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Rapid, sensitive, point-of-care detection of pathogenic bacteria is important for food safety. In this study, we developed a novel quantum dot nanobeads-labelled lateral flow immunoassay strip (QBs-labelled LFIAS) combined with strand displacement loop-mediated isothermal amplification (SD-LAMP) for quantitative Salmonella Typhimurium (ST) detection. Quantum dot nanobeads (QBs) served as fluorescence reporters, providing good detection efficiency. The customizable strand displacement (SD) probe was used in LAMP to improve the specificity of the method and prevent by-product capture. Detection was based on a sandwich immunoassay. A fluorescence strip reader measured the fluorescence intensity (FI) of the test (T) line and control (C) line. The linear detection range of the strip was 102–108 CFU·mL−1. The visual limit of detection was 103 CFU·mL−1, indicating that the system was 10-fold more sensitive than AuNPs-labelled test strips. ST specificity was analyzed in accordance with agarose gel outputs of PCR and SD-LAMP. We detected ST in foods with an acceptable recovery of 85%–110%. The method is rapid, simple, almost equipment-free, and suitable for bacterial detection in foods and for clinical diagnosis.

Escherichia coli O157:H7, Cronobacter spp., and Salmonella spp. are common food-borne pathogens in milk that may cause serious diseases. In the present study, we established a simple, rapid, and specific method to simultaneously detect viable E. coli O157:H7, Cronobacter spp., and Salmonella spp. in milk. Three specific genes, fliC from E. coli O157:H7, cgcA from Cronobacter spp., and invA from Salmonella spp., were selected and used to design primers and probes. False-positive results were eliminated with the use of a combined sodium deoxycholate (SD) and propidium monoazide (PMA) treatment. Using the optimized parameters, this SD-PMA treatment combined with multiplex real-time PCR (SD-PMA-mRT-PCR) detected E. coli O157:H7, Cronobacter spp. and Salmonella spp. respectively, at 102 cfu/mL in pure culture or artificially spiked skim milk samples. A reasonable recovery rate (from 100 to 107%) for detection of viable bacteria using the SD-PMA-mRT-PCR assay was obtained in the presence of dead bacteria at 107 cfu/mL. The SD-PMA-mRT-PCR method developed in this study can accurately detect and monitor combined contamination with E. coli O157:H7, Cronobacter spp., and Salmonella spp. in milk and milk products.

Salmonella infection could come from eating contaminated meat or raw eggs, and drinking milk or water contaminated by Salmonella enteritidis. Therefore, it is necessary to explore a fast and easy method for the detection of S. enteritidis in these diverse samples. For this purpose, a novel particle size sensing tool was designed for ultrasensitive and accurate S. enteritidis detection. This assay consisted of rolling circle amplification (RCA) with dynamic light scattering (DLS) using gold nanoparticles (AuNPs) modified with DNA probe as DNA-AuNPs as the capture surface into a hybrid RCA-DLS assay combined with asymmetric polymerase chain reaction (aPCR) and subsequent detection. Under optimal experimental conditions, the novel hybrid RCA-DLS assay combined with aPCR for S. enteritidis reached a limit of detection (LOD) as low as 3 × 10

To achieve rapid and sensitive detection of aflatoxin B

Staphylococcus aureus, S. epidermidis, S. haemolyticus, and S. hominis are important pathogenic microorganisms that cause food contamination and clinical infections. This study aimed to develop a rapid, accurate, and monitoring technique for simultaneous detection of these Staphylococcus spp. in food. A total of 19 novel candidate species-specific targets for these four Staphylococcus spp. were mined based on pangenome analysis. Four novel targets (comFA for S. aureus, group_14348 for S. epidermidis, group_26190 for S. haemolyticus, and group_26478 for S. hominis) were selected for quantitative polymerase chain reaction (qPCR). The limit of detection in pure culture without pre-enrichment were 102 colony-forming units (CFU)/mL for S. aureus, 103 CFU/mL for S. epidermidis and S. haemolyticus, and 100 CFU/mL for S. hominis, respectively. This method allowed fast, sensitive, and accurate detection of the target Staphylococcus spp. in actual samples and the presence of nontarget bacteria. The feasibility of this method was satisfying in terms of sensitivity, specificity and efficiency after evaluating 100 samples, as well as being consistent with the national standard detection method. Thus, this qPCR assay, based on novel species-specific targets, allows efficient screening for pathogenic Staphylococcus spp., and may help prevent staphylococcal contamination of food.

Rapid, accurate point-of-care testing (POCT) for pathogenic bacteria detection is the key for avoiding foodborne diseases caused by pathogens or their toxins. The lateral flow biosensor (LFB) based on clustered regularly interspaced short palindromic repeats (CRISPR/Cas) has displayed remarkable potential for pathogen diagnosis. In this study, we report a novel CRISPR/Cas12a-based fluorescence enhanced LFB in conjunction with functionalized quantum dots, combined with recombinase-assisted amplification (RAA), to establish low-cost, simple, and sensitive detection of Staphylococcus aureus, namely CRA-LFB (CRISPR/Cas-recombinase-assisted amplification based LFB). The CRA-LFB assay is characterized by Cas12a-mediated trans-cleavage activation induced by the target DNA to digest biotin-DNA probes, caused no complementarity to the capture probe immobilized on the test (T) line, and resulted in an undetectable T line fluorescence signal on LFB. The naked eye or fluorescence strip reader was used to determine the fluorescence intensity of the T and control lines. The limit of detection (at optimal conditions) was as low as 75 aM of genomic DNA, and 5.4 × 102 cfu/mL of S. aureus in pure cultures were detected. Moreover, this CRA-LFB assay can rapidly and accurately detect S. aureus in spiked and natural meat and vegetable samples. The CRA-LFB method yielded high specificity and no interference from other nontargeted bacteria. The results exhibited high-resolution, high-intensity fluorescent signals obtained within 70 min. This bacterial detection platform is simple, low cost, almost equipment-free, and fully qualified for food and clinical diagnosis onsite testing requirements.

The abundant information provided by the pan-genome analysis approach reveals the diversity among Listeria monocytogenes serotypes. The objective of this study was to mine novel target genes using pan-genome analysis for multiplex PCR detection and differentiation of the major L. monocytogenes serotypes present in food. Pan-genome analysis and PCR validation revealed a total of 10 specific targets: one for lineage I, two for serogroup I.1, one for serogroup I.2, two for lineage II, one for serogroup II.1, three for lineage III. Primers for the novel targets were highly specific in individual reactions. The detection limits were 103–104 colony-forming units (CFU)/mL in pure bacterial cultures, meeting the requirements of molecular detection. Based on these novel targets, two new “lineage” multiplex PCR assays were developed to simultaneously distinguish between three lineages (I, II, and III) and five major serotypes (1/2a, 1/2b, 1/2c, 4b, and 4c) of L. monocytogenes. The detection limits of lineage I and lineage II&III mPCRs were 0.771 pg/μL and 1.76 pg/μL genomic DNA, respectively. The specificity of the mPCRs was robustly verified using other L. monocytogenes and non-L. monocytogenes serotypes. These results suggest that the two “lineage” multiplex PCRs based on novel targets offer a promising approach for accurate, sensitive, and rapid identification of L. monocytogenes serotypes.

Emetic Bacillus cereus is a common foodborne pathogen that can cause vomiting after ingesting the contaminated food. Detection of emetic B. cereus plays an important role in food industry. In this study, a flow cytometric bead assay based on hybridization chain reaction (HCR) were developed to detect emetic B. cereus. Forward primer labeled magnetic beads (MBs) could capture single-strand DNA (ssDNA), which were generated through the asymmetric polymerase chain reaction from the partial cereulide-encoded gene of emetic B. cereus. Then the ssDNA initiated HCR to self-assemble with two fluorescent hairpin probes. Flow cytometry was then performed to monitor the HCR products of MBs. Under optimal experimental conditions, the limit of detection (LOD) for emetic B. cereus was 7.6 × 100 CFU/mL with a liner range from 7.6 × 100 CFU/mL to 7.6 × 106 CFU/mL in pure culture. The proposed strategy can be used in spiked milk with LOD of 9.2 × 102 CFU/mL without enrichment. The high specificity of the assay was verified using closely related strains, such as B. subtilis, non-emetic B. cereus. The proposed strategy offers a great promise for simple, low-cost, sensitive, and specific pathogen diagnosis in a real food matrix.

To achieve rapid and sensitive detection of aflatoxin B1 (AFB1), we developed a polydimethylsiloxane gravity-driven cyclic microfluidic chip using the two-signal mode strategy. The structural design of the chip, together with the two-wavelength quantum dot ratio fluorescence, effectively eliminates the influence of environmental factors, improves the signal stability, and ensures that the final detection result positively correlates with the target concentration. Moreover, the theoretical analysis performed for the established physical model of the three-dimensional reaction interface inside the chip confirmed the improved reaction rate of immune adsorption in the microfluidic strategy. Overall, the method exhibited a wide analytic range (0.2–500 ng mL−1), low detection limit (0.06 ng mL−1), high specificity, good precision (coefficient of variation

Cronobacter species are opportunistic pathogens associated with severe infections in immunocompromised individuals. Species differentiation is essential as the sensitivity of different species to chemical reagents, antibiotics, and virulence factors may be diverse. This study aimed to mine new molecular targets for the detection of Cronobacter strains at the species level. Using a bioinformatics approach and the online BLAST software, we obtained serial Cronobacter species-specific target genes. Primers for the selected genes were constructed, and the PCR products were evaluated using various Cronobacter species and non-Cronobacter strains. As a result, 2, 5, 7, and 5 specific target genes were found for C. sakazakii, C. malonaticus, C. dublinensis, and C. turicensis, respectively. Thereafter, 4- and 5-multiplex PCR (mPCR) was successfully developed to identify these four species based on the validated species-specific primers. Overall, our data indicated that PCR and mPCR assays have excellent specificity and sensitivity for the detection of Cronobacter species, suggesting the potential of their use in food inspection and clinical diagnosis.

Salmonella serotyping is a critical step for the identification of pathological isolates in humans and animals. Whole-genome analysis is a proven method for Salmonella serovars identification. In this study, we analysed 1668 genomic sequences of 23 serogroup B serovars and 129 genomic sequences of 80 non-target serovars to detect serovar-specific molecular targets for serogroup B (O:4 epitopes). Serogroup B is the most common serogroup found in China, whose presence is increasing in recent years. We identified five, eight, one, and seven new serovar-specific genes for Indiana, Derby, Typhimurium, and Agona serovars, respectively. We then designed specific PCR primer sets for these genes to validate our results. All target Salmonella serovars could be detected successfully by PCR in different types of food samples. The analytical sensitivity of PCR for 19 out of 21 pairs of primers was 101–103 CFU/mL and 102–105 CFU/g in pure cultures of the bacteria and artificially contaminated fresh pork samples, respectively. Furthermore, we performed a functional analysis of the newly-identified serovar-specific genes. We found that the function was unknown for most of the genes, although for some of them we were able to identify their generic functional categories (membrane protein genes and secreted proteins genes). Our findings revealed that whole-genome analysis is a valuable tool to identify new serovar-specific targets, which could be used to develop rapid and accurate molecular detection methods and provide additional information about different Salmonella serogroup B serovars.

Accurate serotyping of Salmonella is essential for epidemiological investigations and surveillance of Salmonella infections. Compared to traditional methods, polymerase chain reaction (PCR) is more sensitive and identifies Salmonella to the serotype level. Few genes have been used as a target for PCR serotyping; thus, research to identify specific targets of common serotypes is needed. The Salmonella E serogroup contains multiple pathogenic serotypes that have been associated with recent cases of human salmonellosis. Thus, this study mined new molecular targets for rapid detection of the E serogroup of Salmonella serovars. Using pan-genome analysis and the online BLAST program, specific genes of Salmonella serovars Weltevreden, London, Meleagridis, and Senftenberg were obtained. Primer sets were evaluated by PCR using target strains, 41 nontarget Salmonella serotypes and 22 non-Salmonella strains. There were 11, 5, 5, and 1 genes specific to S. Weltevreden, S. London, S. Meleagridis, and S. Senftenberg, respectively, with detection limits as low as 102 or 103 CFU/mL per assay for pure culture. Moreover, the primer sets satisfactorily identified Salmonella serotypes in artificially contaminated foods. These results demonstrated that pan-genome analysis identified new specific targets, and PCR serotyping could be a primary screening method or a supplement to the classical detection method.

Salmonella is a food-borne pathogen that causes worldwide infections in humans and animals. Over 2600 Salmonella serovars have been identified, and serogroup C1 (O:6, 7 epitopes) contains highly diverse serovars associated with high pathogenicity. Here, we aimed to develop a reliable polymerase chain reaction (PCR) method for detecting serovar-specific markers of Salmonella serogroup C1, with greater reliability and lower complexity than current methods. We identified three, two, four, and one novel genes unique to the Infantis, Rissen, Braenderup, and Virchow serovars. The PCR primers targeting these genes were specific for the target serovars. The detection limits of the PCR assays using 9 pairs of primers were 102–103 colony-forming units (CFUs)/mL and 104–105 CFU/g in pure bacterial cultures and artificially contaminated raw pork samples, respectively. No false-positive or false-negative results were obtained with 45 food samples, as determined by comparison with biochemical tests and traditional serotyping methods. The potential functions of all serovar-specific genes identified are unknown, except for one Rissen-specific gene that encodes a secreted protein. Our results imply that all 10 novel, specific genes can be considered as useful targets for detecting the Salmonella Infantis, Rissen, Braenderup, and Virchow serovars in food.

Salmonella is one of the most common pathogens that cause foodborne diseases in humans, resulting in high medical and economical costs worldwide. The aim of this study was to develop a rapid and accurate approach for simultaneous detection of viable Salmonella spp. in raw meat, including Salmonella enterica serovars Paratyphi B, S. Typhimurium, and S. Enteritidis. To reduce detection time and improve sensitivity, immunomagnetic separation (IMS) was used as a pre-concentration and separation method. In addition, false positive and false negative results were removed by combining propidium monoazide (PMA) treatment with internal amplification control. Results showed that the detection limit of IMS-PMA combined with sextuplex polymerase chain reaction (PCR) assay reached as low as 101 CFU/mL in pure culture and 102 CFU/g in spiked raw meat (ground pork and ground beef), and the total assay time took less than 6 h. Thus, the novel IMS-PMA-mPCR assay developed in this study holds promise for routine screening of viable Salmonella serovars in meat and other samples.

Cronobacter sakazakii , Staphylococcus aureus , and Bacillus cereus are important pathogens contaminating infant food products. In this study, we developed a specific, sensitive, and accurate technique for the simultaneous detection of viable C. sakazakii , S. aureus , and B. cereus in infant food products. An internal amplification control (IAC) was added to the multiplex PCR (mPCR) as indicator of false negative results that are mainly caused by the PCR inhibitors in food products. In addition, to detect only the viable bacteria, propidium monoazide (PMA) was applied to selectively suppress the PCR signal from dead cells. The limit of detection (LOD) for the viable cells with or without PMA treatment was 10 4 CFU/mL for C. sakazakii and B. cereus , and 10 2 CFU/mL for S. aureus in pure culture, showing that the PMA treatment did not influence the sensitivity. After 12 h enrichment, the PMA-mPCR-IAC assay could detect as low as 10 1 CFU/g for C. sakazakii and S. aureus , and 10 0 CFU/g for B. cereus in spiked infant food products (infant formula, noodles, milk and rice noodles). This PMA-mPCR-IAC assay we developed hold promise for the simultaneous detection of C. sakazakii , S. aureus , and B. cereus in infant food products.

A rapid, reliable, and sensitive method for the detection of Cronobacter sakazakii, a common foodborne pathogen that may cause serious neonatal disease, has been developed. In this study, a rapid real-time quantitative PCR (qPCR) assay combined with sodium deoxycholate (SD) and propidium monoazide (PMA) was developed to detect C. sakazakii contamination in powdered infant formula (PIF). This method could eliminate the interference from dead or injured bacteria. Optimization studies indicated that SD and PMA at 0.08% (wt/vol) and 5µg/mL, respectively, were the most appropriate. In addition, qPCR, PMA-qPCR, SD-PMA-qPCR, and plate count assays were used to account for the number of viable bacteria in cell suspensions that were exposed to a 55°C water bath at different length of time. As a result, the viable number by PMA-qPCR showed significantly higher than of the number from SD-PMA-qPCR or plate counts. The number of viable bacteria was consistent between SD-PMA-qPCR and traditional plate counts, which indicated that SD treatment could eliminate the interference from dead or injured cells. Using the optimized parameters, the limit of detection with the SD-PMA-qPCR assay was 3.3×102 cfu/mL and 4.4×102 cfu/g in pure culture and in spiked PIF, respectively. A similar detection limit of 5.6×102 cfu/g was obtained in the presence of the Staphylococcus aureus (107 cfu/mL). The combined SD-PMA-qPCR assay holds promise for the rapid detection of viable C. sakazakii in PIF.

Bacillus cereus is a major foodborne pathogen that can cause a type of diarrhea. Diarrheal syndrome results from the ingestion of food products contaminated with enterotoxin-producing B. cereus. This study investigated the presence of four enterotoxins genes in 130 B. cereus isolated from various food products from Jiangxi, China. The expression levels of the enterotoxin genes in three B. cereus strains of different origins were subsequently analyzed. All of the B. cereus strains harbor at least 1 enterotoxin gene, whereas 34 strains harbor 2 enterotoxin genes, 44 strains harbor 3 enterotoxin genes, and 47 strains carry all of the four enterotoxin genes. The detection rates of the cytK, hblD, nheA, and entFM enterotoxin genes in all B. cereus isolates were 71%, 43%, 92%, and 99%, respectively. Moreover, the food matrix significantly affected the expression of these enterotoxin genes. The majority of the enterotoxin genes were also downregulated in four food matrices, indicating that the relative expression of certain enterotoxin genes, especially the entFM gene, was lower in a real food matrix than in laboratory broths. Hence, these data revealed that B. cereus is a serious health hazard and that the food matrix may influence the virulence expression of B. cereus. Our results provide better insights into the physiology of the microorganism grown in food products.

Escherichia coli O157:H7 and Salmonella spp. in milk are 2 common pathogens that cause foodborne diseases. An accurate, rapid, specific method has been developed for the simultaneous detection of viable E. coli O157:H7 and Salmonella spp. in milk. Two specific genes, namely, fliC from E. coli O157:H7 and invA from Salmonella spp., were selected to design primers and probes. A combined treatment containing sodium deoxycholate (SDO) and propidium monoazide (PMA) was applied to detect viable E. coli O157:H7 and Salmonella spp. only. Traditional culture methods and SDO-PMA-multiplex real-time (mRT) PCR assay were applied to determine the number of viable E. coli O157:H7 and Salmonella spp. in cell suspensions with different proportions of dead cells. These methods revealed consistent findings regarding the detected viable cells. The detection limit of the SDO-PMA-mRT-PCR assay reached 102 cfu/mL for Salmonella spp. and 102 cfu/mL for E. coli O157:H7 in milk. The detection limit of SDO-PMA-mRT-PCR for E. coli O157:H7 and Salmonella spp. in milk was significantly similar even in the presence of 106 cfu/mL of 2 nontarget bacteria. The proposed SDO-PMA-mRT-PCR assay is a potential approach for the accurate and sensitive detection of viable E. coli O157:H7 and Salmonella spp. in milk.

At present, the detection of rotary axis is a difficult problem in the errors measurement of NC machine tool. In the paper, a method with laser tracker on the basis of multi-station and time-sharing measurement principle is proposed, and this method can rapidly and accurately detect the rotary axis. Taking the turntable measurement for example, the motion of turntable is measured by laser tracker at different base stations. The redundant equations can be established based on the large amount of measured data concerning the distance or distance variation between measuring point and base station. The coordinates of each measuring point during turntable rotation can be accurately determined by solving the equations with least square method. Then according to the error model of rotary axis, the motion error equations of each measuring point can be established, and each error of turntable can be identified. The algorithm of multi-station and time-sharing measurement is derived, and the error separation algorithm is also deduced and proved feasible by simulations. Results of experiment show that a laser tracker completes the accuracy detection of the turntable of gear grinding machine within 3 h, and each error of the turntable are identified. The simulations and experiments have verified the feasibility and accuracy of this method, and the method can satisfy the rapid and accurate detecting requirements for rotary axis of multi-axis NC machine tool.

A CW optically pumped FIR waveguide laser is demonstrated. An output power of 75 mW at wavelength of 96.5 micrometers has been obtained and harmonic mixing between microwaves and FIR radiation has been shown.