as the electrochemically active substance. The other model was a signal-enhanced ECL immunosensor based on a GN-PAMAM modified electrode for the detection of CdSe/ZnS quantum dots (QDs)-labeled antigens. The ECL signal responses of the reduced and enhanced immunosensors linearly decreased as the increase of the soybean RRS and RRS-QDs content in the range of 0.05% to 1.5% and 0.025% to 1.0%, with the limits of detection of 0.03% and 0.01% (S/N = 3), respectively. Both of the ECL immunosensors showed good specificity, stability, accuracy, and reproducibility in the analysis of real samples. The results indicate that the two immunosensors provide an ultra-sensitive and quantitative approach for the determination of the CP4-EPSPS protein. Due to their outstanding performances, the two ECL immunosensors could be useful tools for achieving the effective regulation of GM crops.

Rapid detection of genetically modified products based on CRISPR-Cas12a combined with recombinase polymerase amplification.

With the large-scale planting of genetically modified (GM) crops, consumers were more aware of biosafety. Onsite rapid diagnostic methods were advantageous to the regulation of GM products. In this study, a rapid, sensitive and portable detection method based on recombinase polymerase amplification were proposed based on RPA reaction and Cas12a cleavage reaction for GM ingredients, named RPA-Cas12a-GM. The results would be displayed by fluorescence signal (FS) and visual bands of lateral flow strip (LFS). RPA-Cas12a-GM method could be completed within 45 min, and the detection limit was as low as 45 copies/µL of the standard plasmid containing CP4-EPSPS gene and Cry1Ab/Ac gene. Furthermore, the detection coincidence rate of RPA-Cas12a-GM method was 100%. In conclusion, the proposed RPA-Cas12a-GM method based FS and LFS were sensitive, specific, rapid and visible for diagnosis of CP4-EPSPS gene and Cry1Ab/Ac gene without complex equipment, which provides technical support for the regulation of GM products in the field.

A nanomaterial-free and thionine labeling-based lateral flow immunoassay for rapid and visual detection of the transgenic CP4-EPSPS protein.

Nanomaterial-based lateral flow immunoassays (LFIAs) have been widely used for the on-site detection of genetically modified components. However, the practical applications are often limited by the complex matrix, such as in red samples. In this study, a thionine (Thi) labeling-based LFIA was developed for the first time to detect CP4-EPSPS protein. The optimal labeling concentration of Thi was 0.5 mg/mL, and the antibody could be rapidly coupled to Thi in 10 min. The visual limit of detection (vLOD) levels for transgenic soybean, sugar beet, and cotton containing the CP4-EPSPS protein reached 0.05%, 0.1%, and 0.1%, respectively, and had no interference from other proteins. After storage at 4 °C for three months, the LFIA sensitivity remained unchanged and showed good stability. This method could be used to screen and detect a variety of transgenic crops containing the CP4-EPSPS protein, and the results were consistent with the current standard assay. This study pioneered the development of an immunochromatographic method using Thi as a marker and applied it to the detection of the CP4-EPSPS protein in herbicide-tolerant transgenic crops. This provides a new method for the rapid immunoassay of Thi as a dye and has good prospects for practical application.

A Label-Free Immunosensor Based on Gold Nanoparticles/Thionine for Sensitive Detection of PAT Protein in Genetically Modified Crops.

Genetically modified (GM) crops containing phosphinothricin acetyltransferase (PAT) protein has been widely planted worldwide. The development of a rapid method for detecting PAT protein is of great importance to food supervision. In this study, a simple label-free electrochemical immunosensor for the ultrasensitive detection of PAT protein was constructed using thionine (Thi)/gold nanoparticles (AuNPs) as signal amplification molecules and electrochemically active substances. Under optimum conditions, the limits of detection of the sensor for soybean A2704-12 and maize BT-176 were 0.02% and 0.03%, respectively. The sensor could detect crops containing PAT protein and had no cross-reaction with other proteins. After storage at 4°C for 33 days, the sensor still retained 82.5% of the original signal, with a relative standard deviation (RSD) of 0.92%. The recoveries of the sensor for soybean A2704-12 and maize BT-176 were 85%-108% and 98%-113%, respectively. The developed PAT-target immunosensor with high sensitivity, specificity, and satisfactory reproducibility and accuracy will be a useful tool in the trace screening of GM crops. Moreover, this design concept can be extended to other proteins by simply changing the antibody.

A sensitive immunosensor based on graphene-PAMAM composites for rapid detection of the CP4-EPSPS protein in genetically modified crops.

A simple electrochemical immunosensor based on nitrogen-doped graphene and polyamide-amine (GN-PAM) composites was proposed for the detection of the CP4-EPSPS protein in genetically modified (GM) crops. In this immunosensor, the amplification of the detection signal was realized through antibodies labeled with gold nanoparticles (AuNPs). The electrochemical responses of the immunosensor were linear (R2 = 0.9935 and 0.9912) when the GM soybean RRS and maize NK603 content ranged from 0.025% to 1.0% and 0.05% to 1.5%, respectively. The limits of detection for the GM soybean RRS and maize NK603 were as low as 0.01% and 0.03%, respectively. The immunosensor also exhibited high specificity, and satisfactory stability, reproducibility, and accuracy. Our findings indicated that the constructed immunosensor provides a new approach for the sensitive detection of the CP4-EPSPS protein. Notably, the sensor may be applied to other proteins or pathogenic bacteria by simply changing the antibodies, and may also be used for multi-component analysis.

Development of a lateral flow test strip for simultaneous detection of BT-Cry1Ab, BT-Cry1Ac and CP4 EPSPS proteins in genetically modified crops.

A colloidal gold immunochromatographic strip (ICS) for simultaneous detection of multiple transgenic proteins, including CP4 EPSPS, BT-Cry1Ab and BT-Cry1Ac, was developed in this study. The sensitivity of the strip to the target protein was 5 ng/mL for CP4 EPSPS, 100 ng/mL for BT-Cry1Ab and Cry1Ac, respectively. Parallel analysis for maize, soybean, sugar beet and cotton showed the strip could detect 1% of transgenic content in crops containing BT-Cry1Ab and Cry1Ac, and, at least, 0.1% of content in crops containing CP4 EPSPS. The detection results for seed samples indicated the multicomponent analysis ICS had good accuracy. The analysis could be completed within 10 min and had the advantages of being high-throughput, easy to operate and visual detection. This is the first report of semi-quantitative ICS for detecting three transgenic proteins simultaneously. The developed approach may provide insights into the development of ICS for analyzing simultaneously multiple components in genetically modified crops.

RPA-Cas12a-FS: A frontline nucleic acid rapid detection system for food safety based on CRISPR-Cas12a combined with recombinase polymerase amplification.

Food analysis to ensure food safety and quality are relevant to all countries. This study aimed to develop a detection technique by combining recombinase polymerase amplification with CRISPR-Cas12a for food safety (termed RPA-Cas12a-FS). Our data showed that this novel method could be detected via fluorescence intensity for the molecular identification of foodborne pathogenic bacteria, genetically modified crops, and meat adulteration. After optimization, the sensitivity and stability of RPA-Cas12a-FS was further enhanced. The RPA-Cas12a-FS system could specifically detect target gene levels as low as 10 copies in 45 min at 37 °C. The RPA-Cas12a-FS system was sensitive both using standard samples in the lab and using samples from the field, which indicated that this detection method was practical. In conclusion, a simple, rapid, and highly sensitive detection method based on CRISPR-Cas12a was developed for molecular identification in the food safety field without requiring technical expertise or ancillary equipment.

Identification and Functional Characterization of a Novel Immunomodulatory Protein From Morchella conica SH.

A novel fungal immunomodulatory protein (FIP) was found in the precious medical and edible mushroom Morchella conica SH, defined as FIP-mco, which belongs to the FIP family. Phylogenetic analyses of FIPs from different origins were performed using Neighbor-Joining method. It was found that FIP-mco belonged to a new branch of the FIP family and may evolved from a different ancestor compared with most other FIPs. The cDNA sequence of FIP-mco was cloned and expressed in the yeast Pichia Pastoris X33. The recombinant protein of FIP-mco (rFIP-mco) was purified by agarose Ni chromatography and determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis. The protein rFIP-mco could significantly suppress the proliferation of A549 and HepG2 cells at the concentration of 15 and 5 µg/ml, respectively, and inhibited the migration and invasion of human A549 and HepG2 cells at the concentration of 15 and 30 µg/ml respectively in vitro. Further, rFIP-mco can significantly reduce the expression levels of TNF-α, IL-1ß, and IL-6 in the THP1 cells (human myeloid leukemia mononuclear cells). In order to explore the potential mechanism of the cytotoxicity effect of rFIP-mco on A549 and HepG2 cells, cell cycle and apoptosis assay in the two cancer cells were conducted. The results demonstrated that G0/G1 to S-phase arrest and increased apoptosis may contribute to the proliferation inhibition by rFIP-mco in the two cancer cells. Molecular mechanism of rFIP-mco's reduction effect on the inflammatory cytokines was also studied by suppression of the NF-κB signaling pathway. It showed that suppression of NF-κB signaling is responsible for the reduction of inflammatory cytokines by rFIP-mco. The results indicated the prospect of FIP-mco from M. conica SH as an effective and feasible source for cancer therapeutic studies and medical applications.

Attenuated Listeria monocytogenes as a Vaccine Vector for the Delivery of OMPW, the Outer Membrane Protein of Aeromonas hydrophila.

Listeria monocytogenes (LM) is a gram-positive facultative intracellular pathogen that could stimulate host to produce inflammatory response, cell-mediated immunity, and humoral immunity. In this study, an attenuated live vector vaccine for Aeromonas hydrophila (AH) named EGDeABdd-dat-ompW was successfully constructed using an attenuated vector named EGDeABdd, in which dal, dat, actA, and inlB genes were deleted from wild-type LM-EGDe. To construct EGDeABdd-dat-ompW, a recombinant plasmid pERL3-dat-ompW obtained by inserting the dat gene from EGDe and outer membrane protein gene ompW from AH into pERL3 plasmid was transformed into EGDeABdd cell. The safety and immunogenicity of EGDeABdd-dat-ompW as an attenuated vector vaccine for delivery of OMPW were assessed through analyzing invasion to Caco-2 cells and mice, cytokine production of macrophagocyte and mouse splenocytes, and T-cell proliferation of mouse splenocytes. Serum titers against AH and the immunoprotective effect of the vaccine to mice were also measured after intravenous injection with vaccine for four times. The results showed that the live vector vaccine EGDeABdd-dat-ompW for AH exhibited high attenuation in invading Caco-2 cells and mice than did EGDe. Real-time PCR (RT-PCR) showed that cytokines (e.g., TNF-α, IL-6, and IL-1ß from macrophages; and IL-6 and IFN-γ from mouse splenocytes) had significantly increased after immunization by EGDeABdd-dat-ompW. Meanwhile, the vaccine could induce the production of CD3+CD4+ and CD3+CD8+ T-cell proliferation of mice and generate effective immunoprotection against lethal challenge of 20 × LD50 AH. All these results indicated that the attenuated EGDeABdd-dat could be used as a live vector for the delivery of the exogenous gene, not only possessing safety but also providing high immunogenicity. The successful application in the AH vaccine further showed that it could be used in other fields such as vaccines in cancer or infectious diseases.

Gut Microbiota and Relevant Metabolites Analysis in Alcohol Dependent Mice.

Alcohol abuse is a major public health crisis. Relative evidences supported that the gut microbiota (GM) played an important role in central nervous system (CNS) function, and the composition of them had changed after alcohol drinking. We sought to explore the changes of GM in alcohol dependence. In our study, the GM of mice with alcohol administration was detected through analyzed 16S rRNA gene sequencing and the fecal metabolites were analyzed by LC-MS. The microbial diversity was significantly higher in the alcohol administration group, the abundance of phylum Firmicutes and its class Clostridiales were elevated, meanwhile the abundance of Lachnospiraceae, Alistipes, and Odoribacter showed significant differences among the three groups. Based on LC-MS results, bile acid, secondary bile acid, serotonin and taurine level had varying degrees of changes in alcohol model. From paraffin sections, tissue damage was observed in liver and colon. These findings provide direct evidence that alcohol intake affects the composition of GM, enable a better understanding of the function of GM in the microbiota-gut-brain (MGB) axis, and give a new thought for alcohol addiction treatment.

Evaluation of Caco-2 cells response to Listeria monocytogenes virulence factors by RT-PCR.

Listeria monocytogenes expresses various virulence factors enabling the invasion and multiplying in host cells, and together induces cytokines transcription. In order to explore the relationship between virulence factors of L. monocytogenes wild-type EGD-e and cellular response in human colonic epithelial cell line(Caco-2), we constructed mutant strains with in-frame deletions of critical virulence genes of inlA, inlB, hly, actA and virulence regulatory factor prfA from EGD-e, respectively. Compared with EGD-e, mutant strains showed significantly decreased invasion and apoptosis in Caco-2 cells. However, mutant strains were capable to evoke cytokines transcription of interleukin-8 (IL-8), mononuclear chemoattractant protein-1 (MCP-1), tumor necrosis factor-a (TNF-a), interleukin-1ß (IL-1ß), interleukin-6 (IL-6) and CXCL-2 production in Caco-2 cells. Interestingly, EGD-e Δhly-infected Caco-2 cells showed a significant decrease of IL-6, IL-8 and MCP-1 transcription compared with EGD-e at 1 h post-infection. Simultaneously, EGD-e ΔinlB-infected cells showed a decrease in IL-6 transcription, while EGD-e ΔactA-infected cells reflected a decrease in MCP-1 transcription. Virulence genes play a role in inflammatory transcription, but the interaction between pathogenic bacteria and the host cells predominates in inflammatory transcription. Overall, the data showed cellular response of Caco-2 cells infected with EGD-e mutant strains.

Enhancing the immunofluorescent sensitivity for detection of Acidovorax citrulli using fluorescein isothiocyanate labeled antigen and antibody.

A rapid lateral flow immunochromatographic strip (ICS) using fluorescein isothiocyanate (FITC) labeled antigen and antibody was developed for the detection of Acidovorax citrulli (Ac) in melons and vegetable samples. In the ICS, signal amplification was realized based on antigen Ac and anti-Ac monoclonal antibody (McAb) 4F conjugated with FITC, respectively, which were forming two probes. The control line and the test line were obtained by immobilizing the goat anti-mouse IgG antibody and anti-Ac McAb 6D on both sides of the nitrocellulose membrane. The visual detection limit of the strip was 105 CFU/mL, which was 10-fold sensitive compared to the strip of FITC only labeling antigen or antibody. Signal amplification ICS was successfully applied to the detection of Ac in melon and vegetable samples with less detection time and operation procedures compared to the traditional enzyme-linked immunosorbent assay (ELISA) and PCR methods. This is the first report of using FITC labeled antigen and McAb as dual fluorescent probes to develop a direct-type immunofluorescence strip for the rapid and sensitive detection of Ac, which demonstrates a powerful tool for rapidly screening Ac in plant materials and other samples. Graphical abstract The schematic presentation of the test strip (a) and the positive result (b) or negative result

Fluorescein Isothiocyanate Labeling Antigen-Based Immunoassay Strip for Rapid Detection of Acidovorax citrulli.

A simple and fast immunoassay strip to detect Acidovorax citrulli (Ac) using fluorescein isothiocyanate as a marker was developed. Fluorescein isothiocyanate (FITC) was added to sample culture medium for bacteria incubation, and the bacteria could emit a yellow-green fluorescence under ultraviolet light and become a fluorescent probe. This immunofluorescence strip (IFS) was based on the binding between fluorescent bacteria and the unlabeled monoclonal antibody (McAb) immobilized on the test area in nitrocellulose membrane. The detection limit of the strip was 106 CFU/ml with a result that could be observed within 10 min. The IFS could detect eight strains of Ac and display no cross-reactions with 30 other pathogenic strains. The detection results would not be affected by impurities in plant or unknown microorganisms in natural field samples and were consistent with PCR results, indicating that the IFS has high accuracy. This is the first report of using only one unlabeled McAb to develop a direct-type immunofluorescence strip for the rapid detection of Ac. The IFS reduced detection time and simplified operation procedures compared with the traditional enzyme-linked immunosorbent assay (ELISA) and PCR methods. In addition, this simple and inexpensive method will play a significant role in monitoring plant pathogens on field detection.

Self-paired monoclonal antibody lateral flow immunoassay strip for rapid detection of Acidovorax avenae subsp. citrulli.

We screened a highly specific monoclonal antibody (McAb), named 6D, against Acidovorax avenae subsp. citrulli (Aac). Single McAb 6D was used as both nanogold-labeled antibody and test antibody to develop a single self-paired colloidal gold immunochromatographic test strip (Sa-GICS). The detection limit achieved using the Sa-GICS approach was 10(5) CFU/mL, with a result that can be observed by the naked eye within 10 min. Moreover, Sa-GICS can detect eight strains of Aac and display no cross-reactions with other pathogenic plant microorganisms. Artificial contamination experiments demonstrated that Sa-GICS would not be affected by impurities in the leaves or stems of the plants and were consistent with the PCR results. This is the first report on the development of a colloidal gold immunoassay strip with self-paired single McAb for the rapid detection of Aac. Graphical Abstract Schematic representation of the test strip.

Simultaneous quantification of Escherichia coli O157:H7 and Shigella boydii using a visual-antibody-macroarray.

Being high throughput, rapid, automated, economical, convenient to operate and highly sensitive, protein arrays have been widely used in the analysis of tumor markers and veterinary drug residues. Pathogenic microbes also can be detected qualitatively by DNA array or protein array; however, their high throughput detection and quantification remains a big obstacle. To evaluate the potentiality of protein arrays for multiple quantitative detection of microorganisms with naked eye examination without the help of any equipment, here we developed a visual-antibody-macroarray (VAMA) aiming at rapid and simultaneous quantification of Escherichia coli O157:H7 and Shigella boydii. The results show that this VAMA is highly specific and is able to distinguish mixed Escherichia coli O157:H7 and Shigella boydii synchronously. The detection limits are equivalent to 3.4 × 10(5) CFU mL(-1) and 3.2 × 10(5) CFU mL(-1), respectively, which conform to the results of plate counting and ELISA. Importantly, the examination can be solely performed with the naked eye. Therefore, we provide an easy, reliable and rapid method for quantitative analysis of microorganisms.