•A natural E. coli O157:H7 specific bacteriophage was isolated and purified.•No interference from S. aureus was observed.•The method was applied to detect E. coli O157:H7 in spiked skim milk samples.•It was the first time to utilize a natural phage for E. coli O157:H7 detection.Mammal IgG antibodies are normally used in conventional immunoassays for E. coli O157:H7, which could lead to false positive results from the presence of protein A producing S. aureus. In this study, a natural specific bacteriophage was isolated and then conjugated with magnetic beads as a capture element in a sandwich format for the rapid and selective detection of E. coli O157:H7. To the best of our knowledge, it was the first time to utilize a natural bacteriophage to develop a phagomagnetic separation combined with colorimetric assay for E. coli O157:H7. The method has an overall time less than 2 h with a detection limit of 4.9 × 104 CFU/mL. No interference from S. aureus was observed. Furthermore, the proposed method was successfully applied to detect E. coli O157:H7 in spiked skim milk. The proposed detection system provided a potential method for E. coli O157:H7 and other pathogenic bacteria in food samples.

•A broad-host-range S. aureus-specific bacteriophage was isolated and purified.•The method circumvented the interference of other bacteria such as Streptococcus.•The method was successfully applied to detect S. aureus in spiked apple juice.•S. aureus was double-recognized by phage and IgG sensitively and specifically.A Staphylococcus aureus (S. aureus)-specific lytic bacteriophage P-S. aureus-9, isolated from an environmental water sample, was assembled on magnetic beads for capturing S. aureus from samples through magnetic separation. Horseradish Peroxidase (HRP) labeled immunoglobulin (IgG) antibodies were used to detect the captured S. aureus by reacting with protein A on S. aureus followed by colorimetric signals, which were generated from the catalytic reaction between HRP and the substrate 3,3′,5,5′-Tetramethylbenzidine (TMB). Under optimal conditions, the calibration curve was linear from 1.0×104 to 1.0×106 CFU mL−1. The limit of detection (LOD) for the assay was 2.47×103 CFU mL−1 and 8.86×103 CFU mL−1 of S. aureus in PBS and apple juice, respectively. Moreover, the whole assay revealed outstanding specificity towards S. aureus, without any interference of common pathogenic bacteria, and can be completed within 90 min without any pre-enrichment. As far as known, it was the first time to detect S. aureus based on the double site recognition of bacteriophage and mammal IgG. The novel approach has shown good potentials for a rapid, specific, cheap and simple detection of S. aureus in food samples.Download high-res image (205KB)Download full-size image

Extracting DNA from Staphylococcus aureus cells is important for detecting MRSA by PCR. However, S. aureus cells are known to be difficult to disrupt due to their compact cell walls. Here, we systematically studied the efficiency of a highly active lysin ClyH for extracting DNA of S. aureus in comparison with commonly used enzymes, such as lysostaphin and achromopeptidase (ACP), and its compatibility in quantitative PCR (qPCR) detection of MRSA. qPCR analysis of S. aureus specific gene femB showed that ClyH was much faster than lysostaphin, ACP and lysozyme for releasing DNA. Five minutes disruption with ClyH at room temperature was enough to release all the DNA from S. aureus. Analysis of the spiked nasal swabs by a dual qPCR assay of the β-lactam resistance mecA gene and the staphylococcal cassette chromosome (SCCmec)–open reading frame X (orfX) junction (SCCmec–orfX) after ClyH lysis showed 100 % sensitivity and specificity to the commercial BD GeneOhm™ MRSA test with ACP lysis, but the lysis time was reduced from 20 min by ACP to 5 min by ClyH. Our research shows that ClyH could be a better option than the currently used enzymes for DNA extraction from S. aureus, which can provide simpler and faster PCR detection of MRSA.

•A method was developed for detecting S. aureus in milk by coupling immunomagnetic separation with enzyme linked cell binding domain (CBD) of lysin.•Millions of binding sites present on S. aureus for CBD greatly improved the detection sensitivity.•The method is rapid with the testing time only 1.5 h.•By this method, 400 CFU of S. aureus can be detected in 100 μl milk.•The method is simple, rapid and sensitive, and could be used for the detecting of S. aureus in various food samples.Staphylococcus aureus (S. aureus) is an important food-borne pathogen in dairy products contaminated through raw ingredients or improper food handling. Rapid detection of S. aureus with high sensitivity is of significance for food quality and safety. In this study, a new method was developed for detecting S. aureus in milk by coupling immunomagnetic separation with enzyme linked cell wall binding domain (CBD) of lysin plyV12, which can bind to S. aureus with high affinity. There are millions of binding sites present on the cell surface of S. aureus for the CBD attachment, which greatly improves the detection sensitivity. The method has the overall testing time of only 1.5 h with the detection limit of 4×103 CFU/mL in spiked milk. Because it is simple, rapid and sensitive, this method could be used for the detection of S. aureus in various food samples.

The novel phage lysin PlySs2, is reported to be highly active against various bacteria, including staphylococci, streptococci and Listeria. However, the molecular mechanisms underlying its broad lytic spectrum remain to be established. In the present study, the lytic activity of the catalytic domain (CD, PlySc) and binding specificity of the cell wall binding domain (CBD, PlySb) of PlySs2 were examined. Our results showed that PlySc alone maintains very limited lytic activity. Enhanced green fluorescent protein (EGFP)-fused PlySb displayed high binding affinity to the streptococcal strains tested, including S. suis, S. dysgalactiae, and S. agalactiae, but not staphylococci, supporting its utility as a good CBD donor for streptococcal-targeted lysin engineering. EGFP-fused intact PlySs2 similarly displayed high affinity for streptococci, but not staphylococci. Notably, four truncated PlySb fragments showed no binding capacity. These findings collectively indicate that integrity of the PlySc and PlySb domains is an essential determinant of the broad lytic activity of PlySs2.

There is an urgent need for convenient, sensitive, and specific methods to detect the spores of Bacillus anthracis, the causative agent of anthrax, because of the bioterrorism threat posed by this bacterium. In this study, we firstly develop a super-paramagnetic lateral-flow immunological detection system for B. anthracis spores. This system involves the use of a portable magnetic assay reader, super-paramagnetic iron oxide particles, lateral-flow strips and two different monoclonal antibodies directed against B. anthracis spores. This detection system specifically recognises as few as 400 pure B. anthracis spores in 30 min. This system has a linear range of 4×103–106 CFU ml−1 and reproducible detection limits of 200 spores mg−1 milk powder and 130 spores mg−1 soil for simulated samples. In addition, this approach shows no obvious cross-reaction with other related Bacillus spores, even at high concentrations, and has no significant dependence on the duration of the storage of the immunological strips. Therefore, this super-paramagnetic lateral-flow immunological detection system is a promising tool for the rapid and sensitive detection of Bacillus anthracis spores under field conditions.

Due to the complicated media, monitoring proteases in real physiological environments is still a big challenge. Bioluminescence resonance energy transfer (BRET) is one of the promising techniques but its application is limited by the susceptibility to buffer composition, which might cause serious errors for the assay. Herein we report a novel combination of BRET pair with humanized Gaussia luciferase (hGluc) and highly bright red fluorescence protein tdTomato for sensitive and robust protease activity determination. As a result, the hGluc/tdTomato BRET pair showed much better tolerance to buffer composition, pH and sample matrices, and wide spectral separation (Δλ:∼110 nm). With the protease sensor built with this pair, the detection limit for enterokinase reached 2.1 pM in pure buffer and 3.3 pM in 3% serum. The proposed pair would find broad use in both in vitro and in vivo assays, especially for samples with complicated matrix.Highlights► Highly bright hGluc and tdTomato were genetically linked as a BRET pair. ► hGluc/tdTomato BRET pair showed high tolerance to buffer components and pH. ► The pair possessed good spectral resolution. ► The BRET sensor showed high sensitivity when detecting enterokinase in vitro. ► The BRET sensor sensitively detected enterokinase in serum.

The effects of two C60 derivatives: C60[C(COOH)2]3 and C60(NH2)x(OH)y, have been investigated on the bioluminescence of humanized Gaussia luciferase (hGluc) for the first time. Utilizing the dual properties of carboxyl C60 with luminescence quenching and free radicals scavenging, a novel BRET system was constructed for protease detection with high sensitivity.

Bioluminescence resonance energy transfer (BRET) has gained favors in recent years as a detection technology for protease activity due to its extreme reliability, high sensitivity and low intrinsic backgrounds. Because of the sensitivity of the donors, substrates and the acceptors, it is expected that BRET systems are sensitive to buffer environments. However, no systematic study has been reported on how buffer components would affect the BRET ratio, and thus affect the determination of protease activity based on BRET. We present here that several environmental factors, including buffer agents, pH and divalent metal ions, influenced BRET ratio significantly, when humanized Gaussia luciferase (hGluc) was utilized as the donor and enhanced yellow fluorescence protein (EYFP) as the acceptor. Based on these findings, an enhancing solution was optimized to improve the performance of the BRET sensor for analysis of enterokinase activity in vitro, resulting in 10-fold and 7-fold improvement of the sensitivity and the detection limit, respectively. We anticipate the system will be applicable for improving performance of other in vitro BRET protease sensors, especially when the optimal conditions for protease activity would severely affect the BRET signal.Graphical abstract.Highlights► We studied some buffer factors affecting BRET ratio for the first time. ► hGluc and EYFP were genetically linked by protease cleavable fragments. ► An optimized buffer improved 10-fold sensitivity, 7-fold limit of protease detection. ► The enhancing buffer optimized the protease reaction and the BRET simultaneously.

Ebola virus (EBOV) and Marburg virus (MARV), belonging to the Filoviridae family, emerged four decades ago and caused severe viral hemorrhagic fever in human and other primates. As high as 50–90% mortality, filoviruses can cause significant threats to public health. However, so far no specific and efficient vaccine has been available, nor have other treatment methods proved to be effective. It is of great importance to detect these pathogens specific, rapidly and sensitively in order to control future filovirus outbreaks. Here, recent progresses in the development of detection and diagnosis methods for EBOV and MARV are summarized.

Biosynthesis of nanoparticles inside S. aureus cells has enhanced the sensitivity of immunoassays based on the S. aureus nanoparticles. However, the current methods are limited to antigen detection by conjugating IgG antibodies on S. aureus nanoparticles. In this study, a simple way to conjugate antigens to the S. aureus nanobioparticles was developed by utilizing a cell wall binding domain (CBD) from a bacteriophage lysin PlyV12. Based on this novel design, simple agglutination tests of the IgG antibodies of Ebola virus (EBOV) nucleoprotein (NP) and Middle East Respiratory Virus (MERS) NP in rabbit sera were successfully developed by conjugating the S. aureus nanobioparticles with two fusion proteins EBOV NP- CBD and MERS NP-CBD, respectively. The conjugation was done easily by just mixing the fusion proteins with the S. aureus nanoparticles. The detection time was within 20 min without any special equipment or expertise. As far as we know, this is the first time to realize the detection of viral antibodies based on S. aureus nanoparticles.

•Red and fluorescent S. aureus nanobioparticles were simply synthesized.•The novel particles are dispersed and naturally homogenous with a size of 800 nm.•100-fold sensitivity enhancement was achieved in agglutination test based on the particles.•The E. coli O157:H7 agglutination test LOD lowered to 2.5 ∗ 104 CFU/ml by fluorescence detection.In this study, a simple and rapid method was developed to transform protein A producing Staphylococcus aureus cells into red color and red fluorescent nanobioparticles, which were homogeneous, dispersive and relatively stable with a uniform size of 800 nm. The method consists of reaction with a monotetrazolium redox dye at 25 °C for 15 min and heat inactivation at 65 °C for 30 min. This method provided the first S. aureus nanobioparticles with the dual property of red color and red fluorescence. Attributed to the IgG binding site known as protein A on their surface, the nanobioparticles could be used as vectors for immunoassays of many bacteria and viruses. Coagglutination test of Escherichia coli O157:H7 observed by naked eyes showed that the detection limitation of the nanobioprobes was 1 ∗ 106 CFU/ml, which was about 100 times more sensitive than the natural uncolored S. aureus bioprobes. Red fluorescence detection and analysis of the coagglutination product by a microplate reader lowered the detection limit to 2.5 ∗ 104 CFU/ml.

The effects of two C60 derivatives: C60[C(COOH)2]3 and C60(NH2)x(OH)y, have been investigated on the bioluminescence of humanized Gaussia luciferase (hGluc) for the first time. Utilizing the dual properties of carboxyl C60 with luminescence quenching and free radicals scavenging, a novel BRET system was constructed for protease detection with high sensitivity.