Red and far-red light improve the antagonistic ability of Trichoderma guizhouense against phytopathogenic fungi by promoting phytochrome-dependent aerial hyphal growth.

Light as a source of information regulates morphological and physiological processes of fungi, including development, primary and secondary metabolism, or the circadian rhythm. Light signaling in fungi depends on photoreceptors and downstream components that amplify the signal to govern the expression of an array of genes. Here, we investigated the effects of red and far-red light in the mycoparasite Trichoderma guizhouense on its mycoparasitic potential. We show that the invasion strategy of T. guizhouense depends on the attacked species and that red and far-red light increased aerial hyphal growth and led to faster overgrowth or invasion of the colonies. Molecular experiments and transcriptome analyses revealed that red and far-red light are sensed by phytochrome FPH1 and further transmitted by the downstream MAPK HOG pathway and the bZIP transcription factor ATF1. Overexpression of the red- and far-red light-induced fluffy gene fluG in the dark resulted in abundant aerial hyphae formation and thereby improvement of its antagonistic ability against phytopathogenic fungi. Hence, light-induced fluG expression is important for the mycoparasitic interaction. The increased aggressiveness of fluG-overexpressing strains was phenocopied by four random mutants obtained after UV mutagenesis. Therefore, aerial hyphae formation appears to be a trait for the antagonistic potential of T. guizhouense.

Fungal phytochrome chromophore biosynthesis at mitochondria.

Mitochondria are essential organelles because of their function in energy conservation. Here, we show an involvement of mitochondria in phytochrome-dependent light sensing in fungi. Phytochrome photoreceptors are found in plants, bacteria, and fungi and contain a linear, heme-derived tetrapyrrole as chromophore. Linearization of heme requires heme oxygenases (HOs) which reside inside chloroplasts in planta. Despite the poor degree of conservation of HOs, we identified two candidates in the fungus Alternaria alternata. Deletion of either one phenocopied phytochrome deletion. The two enzymes had a cooperative effect and physically interacted with phytochrome, suggesting metabolon formation. The metabolon was attached to the surface of mitochondria with a C-terminal anchor (CTA) sequence in HoxA. The CTA was necessary and sufficient for mitochondrial targeting. The affinity of phytochrome apoprotein to HoxA was 57,000-fold higher than the affinity of the holoprotein, suggesting a "kiss-and-go" mechanism for chromophore loading and a function of mitochondria as assembly platforms for functional phytochrome. Hence, two alternative approaches for chromophore biosynthesis and insertion into phytochrome evolved in plants and fungi.

Phage-Modified Clear Hydrogel for Simultaneous Detection of Multiple Bacteria.

The proliferation speed of live foodborne pathogens is fast. A small number of pathogens will have a great impact on food and the environment if positive samples are not detected timely. In this study, transparent porous hydrogel stir bars, modified by two different phages (corresponding to two different bacteria (Escherichia coli and Hafnia sp)), have been developed for rapid detection of foodborne bacteria. A large number of samples can be analyzed simultaneously with a small animal live imager device to screen out the positive samples, while an adenosine triphosphate (ATP) bioluminescence sensor can be used to quantify the number of bacteria in the positive samples. The phage has good specificity and capture ability to bacteria, which makes the method highly sensitive. In addition, the use of multiple phages also enables the method to detect multiple bacteria simultaneously. The three-dimensional structure of the hydrogel allows it to modify more phages, and its transparent nature also allows the inside bioluminescence to be detected. Both can enhance the sensitivity of the detection. Finally, the reagents needed for bioluminescence, such as d-luciferin, can also be preencapsulated in the hydrogel, thus simplifying the detection step. Under the best conditions, the detection range of the method is 102-108 CFU·mL-1, and the limit of detection is 30 CFU·mL-1 within 11 min. The test results of actual samples show that there is no difference between using the method developed through this study and the traditional plate counting method, but the detection time is greatly shortened.

Versatile Platinum Nanoparticles-Decorated Phage Nanozyme Integrating Recognition, Bacteriolysis, and Catalysis Capabilities for On-Site Detection of Foodborne Pathogenic Strains Vitality Based on Bioluminescence/Pressure Dual-Mode Bioassay.

Sensitive and on-site discrimination of live and dead foodborne pathogenic strains remains a significant challenge due to the lack of appropriate assay and signal probes. In this work, a versatile platinum nanoparticle-decorated phage nanozyme (P2@PtNPs) that integrated recognition, bacteriolysis, and catalysis was designed to establish the bioluminescence/pressure dual-mode bioassay for on-site determination of the vitality of foodborne pathogenic strains. Benefiting from the bacterial strain-level specificity of phage, the target Salmonella typhimurium (S.T) was specially captured to form sandwich complexes with P2@PtNPs on another phage-modified glass microbead (GM@P1). As the other part of the P2@PtNPs nanozyme, the introduced PtNPs could not only catalyze the decomposition of hydrogen peroxide to generate a significant oxygen pressure signal but also produce hydroxyl radicals around the target bacteria to enhance the bacteriolysis of phage and adenosine triphosphate release. It significantly improved the bioluminescence signal. The two signals corresponded to the total and live target bacteria counts, so the dead target could be easily calculated from the difference between the total and live target bacteria counts. Meanwhile, the vitality of S.T was realized according to the ratio of live and total S.T. Under optimal conditions, the application range of this proposed bioassay for bacterial vitality was 102-107 CFU/mL, with a limit of detections for total and live S.T of 30 CFU/mL and 40 CFU/mL, respectively. This work provides an innovative and versatile nanozyme signal probe for the on-site determination of bacterial vitality for food safety.

Genome-wide analyses of light-regulated genes in Aspergillus nidulans reveal a complex interplay between different photoreceptors and novel photoreceptor functions.

Fungi sense light of different wavelengths using blue-, green-, and red-light photoreceptors. Blue light sensing requires the "white-collar" proteins with flavin as chromophore, and red light is sensed through phytochrome. Here we analyzed genome-wide gene expression changes caused by short-term, low-light intensity illumination with blue-, red- or far-red light in Aspergillus nidulans and found that more than 1100 genes were differentially regulated. The largest number of up- and downregulated genes depended on the phytochrome FphA and the attached HOG pathway. FphA and the white-collar orthologue LreA fulfill activating but also repressing functions under all light conditions and both appear to have roles in the dark. Additionally, we found about 100 genes, which are red-light induced in the absence of phytochrome, suggesting alternative red-light sensing systems. We also found blue-light induced genes in the absence of the blue-light receptor LreA. We present evidence that cryptochrome may be part of this regulatory cue, but that phytochrome is essential for the response. In addition to in vivo data showing that FphA is involved in blue-light sensing, we performed spectroscopy of purified phytochrome and show that it responds indeed to blue light.

Ultrasensitive and Specific Phage@DNAzyme Probe-Triggered Fluorescent Click Chemistry for On-Site Detection of Foodborne Pathogens Using a Smartphone.

Rapid, specific, and on-site detection of virulent foodborne pathogenic strains plays a key role in controlling food safety. In this work, an ultrasensitive and specific Phage@DNAzyme signal probe was designed to detect foodborne pathogens. The proposed sensing probe was composed of the selected phage and functionalized DNAzyme, which realized the specific recognition of target foodborne pathogens at the strain level and the efficient catalysis of copper(II) based azide-alkyne cycloaddition (CuAAC) click reaction with fluorescent signal, respectively. As a proof of concept, the virulent Escherichia coli O157:H7 (E. coli O157:H7) as the representative analyte was first enriched and purified from the complex food samples by a 4-mercaptophenylboronic acid-modified gold slide. Following, the Phage@DNAzyme probes were specifically combined with the captured E. coli O157: H7 and catalyzed the click reaction between 3-azido-7-hydroxycoumarin and 3-butyn-1-ol with the assistance of Cu(II) to generate a visual fluorescent signal. Finally, the corresponding fluorescent signals were measured by a smartphone to quantify the target concentrations. Under optimized conditions, the bioassay exhibited a wide linear range from 102 to 108 CFU/mL and the detection limit was 50 CFU/mL (S/N = 3). It was further extended to the detection of another foodborne pathogen Salmonella typhimurium with satisfying sensing performances. This work gives a new path for developing rapid, specific, and on-site detection methods for trace levels of pathogenic strains in foods.

A new atypical short-chain dehydrogenase is required for interfungal combat and conidiation in Trichoderma guizhouense.

Hypocrealean Trichoderma are the most extensively studied facultative mycoparasites against phytopathogenic fungi. Aerial hyphae of Trichoderma guizhouense can rapidly proliferate over Fusarium oxysporum hyphae, cause sporadic cell death and arrest the growth of the host. The results of the present study demonstrated that a unique short-chain dehydrogenase/reductase (SDR), designated as TgSDR1, was expressed at a high level in T. guizhouense challenged by the hosts. Similar to other SDRs family members, the TgSDR1 protein contains a cofactor-binding motif and a catalytic site. The subcellular localization assay revealed that the TgSDR1::GFP fusion protein translocated to lipid droplets in mycelia and conidia. The data obtained using reverse genetic approach indicated that TgSDR1 is associated with antifungal ability, plays an important role in providing reducing equivalents in the form of NADPH and regulates the amino sugar and nucleotide sugar metabolism in T. guizhouense upon encountering a host. Moreover, the TgSDR1 deletion mutant was defective in conidiation. Thus, TgSDR1 functions as a key metabolic enzyme in T. guizhouense to regulate mycotrophic interactions, defence against other fungi, such as F. oxysporum, and conidiation.

Comprehensive analysis of the regulatory network of blue-light-regulated conidiation and hydrophobin production in Trichoderma guizhouense.

Conidia of Trichoderma guizhouense (Hypocreales, Ascomycota) are frequently applied to the production of biofertilizers and biocontrol agents. Conidiation of some Trichoderma species depends on blue light and the action of different blue light receptors. However, the interplay between different blue-light receptors in light signalling remained elusive. Here, we studied the functions of the blue light receptors BLR1 and ENV1, and the MAP kinase HOG1 in blue light signalling in T. guizhouense. We found that the BLR1 dominates light responses and ENV1 is responsible for photoadaptation. Genome-wide gene expression analyses revealed that 1615 genes, accounting for ~13.4% of the genes annotated in the genome, are blue-light regulated in T. guizhouense, and remarkably, these differentially expressed genes (DEGs) including 61 transcription factors. BLR1 and HOG1 are the core components of the light signalling network, which control 79.9% and 73.9% of the DEGs respectively. In addition, the strict regulation of hydrophobin production by the blue light signalling network is impressive. Our study unravels the regulatory network based on the blue light receptors and the MAPK HOG pathway for conidiation, hydrophobin production and other processes in T. guizhouense.

Pressure-Based Biosensor Integrated with a Flexible Pressure Sensor and an Electrochromic Device for Visual Detection.

This work demonstrated a pressure-based biosensor integrated with a flexible pressure sensor and an electrochromic device for visual detection. Initially, a sandwich-type immunoreaction for target carcinoembryonic antigen (CEA, as a model analyte) was carried out using the capture antibody (cAb) and platinum nanoparticles-labeled detection antibody (PtNPs-dAb) in a reaction cell. The added hydrogen peroxide (H2O2) could be catalyzed by the PtNPs to generate oxygen (O2). In a sealed chamber, the pressure increased with the overflowing O2. Meanwhile, a skin-inspired flexible pressure sensor with excellent sensing performance was fabricated to monitor the pressure change in real time. Thus, the electrical signal of the pressure sensor could reveal the target concentration. Moreover, a voltage-regulated electrochromic device based on polyaniline (PANI) and tungsten oxide (WO3) was integrated into the platform to provide a visualized readout. According to the electrical signal of the pressure sensor, the electrochromic device would change its color from green to blue, which also revealed the target concentration and could be observed by the naked eye. Under optimal conditions, the biosensor presented a high sensitivity for CEA in a detectable range of 0.2-50 ng/mL. The limit of detection (LOD) was 94 pg/mL. The selectivity, reproducibility, and accuracy were also satisfying. Furthermore, this immunoassay gives a path for developing visualized biosensors in point-of-care settings.

Two hybrid histidine kinases, TcsB and the phytochrome FphA, are involved in temperature sensing in Aspergillus nidulans.

The adaptation of microorganisms to different temperatures is an advantage in habitats with steadily changing conditions and raises the question about temperature sensing. Here we show that in the filamentous fungus Aspergillus nidulans, the hybrid histidine kinase TcsB and phytochrome are involved in temperature-induced gene transcription. Temperature-activated phytochrome fed the signal into the HOG MAP kinase pathway. There is evidence that the photoreceptor phytochrome fulfills a temperature sensory role in plants and bacteria. The effects in plants are based on dark reversion from the active form of phytochrome, Pfr, to the inactive form, Pr. Elevated temperature leads to higher dark reversion rates, and hence, temperature sensing depends on light. In A. nidulans and in Alternaria alternata, the temperature response was light-independent. In order to understand the primary temperature response of phytochrome, we performed spectral analyses of recombinant FphA from both fungi. Spectral properties after heat stress resembled the spectrum of free biliverdin, suggesting conformational changes and a softening of the binding pocket of phytochrome, possibly mimicking photoactivation. We propose a novel function for fungal phytochrome as temperature sensor.

Azaphilones biosynthesis complements the defence mechanism of Trichoderma guizhouense against oxidative stress.

Filamentous fungi are known as producers of a large array of diverse secondary metabolites (SMs) that aid in securing their environmental niche. Here, we demonstrated that the SMs have an additional role in fungal defence against other fungi: Trichoderma guizhouense, a mycoparasite, is able to antagonize Fusarium oxysporum f. sp. cubense race 4 (Foc4) by forming aerial hyphae that kill the host with hydrogen peroxide. At the same time, a gene cluster comprising two polyketide synthases is strongly expressed. Using functional genetics, we characterized this cluster and identified its products as azaphilones (termed as trigazaphilones). The trigazaphilones were found lacking of antifungal toxicity but exhibited high radical scavenging activities. The antioxidant property of trigazaphilones was in vivo functional under various tested conditions of oxidative stress. Thus, we conclude that the biosynthesis of trigazaphilones serves as a complementary antioxidant mechanism and defends T. guizhouense against the hydrogen peroxide that it produces to combat other fungi like Foc4.

Paper Electrode-Based Flexible Pressure Sensor for Point-of-Care Immunoassay with Digital Multimeter.

A novel paper electrode-based flexible pressure sensor modified with multiwalled carbon nanotubes was designed for point-of-care (POC) immunoassay of carcinoembryonic antigen (CEA) with digital multimeter readout. The portable POC testing device consisted of flexible pressure sensor equipped with a paper electrode and connected through syringe tubing to a single-break microplate. The immunoreaction was initially carried out on the microplate with a sandwich-type assay format using platinum nanozyme-labeled secondary antibody for the gas generation. Upon addition of hydrogen peroxide (H2O2), platinum nanozyme (catalase-like mimic) reduced it into hydrogen oxide and oxygen (O2). The overflowing oxygen gas increased the pressure of the multiwalled carbon nanotube-functionalized paper electrode in a homemade pressure-tight system, and the increased pressure could be readily monitored using the paper electrode-based flexible pressure-tight sensor with a digital multimeter readout. The detectable signal mainly derived from the resistance change of pressure sensor because of its deformation with the assistance of the as-generated gas, and the shift in the resistance could be allowed to detect the gas pressure even as low as 80 Pa. Under optimum conditions, pressure sensor-based immunoassay exhibited good resistance responses toward target CEA within a linear range of 0.5-60 ng/mL at a detection limit of 167 pg/mL. Moreover, our strategy provided acceptable reproducibility, precision, high specificity, and good accordance with the commercial CEA ELISA kit for detecting human serum specimens.

The Aspergillus nidulans Velvet-interacting protein, VipA, is involved in light-stimulated heme biosynthesis.

Filamentous fungi are able to differentiate morphologically and adapt the metabolism to internal and external cues. One major regulator is the so-called velvet protein, VeA, best studied in Aspergillus nidulans. The protein interacts with several other proteins to regulate light sensing, the balance between asexual and sexual development, penicillin biosynthesis or mycotoxin production. Here, we characterized a novel VeA-interacting protein, VipA. The 334 amino acid long protein comprises a FAR1-like DNA-binding domain, known from plant transcription factors like FHY3 (Far-red elongated hypocotyl 3). VipA interacted not only with VeA, but also with the WC orthologue LreA in the nuclei and with the phytochrome FphA in the cytoplasm. Conidia and cleistothecia formation was similarly affected in a vipA-deletion strain as in an fphA mutant. However, the effect was less pronounced, suggesting a modulating and not an essential role in light sensing. In addition, VipA modulated heme biosynthesis in response to light through association with the hemB promoter, the gene encoding 5-aminolevulinic acid dehydratase. After illumination of A. nidulans mycelia with white light the intracellular heme concentration increased by 30% in comparison to a vipA-deletion mutant. Hence, VipA couples heme biosynthesis to the illumination conditions.

Metal-Polydopamine Framework: An Innovative Signal-Generation Tag for Colorimetric Immunoassay.

In this work, an innovative enzyme-free colorimetric immunoassay was proposed for the sensitive detection of alpha-fetoprotein (AFP) by introducing thymolphthalein-modified metal-polydopamine framework (MPDA@TP) for the signal generation and amplification. Using zeolitic imidazolate framework (ZIF-67) as the template, the hollow-structured metal-polydopamine framework (MPDA) with high surface recovery and abundant groups was synthesized and functionalized with thymolphthalein (TP) molecules via typical π-stacking reaction. In the presence of target AFP, an MPDA@TP-linked immunosorbent assay (MLISA) was implemented on the capture antibody-modified microplate by using detection antibody-labeled MPDA@TP as the secondary antibody. Upon alkaline solution introduction, the coated hydrophobic TP on the MPDA was deprotonated into hydrophilic TP2- ion and dissolved in the solution, thereby resulting in the color change of the solution from nearly colorless to deep blue, and the increasing absorbance of the solution at 595 nm. Importantly, the MPDA@TP-based immunoassay could exhibit high sensitivity for the quantitative detection of target AFP on the basis of the absorbance within a linear range of 10-1000 pg mL-1 at a low detection limit of 2.3 pg mL-1. Furthermore, this system was validated preliminarily to screen human serum specimens with well-matched results for the referenced AFP ELISA kit. Taking advantage of simplicity, enzyme-free, convenience, and sensitivity, MPDA@TP-linked immunosorbent assay has the potential for the application in scientific research and clinical diagnosis.

A new enzyme immunoassay for alpha-fetoprotein in a separate setup coupling an aluminium/Prussian blue-based self-powered electrochromic display with a digital multimeter readout.

A new enzyme immunosensing platform was designed for the simple, rapid and sensitive detection of disease-related biomarkers (alpha-fetoprotein, AFP, was used as a model in this case), coupling an aluminium (Al)/Prussian blue-based self-powered electrochromic display with a digital multimeter readout.

Light-dependent gene activation in Aspergillus nidulans is strictly dependent on phytochrome and involves the interplay of phytochrome and white collar-regulated histone H3 acetylation.

The ability for light sensing is found from bacteria to humans but relies only on a small number of evolutionarily conserved photoreceptors. A large number of fungi react to light, mostly to blue light. Aspergillus nidulans also responds to red light using a phytochrome light sensor, FphA, for the control of hundreds of light-regulated genes. Here, we show that photoinduction of one light-induced gene, ccgA, occurs mainly through red light. Induction strictly depends on phytochrome and its histidine-kinase activity. Full light activation also depends on the Velvet protein, VeA. This putative transcription factor binds to the ccgA promoter in an fphA-dependent manner but independent of light. In addition, the blue light receptor LreA binds to the ccgA promoter in the dark but is released after blue or red light illumination and together with FphA modulates gene expression through histone H3 modification. LreA interacts with the acetyltransferase GcnE and with the histone deacetylase HdaA. ccgA induction is correlated to an increase of the acetylation level of lysine 9 in histone H3. Our results suggest regulation of red light-induced genes at the transcriptional level involving transcription factor(s) and epigenetic control through modulation of the acetylation level of histone H3.

Genetically shaping morphology of the filamentous fungus Aspergillus glaucus for production of antitumor polyketide aspergiolide A.

BACKGROUND: For filamentous fungi, the basic growth unit of hyphae usually makes it sensitive to shear stress which is generated from mechanical force and dynamic fluid in bioreactor, and it severely decreases microbial productions. The conventional strategies against shear-sensitive conundrum in fungal fermentation usually focus on adapting agitation, impeller type and bioreactor configuration, which brings high cost and tough work in industry. This study aims to genetically shape shear resistant morphology of shear-sensitive filamentous fungus Aspergillus glaucus to make it adapt to bioreactor so as to establish an efficient fermentation process. RESULTS: Hyphal morphology shaping by modifying polarized growth genes of A. glaucus was applied to reduce its shear-sensitivity and enhance aspergiolide A production. Degenerate PCR and genome walking were used to obtain polarized growth genes AgkipA and AgteaR, followed by construction of gene-deficient mutants by homologous integration of double crossover. Deletion of both genes caused meandering hyphae, for which, ΔAgkipA led to small but intense curves comparing with ΔAgteaR by morphology analysis. The germination of a second germ tube from conidiospore of the mutants became random while colony growth and development almost maintained the same. Morphology of ΔAgkipA and ΔAgteaR mutants turned to be compact pellet and loose clump in liquid culture, respectively. The curved hyphae of both mutants showed no remarkably resistant to glass bead grinding comparing with the wild type strain. However, they generated greatly different broth rheology which further caused growth and metabolism variations in bioreactor fermentations. By forming pellets, the ΔAgkipA mutant created a tank environment with low-viscosity, low shear stress and high dissolved oxygen tension, leading to high production of aspergiolide A (121.7 ± 2.3 mg/L), which was 82.2% higher than the wild type. CONCLUSIONS: A new strategy for shaping fungal morphology by modifying polarized growth genes was applied in submerged fermentation in bioreactor. This work provides useful information of shaping fungal morphology for submerged fermentation by genetically modification, which could be valuable for morphology improvement of industrial filamentous fungi.

Integrated navigation fusion strategy of INS/UWB for indoor carrier attitude angle and position synchronous tracking.

In some GPS failure conditions, positioning for mobile target is difficult. This paper proposed a new method based on INS/UWB for attitude angle and position synchronous tracking of indoor carrier. Firstly, error model of INS/UWB integrated system is built, including error equation of INS and UWB. And combined filtering model of INS/UWB is researched. Simulation results show that the two subsystems are complementary. Secondly, integrated navigation data fusion strategy of INS/UWB based on Kalman filtering theory is proposed. Simulation results show that FAKF method is better than the conventional Kalman filtering. Finally, an indoor experiment platform is established to verify the integrated navigation theory of INS/UWB, which is geared to the needs of coal mine working environment. Static and dynamic positioning results show that the INS/UWB integrated navigation system is stable and real-time, positioning precision meets the requirements of working condition and is better than any independent subsystem.

Integrated positioning for coal mining machinery in enclosed underground mine based on SINS/WSN.

To realize dynamic positioning of the shearer, a new method based on SINS/WSN is studied in this paper. Firstly, the shearer movement model is built and running regularity of the shearer in coal mining face has been mastered. Secondly, as external calibration of SINS using GPS is infeasible in enclosed underground mine, WSN positioning strategy is proposed to eliminate accumulative error produced by SINS; then the corresponding coupling model is established. Finally, positioning performance is analyzed by simulation and experiment. Results show that attitude angle and position of the shearer can be real-timely tracked by integrated positioning strategy based on SINS/WSN, and positioning precision meet the demand of actual working condition.

A universal dual-mode hydrogel array based on phage-DNA probe for simultaneous rapid screening and precisely quantitative detection of Escherichia coli O157:H7 in foods by the fluorescent/microfluidic chip electrophoresis methods.

Rapid and specific detection of virulent bacterial strains is a great challenge for food safety regarding large amounts of contaminated samples. Herein, a dual-mode hydrogel array biosensor was constructed to simultaneously rapidly screen and precisely quantitatively detect virulent Escherichia coli O157:H7 (E. coli O157:H7) based on a novel DNA-modified phage probe. First, E. coli O157:H7 was incubated with alginate to form the E. coli O157:H7/hydrogel premix complex. Subsequently, hydrogel formation by cross-linking upon the addition of calcium ions and phages for E. coli O157:H7 modified with a DNA primer (phage-DNA) was added to the alginate hydrogel. The DNA on the complex could trigger rolling circle amplification (RCA) to form a phage probe containing a long-chain DNA skeleton (phage@RCA-DNA). The RCA-DNA was then hybridized with the complementary DNA (cDNA) to form double-stranded DNA fragments (phage@RCA-dsDNA), which could be stained by the SYBR Green dye to emit visual green fluorescence (FL) and determined by a smartphone for rapid screening. Meanwhile, the unreacted cDNA in the supernatant could be quantitatively detected by microfluidic chip electrophoresis (MCE). The signal decrement was also proportional to the bacterial concentration. The detection limit values of E. coli O157:H7 were 50 CFU mL-1 by the FL signal and 6 CFU mL-1 by the MCE signal. The two results could be mutually corrected to decrease the false-positive results. This assay was also employed to detect virulent Salmonella Typhimurium (S. Typhimurium) using the corresponding S. Typhimurium phage@RCA-DNA probe. All these results demonstrated that the universal bioassay was suitable for simultaneous rapid screening and precisely quantitative detection of virulent bacterial strains.