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Lanthanide-labeled fluorescent-nanoparticle immunochromatographic strips enable rapid and quantitative detection of Escherichia coli O157:H7 in food samples
Food Control 109 (2020) 106894
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Lanthanide-labeled fluorescent-nanoparticle immunochromatographic strips enable rapid and quantitative detection of Escherichia coli O157:H7 in food samples
T
Quan Wanga, MengYao Longa, CaiYun Lvb, SiPei Xina, XianGan Hana, Wei Jianga,∗ a b
Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, 518 Ziyue Road, Minhang, Shanghai, 200241, PR China School of Life and Environmental Sciences, Huangshan University, 44 Daizheng Road, Tunxi, Huangshan, 245041, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Lanthanide-labeled fluorescent nanoparticles Fluorescence immunochromatographic strips Escherichia coli O157:H7 Quantitative detection
A simple, rapid, highly sensitive, and quantitative fluorescence-based immunochromatographic test (ICT) was successfully developed for the determination of Escherichia coli O157:H7 in food samples. Europium (III) chelated nanoparticles (EuNPs) conjugated to monoclonal antibodies specific to the O-specific polysaccharide fractions of E. coli O157:H7 were used as the fluorescence reporter. The fluorescence was visually observable within 15 min under dark conditions with an ultraviolet light source, and a test-strip reader was used for quantitative analysis. Under optimal conditions, the visual limit of detection (LOD) of the strip was 104 CFU/mL, and the LOD for quantitative detection was as low as 5 × 102 CFU/mL with the test-strip reader. No crossreaction was observed in six E. coli strains with different somatic (O) antigens and five other major foodborne pathogenic strains, thus indicating the good specificity of this method. Additionally, the detection sensitivity of E. coli O157:H7 was improved to 1 CFU/mL of the original bacterial content by pre-incubation for 8 h in meat (beef, pork, and chicken) and 6 h in milk samples. The average recovery of E. coli O157:H7 spiked in pork was 84.5–103%, thus indicating the accuracy and reliability of the novel test strips. The developed EuNP-ICT strip assay is highly sensitive, specific, and replicable. This detection system provides a tool that can potentially be used to detect E. coli O157:H7 and other pathogenic bacteria in food samples.
1. Introduction Food contaminated by pathogenic bacteria causes foodborne illnesses, which pose a serious threat to human health and are therefore a major public health concern (Yang, Lin, Aljuffali, & Fang, 2017). Escherichia coli O157:H7 is listed by the United States Centers for Disease Control and Prevention (CDC) as one of the top five pathogens resulting in hospitalization. Infection with E. coli O157:H7 often leads to hemolytic uremic syndrome, bloody diarrhea, and possibly death; as many as 100 deaths are caused by E. coli O157:H7 annually in the United States alone (Cho, Bhunia, & Irudayaraj, 2015; Song, Liu, Li, & Liu, 2016; Zhang, Yan, Yang, Yu, & Wei, 2017). This pathogen enters the host via ingestion of contaminated food, such as meat, milk, and water, and the dose necessary for E. coli O157:H7 infection is extremely low (10–100 CFU of viable bacteria) (MacDonald et al., 1988; Saeedi et al., 2017). Therefore, sensitive and selective detection of E. coli O157:H7 in food is important to promote food quality and safety. Traditional culturing and colony counting methods are gold-
∗
standard approaches for detecting E. coli O157:H7; however, these methods are inconvenient, because they require considerable time and highly trained personnel (Cho et al., 2015). Other detection methods, such as polymerase chain reaction (PCR), quantitative real-time PCR (qPCR), enzyme-linked immuno-sorbent assay (ELISA), PCR-ELISA, and electrochemical immunosensors (Blais, Leggate, Bosley, & MartinezPerez, 2004; Donhauser, Niessne, & Seidel, 2011; Gordillo, Rodríguez, Werning, Bermúdez, & Rodríguez, 2014; Hu et al., 2018; Huang et al., 2016; Malorny et al., 2003), are less time consuming but require expensive equipment and involve complex procedures and relatively extended analysis times, thus restricting their application in on-site detection of E. coli O157:H7. Therefore, methods enabling faster detection with greater sensitivity should be developed. Immunochromatographic tests (ICTs) are useful tools because of their simplicity, rapidity, and low cost, as well as their efficacy in detecting single or multiple analytes ((C. Wang et al., 2018; Q. Wang, Liu, Wang, Chen, & Jiang, 2018)). The most widely used format involving strip sensors uses gold nanoparticles (AuNPs) as reporters for
Corresponding author. E-mail addresses: [email protected], [email protected] (W. Jiang).
https://doi.org/10.1016/j.foodcont.2019.106894 Received 5 July 2019; Received in revised form 11 September 2019; Accepted 13 September 2019 Available online 14 September 2019 0956-7135/ © 2019 Elsevier Ltd. All rights reserved.
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2.2. Preparation of monoclonal antibodies (McAbs) against E. coli O157:H7
colorimetric detection (Jiang et al., 2015; Lin, Pillai, Lee, & Jemere, 2019). However, AuNP-based strips have severe limitations in high sensitivity quantification, mainly because of the insufficient signal intensity of ICTs with 20–30 nm AuNPs (Huang, Aguilar, Xu, Lai, & Xiong, 2016; Omidfar, Khorsand, & Darziani, 2013). Moreover, traditional AuNP-based ICTs provide only qualitative or semi-quantitative results (yes/no or positive/negative), and visual interpretation of bands on a test strip with the naked eye is prone to human error. Recently, fluorescent nanoparticles labeled with multiple lanthanide chelates have attracted increasing research attention because of their excellent fluorescence properties, including long fluorescence lifetimes, long emission wavelengths, narrow emission spectra, large Stokes shifts, and excellent photostability (Zhang et al., 2014). These properties make lanthanide-labeled nanoparticles promising for improving the sensitivity and multiplexing capabilities of bioassays and biosensors. Europium (III)-chelated nanoparticles (EuNPs) bearing modified carboxylic acid groups perform effectively in these assays, because they can covalently conjugate with proteins, increase the stability of the fluorescent label, and decrease interference (Liang et al., 2015). Additionally, EuNPs contain thousands of fluorescent chelates within a single polystyrene shell, thereby providing a high emission fluorescence intensity and enhanced labeling efficiency (Lai et al., 2016). Here, we successfully prepared a sandwich format fluorescence strip using an EuNP as a reporter for detecting E. coli O157:H7. Use of this method together with a portable strip reader provided both qualitative and quantitative results. We evaluated the performance of the EuNPbased ICT in terms of sensitivity, linearity, specificity, and reproducibility. We also compared the accuracy of the fabricated strips with an ELISA method for E. coli O157:H7 detection.
For identification of the O157 antigen essential for the detection of E. coli O157:H7, McAbs specific to E. coli O157:H7 lipopolysaccharide (LPS) were produced as previously described (Westerman, He, Keen, Littledike, & Kwang, 1997), with modifications. Briefly, LPS was extracted from E. coli through the hot phenol-water method, and O-specific polysaccharide (O-SP) fractions were prepared by acid hydrolysis from the phenol phase of the LPS fraction, collected, and lyophilized (concentration: 40 μg/mL). Female BALB/c mice (8–10 weeks old; Slack Shanghai Laboratory Animal Co., Ltd., Shanghai, China) were injected with inactivated E. coli O157:H7 emulsified in Montanide ISA 50 V (SEPPIC, Paris, France). When the PcAb titer reached 1:10,000, the mice were sacrificed, and the spleens were collected and fused with Sp2/0 cells for hybridoma screening. E. coli O157:H7 and O-SPs were used as the coating antigens to screen positive putative hybrids by indirect-ELISA (i-ELISA), and several non-O157:H7 E. coli or other Gramnegative strains were used as negative controls. Positive hybridomas were subcloned more than three times to ensure stable antibody production. The ascitic fluid was collected 7–14 days after injection of the hybridoma cells into the BALB/c mice, and McAbs were purified with a HiTrap Protein G purification column (GE Healthcare, Pittsburgh, PA, USA). The titers of the purified McAbs were determined separately by iELISA, and concentrations were determined with a BCA protein assay kit (Thermo Fisher Scientific). 2.3. Preparation of EuNPs coupled with anti-E. coli O157:H7 McAbs EuNP and anti-E. coli O157:H7 McAb conjugates were prepared as follows. Activation of EuNPs (2 mg) was performed in 1 mL of 25 mM MES solution (pH 7.2) supplemented with EDC (0.2 mg/mL) and sulfoNHS (0.4 mg/mL) for 30 min at room temperature with gentle shaking; this was followed by centrifugation at 12,000g for 15 min at 4 °C. After removal of the supernatant, the activated EuNPs were resuspended in 200 μL of 50 mM boric acid buffer by sonication. Subsequently, 0.1 mg of the purified anti-E. coli O157:H7 McAb was mixed with the activated EuNPs, and the reaction mixture was agitated for 24 h at 4 °C. Uncoupled Ab was removed by centrifugation at 12,000g for 20 min at 4 °C. After two washes with 0.01 M phosphate-buffered saline (PBST), McAb-linked EuNPs were blocked by the addition of an equal volume of blocking solution (50 mM Tris-HCl buffer containing 0.5% BSA and 0.2% sodium azide [pH 8.0]) for 6 h at 4 °C with shaking. The conjugates were then centrifuged, dissolved in 300 μL of dilution buffer (50 mM Tris-HCl buffer containing 0.5% BSA, 0.2% sodium azide, and 0.5% polyvinyl pyrrolidone [pH 8.0]), and stored at 4 °C until use. Conjugated and unconjugated EuNPs were characterized by transmission electron microscopy (TEM).
2. Materials and methods 2.1. Bacterial strains and reagents The microorganisms used in this study were E. coli O157:H7 (ATCC35150, ATCC43889), E. coli O6 (ATCC25922), Yersinia enterocolitica (ATCC23715), Staphylococcus aureus (ATCC6538), Listeria monocytogenes (ATCC19115), and Salmonella Typhimurium (ATCC13311), which were obtained from the American Type Culture Collection Center. E. coli O1 (CVCC249), E. coli O2 (CVCC1565), E. coli O78 (CVCC1490), E. coli O26 (CVCC1540), and E. coli O111 (CVCC1450) were purchased from the China Veterinary Culture Collection Center. Streptococcus suis (HA9801) was provided by Dr. Xiangan Han (Chinese Academy of Agricultural Sciences, Shanghai Veterinary Research Institute). E. coli O157:H7 strain 038 is a clinical strain isolated from pigs; the E. coli O157 strains C017, C029, C041, and C047 were isolated from pork or pig fecal samples, and were provided by Professor Zhiyong Ma and Dr. Shaohui Wang (Chinese Academy of Agricultural Sciences, Shanghai Veterinary Research Institute). The rabbit polyclonal antibody (PcAb) against E. coli O157:H7 was produced in our laboratory. Nitrocellulose (NC) membranes, glass fibers, absorbent papers, and polyvinyl chloride membranes were purchased from Shanghai Kinbio Co., Ltd. (Shanghai, China); 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) was purchased from Thermo Fisher Scientific (Waltham, MA, USA); and N-hydroxy succinyl amine (NHS) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Glycine, goat anti-mouse IgG, 2-(N-morpholino)ethanesulfonic acid (MES), and albumin from bovine serum (BSA) were purchased from Sigma-Aldrich. EuNPs were purchased from Chengdu Micro-Rui Biotechnology Co., Ltd. (Chengdu, China). All solvents and other chemicals were of analytical reagent grade, and all solutions used in this study were prepared with ultrapure water (> 18 MΩ).
2.4. Preparation of the EuNP-based test strips The lanthanide-labeled fluorescent-NP-based lateral-flow test strips comprised four components. The sample pad, NC membrane, and absorbent pad were assembled sequentially onto a plastic backing sheet. Images of the ready-to-use test strip and the opened cassette are presented in Fig. 1a. The sample pad (20 × 200 mm) made from glass fiber was impregnated with the optimal sample-pad buffer solution (0.2% BSA and 0.2% Tween-20 in 0.01 M PBST) and dried for 24 h at 37 °C. The NC membrane (25 × 300 mm) was spotted with an XYZ biostrip dispenser (Bio-Dot, Irvine, CA, USA), and anti-E. coli O157:H7 PcAb and goat anti-mouse IgG served as the test and control lines, respectively. The absorbent pad, which was made from 100% pure cellulose fiber, was cut into strips (20 × 300 mm), and all three components were assembled on a backing plate in the correct order, with the ends of the components overlapped to achieve continuous flow from the sample pad to the absorbent pad by capillary action. The assembled product 2
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Fig. 1. (a) Components of the EuNP-based strip test and the opened cassette. (b) The fluorescence strip reader integrated with the strip shown in (a).
7.2) containing 1% BSA and tested them with the strips. The visual limit of detection (LOD) of the strip was defined as the minimum target concentration producing a fluorescence intensity at the test line that differed significantly from that produced by a blank-control strip in the absence of target bacteria. The fluorescence intensity of the test line (FIT) was recorded by the strip reader to quantitatively determine E. coli O157:H7. A standard calibration curve was established by plotting the FIT from the standard bacterial solution against the logarithmic concentrations, and the LOD for quantitative detection by the strip reader was defined as the minimum concentration of E. coli O157:H7 in the sample solution capable of producing a FIT ≥2.1 × the FIT of the blank control (solution without E. coli O157:H7). The specificity of the test strip was determined by individually testing E. coli strains with different somatic (O) antigens, including O1, O2, O6, O26, O78, O111, and O157. Additionally, we tested other bacterial non-E. coli strains, including L. monocytogenes, S. Typhimurium, Y. enterocolitica, S. suis, and S. aureus, which were prepared in sterile PBST at a high concentration of 108 CFU/mL to avoid false-negative results. E. coli O157:H7 (106 CFU/mL) was used as a positive control, and sterile PBS solution was used as the blank control. 2.7. Simulated sample detection and comparison with ELISA To estimate the applicability of the EuNP-based test strips, we first used PCR methods to validate the absence of E. coli O157:H7 in milk, beef, pork, and chicken samples purchased from local food markets. Then meat slices from the beef, pork, and chicken samples were homogenized in a blender, and all four samples were artificially contaminated with 1–10 CFU/mL E. coli O157:H7, then analyzed by ELISA and the test strips after incubation in enrichment broth. Aliquots (5 mL) from each bottle at various incubation times (0, 2, 4, 6, 8, 10, and 12 h) were collected and centrifuged at 10,000g for 5 min at 4 °C, and the pellets were re-suspended in 1 mL of sterile PBST solution. Pork samples spiked with E. coli O157:H7 at different concentrations were quantitatively analyzed with ELISA and the test strips. Additionally, strain ATCC35150 and the clinical strain 038 were added to the homogenized pork samples at a ratio of 1:20 (w/v), and 5 mL of each mixed solution was centrifuged. The pellets were re-suspended in 1 mL of sterile PBST, tested with the EuNP-ICT strips, and quantified with the strip reader. Fifty beef samples and 50 pork samples with unknown target bacteria concentrations were sourced from retail outlets in Shanghai, China. All collected samples were assayed with the EuNP-ICT strips and ELISA. Sandwich ELISA was performed as follows. First, high-binding 96well microtiter plates (Shanghai GenoMintel Medical Instrument Co., Ltd., Shanghai, China) were coated with 100 μL of anti-E. coli O157:H7 PcAb diluted in carbonate buffer (pH 9.6) and incubated overnight at 4 °C. After the wells were washed three times and blocked with 1% gelatin, standard bacterial solutions or sample solutions were added to the microtiter plates, incubated for 45 min at 37 °C, and then washed;
Fig. 2. TEM images of EuNPs solution (a) and EuNP-McAb conjugate solution (b).
was cut into 4-mm wide strips with a CM4000 cutter (Bio-Dot) and maintained as strip cassettes with silica desiccant gel, which were stored under dry conditions at room temperature until use. 2.5. Analytical procedures The EuNP-based test strips were used by transferring 100 μL of the sample solution and 50 μL of the McAb-EuNP conjugates into the sample well. After incubation for 15 min, the strip results were visualized in a dark room under an ultraviolet light source with a 365-nm filter, and the fluorescence intensity of the test line was determined with a test-strip reader (Micro-Rui, Ltd., Chengdu, China) for quantitative analysis (Fig. 1b). The reader was a portable, single-channel fluorescence lateral flow instrument, and a fixed excitation wavelength of 420 nm and an emission wavelength of 615 nm were used for measuring the EuNPs. 2.6. Evaluation of the sensitivity and specificity of the EuNP-ICT strips To confirm the sensitivity of the EuNP-ICT strips, we diluted E. coli O157:H7 samples from 107 CFU/mL to 101 CFU/mL in 0.01 M PBST (pH 3
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Fig. 3. Schematic diagram of process of detecting E. coli O157:H7 using the strip sensor. (a) Schematic illustration of the assay procedure. (b) Fluorescence detection by the strip reader. Positive: peaks appear in the control and test regions; negative: a single peak appears in the control zone.
subsequently, 100 μL of horseradish peroxidase-labeled anti-E. coli O157 McAb was added to each well and incubated at 37 °C for 45 min. After the plates were washed, reactions were detected with 100 μL of 3,3′,5,5′-tetra-methylbenzidine for 15 min, and the enzyme reaction was stopped by addition of 50 μL of 1 M H2SO4. Absorbance was measured with a microplate reader at an optical density of 450 nm (OD450), and a standard curve was obtained by plotting the OD450 values from the standard bacterial solution against the logarithmic concentrations. The visual LOD of the sandwich ELISA was defined as the minimum bacterial concentration necessary to produce an OD450 > 2.1 × that of
the blank control.
3. Results and discussion 3.1. Preparation of the McAbs and EuNP-McAb conjugates We used i-ELISA to detect hybridoma McAb production on day 8 after cell fusion. Hybridomas producing Abs capable of recognizing both E. coli O157:H7 and O-SPs were subcloned three times through the limiting-dilution method, and three hybridoma cells displaying a high 4
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Fig. 4. Optimization of immunosensor parameters. (a) Effects of PcAb concentration and dilution time of EuNP-McAb conjugates on FITs/FITb. (b) Effect of immunoreaction time on fluorescence-peak heights (left) and FITs/FITb (right).
antigen–antibody reaction, thus forming an EuNP-McAb-E. coli O157:H7-PcAb complex and resulting in visible fluorescence in the test zone (marked “T”) under an ultraviolet light source. The density of the fluorescent band is proportional to the concentration of E. coli O157:H7 in the sample. Additionally, excess EuNP–McAb conjugates flow over the test line and bind purified goat anti-mouse antibodies on the control line (marked “C”), thus forming another fluorescent band at the control line of the strip. The results can be assessed with the naked eye within 15 min under an ultraviolet light source (Fig. 3b; right): a positive result is indicated by the appearance of two red lines in the test and control regions, and a negative result is indicated by a single line in the control region. The “C” line should always be present; if no line is observed at the control position, the test should be considered invalid. For quantitative detection, the strip is subjected to fluorescence detection with the strip reader, and positive and negative results are differentiated according to the appearance of peaks in the test regions. The higher the concentration of E. coli O157:H7 in the sample solution, the more bacteria are subsequently captured by the EuNP-McAbs and the anti-E. coli O157:H7 PcAbs, thus leading to increased complex binding on the “T” line and an increased fluorescence signal, which is inversely proportional to target-bacteria concentrations in the sample (Fig. 3b; left). Therefore, the E. coli O157:H7 concentration is directly proportional to the ratios of the heights of the fluorescence peaks of the test lines produced by the sample solution and the blank control (the FITs/FITb
antibody titer (1F1, 8D3, and 6H12) were found to stably secrete McAbs. One of these hybridoma cell lines (1F1) was used for McAb preparation, yielding 11.8 mg McAb per mL. E. coli O157:H7 at 1 × 107 CFU/well resulted in highly sensitive detection of the 1F1 McAb at a titer of 1:1.6 × 105, according to i-ELISA. TEM images revealed that the EuNPs were round with a homogeneous size distribution, with an average size of 103 nm ± 1.4 nm (Fig. 2a). The carboxylic acid on the EuNPs was activated with EDC and NHS to produce an active ester, which then reacted with the amine groups on the antibody and formed an amide bond, thereby conjugating the antibodies to the EuNPs. After conjugation, the EuNP size increased, and the hydrodynamic diameter of the EuNP-McAb reached 114 nm ± 1.7 nm (Fig. 2b), thus indicating successful preparation of the EuNP-McAb conjugates.
3.2. Detection procedure and principles of EuNP-ICT Use of the designed strip sensor based on a sandwich-immunoassay format is outlined in Fig. 3a. Detection was initially performed by placing an aliquot (100 μL) of sample solution and 50 μL of anti-E. coli O157: H7 McAb-EuNP conjugate reagents onto the sample pad; the liquid moved along the NC membrane via capillary action. When E. coli O157:H7 is present in the sample, the target bacteria are captured by the anti-E. coli O157:H7 PcAb coated on the test line via an 5
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Fig. 5. Sensitivity of the EuNP-ICT strip sensor. (a) Image of the test strip (membrane area) in the presence of E. coli O157:H7 samples diluted from 107 to 101 CFU/ mL and the blank control (solution without E. coli O157:H7). (b) EuNP-ICT-detection curve for E. coli O157:H7 according to the fluorescence-strip reader. The curve illustrates the linear relationship between relative fluorescence intensity (FITs/FITb) and different concentrations of E. coli O157:H7.
sample in parallel. Each value was based on quintuplicate measurements, and the error was represented by the standard deviation. The kinetic reaction curve showed that the FITs and FITb sharply increased within the first 15 min, then equilibrated after 21 min. Similarly, the FITs/FITb ratio increased rapidly 12 min before equilibration and plateaued at 15 min, after which the ratio stabilized. Therefore, we selected 15 min as the optimum immunoreaction time for this system. These results indicate that the FITs/FITb ratio is a better parameter for quantifying E. coli O157:H7 concentration than FITs alone, on the basis of its ability to eliminate the effects of differences in immunoreaction kinetics and to shorten turnaround time.
ratio), as well as the FITs. However, the intrinsic heterogeneity of the lateral-flow test strip might adversely affect assay reliability and reproducibility when FITs alone is used for signal quantification. On the basis of this process, we used the FITs/FITb ratio for measurement. 3.3. Optimization of experimental parameters Because strip sensitivity is dependent on the fluorescence intensity of the test line, strip performance is primarily affected by the concentrations of the EuNP-McAb conjugates and the PcAbs used for coating. To increase sensitivity, we tested different dilution times for the fluorescence conjugates (from 25 to 100-fold) and different PcAb concentrations (from 0.5 to 2.5 mg/mL) and optimized strip performance according to the checkerboard-titration method, using a 106 CFU/mL E. coli O157:H7 sample and a negative sample in parallel. These parameters were selected on the basis of the ratio of the fluorescence intensity (FITs/FITb) of the E. coli O157:H7 standard sample (106 CFU/mL) to that of a blank sample without E. coli O157:H7. The FITs/FITb ratio peaked at a 35-fold dilution of EuNP-McAb conjugates and PcAb at 1.0 mg/mL (Fig. 4a); therefore, this dilution was used to coat the test line, and a 35-fold dilution of the conjugates was used in subsequent experiments. We then optimized the immunoreaction time to maximize the fluorescence signal. As shown in Fig. 4b, immunoreaction time was evaluated by monitoring FITs, FITb, and FITs/FITb over a 3- to 42- min incubation with a 106 CFU/mL E. coli O157:H7 sample and a negative
3.4. Sensitivity of the EuNP-ICT strips We evaluated the sensitivity of the EuNP-ICT strips by analyzing a series of diluted E. coli O157:H7 solutions in quintuplicate (Fig. 5a). We observed that the fluorescence intensity of the test lines increased with increasing E. coli O157:H7 concentration. Additionally, the fluorescence intensity of the test line at 104 CFU/mL of E. coli O157:H7 differed significantly from that of the blank control; therefore, we established 104 CFU/mL as the visual LOD for the EuNP-ICT strip sensor. The calibration curve obtained by plotting the relative fluorescence intensity (FITs/FITb) against the logarithm of the E. coli O157:H7 concentration (101–107 CFU/mL) suggested a good linear range for the fluorescence strips between 104 and 107 CFU/mL, according to the equation: y = 17.73x − 61.01, R2 = 0.9954 (Fig. 5b). The LOD for 6
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Fig. 6. Specificity of the EuNP-ICT strip sensor. (a) Image of fluorescence strips (membrane area) prepared using 12 standard strains:1. E.coli O157; 2. E.coli O1; 3. E.coli O2; 4. E.coli O78; 5. E.coli O6; 6. E.coli O26; 7. E.coli O111; 8. L.monocytogenes;9. S.typhimurium;10. Y.enterocolitica;11. S. suis;12. S. aureus; 13. Blank control (PBST solution). (b) Relative fluorescence intensity (FITs/FITb) of the immunosensor in the presence of 12 standard strains and blank control.
effective up to 48 weeks under proper storage.
quantitative detection was defined as the concentration of E. coli O157:H7 capable of inducing a fluorescence signal 2.1-fold higher than that of the blank (negative) control. We established the LOD for quantitative detection at 5 × 102 CFU/mL, thus suggesting that the EuNP-ICT strips had higher sensitivity than colloidal gold-based or traditional ELISA methods (Chen et al., 2015; Pang et al., 2018; Wang et al., 2016).
3.6. Sample analysis and comparison with ELISA The sensitivities of the EuNP strips and sandwich ELISA were compared with the same pair of antibodies. A standard curve was observed for the sandwich ELISA, and the LOD was found to be 105 CFU/ mL (Fig. S2), which was 200 times higher than that of the EuNP-ICT strips. Bacterial-microbiological assays are often preceded by culture enrichment to allow viable cells to grow, thereby enabling the detection of very low levels of bacteria. To verify the applicability of the EuNPICT strips, we detected simulated samples spiked with E. coli O157:H7 after different incubation periods. Generally, we observed a good correlation between the techniques. As shown in Table 1, beef, milk, pork, and chicken samples showed positive results after incubation for 8 h, 6 h, 8 h, and 8 h, respectively, and the detection sensitivity of the EuNPICT strips increased to 1 CFU/mL. The test results from three non-spiked samples incubated > 12 h were all negative. The enrichment times required to achieve positive results were 2 h longer for ELISA than the EuNP-ICT strips. Culture enrichment is time consuming and labor intensive. Recently, simple and time-saving methods, such as immunomagnetic separation, have been applied to achieve efficient and simple isolation or concentration of target bacteria from complex samples (such as food matrices), thereby decreasing total experimental
3.5. Specificity of the EuNP-ICT strips We confirmed the specificity of EuNP-ICT strips with 106 CFU/mL E. coli O157:H7 and 11 non-E. coli O157: H7 strains (108 CFU/mL). Only E. coli O157:H7 strains resulted in fluorescence in the test region, whereas no fluorescence was observed for the other non-E. coli O157:H7 strains or the blank control (PBS) (Fig. 6a). Moreover, the relative fluorescence intensity ratio (FITs/FITb) of E. coli O157:H7 was significantly higher than those of the 11 non-E. coli O157:H7 strains, which were near that of the blank control (Fig. 6b). Furthermore, six other standard and food/animal isolates of E. coli O157 were also tested with the EuNP-ICT strips and yielded a positive signal at the test line (Fig. S1). These results indicated that the EuNP-ICT strips are highly specific for detecting E. coli O157 without cross-reactivity with other strains, even at high concentrations. We investigated the strips via periodic testing of control samples and found that the strips remained 7
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concentrations of target bacteria were analyzed with the EuNP-ICT strips and the ELISA method. We obtained no positive results through either method, thus indicating that the data obtained with these two methods correlated well. ELISA is widely considered an effective tool for detecting foodborne pathogenic bacteria, such as E. coli O157:H7, in regulatory and industrial laboratories (Guo et al., 2016; Long et al., 2018; Pang et al., 2018). However, it requires a laboratory, skilled technicians, and special instruments. Moreover, measurements take ~2 h to complete, thus making rapid on-site detection difficult. Compared with ELISA, the EuNP-ICT strips described here had higher specificity and sensitivity without a need for special equipment, and the results were obtained within 15 min with a test-strip reader.
Table 1 Sample detection for E. coli O157:H7 over different incubation periods by EuNP-ICT strips and ELISA. Samples
Time
beef
0h 2h 4h 6h 8h 10 h 12 h 0h 2h 4h 6h 8h 10 h 12 h 0h 2h 4h 6h 8h 10 h 12 h 0h 2h 4h 6h 8h 10 h 12 h
milk
Pork
chicken
Method EuNP-ICT strip (n = 3) – – – – + + + – – – + + + + – – – – + + + – – – – + + +
ELISA (n = 3) – – – – – + + – – – – + + + – – – – – + + – – – – – + +
4. Conclusions Lateral-flow immunoassays are an accepted point-of-care testing technique for pathogen detection that is based on generating colorimetric signals from AuNP tracers that can be perceived by the naked eye. However, these methods often have the drawbacks of poor quantitative discrimination and low analytical sensitivity. To address these limitations, we used EuNPs as fluorescent labels conjugated to an McAb specific for the O-SP of E. coli O157:H7. Using a strip reader as a quantitative test system, we detected concentrations as low as 5 × 102 CFU/mL E. coli O157:H7 within 15 min, representing a 200fold increase in the LOD, obtained with a 2 h shorter processing time, as compared with ELISA. Additionally, the EuNP method was successfully used to detect E. coli O157:H7 in beef, milk, pork, and chicken samples, and it can potentially be used for other foods. These results suggest the efficacy of the EuNP-based ICT strips for quantitative determination of foodborne pathogens. The method might also be useful for applications such as point-of-care clinical analysis and food and agricultural field operations.
Table 2 Sample detection for different concentrations of E. coli O157:H7 by EuNP-ICT strips and ELISA. Spiked E. coli O157:H7 stains
Spiked concentration (CFU/mL) 4
ATCC35150
1.0 × 10 1.0 × 105 1.0 × 106 1.0 × 107
038
1.0 × 104 1.0 × 105 1.0 × 106 1.0 × 107
Conflicts of interest
Numbers of E. coli O157:H7 detected by the two methods (CFU/mL) EuNP-ICT strip ELISA (n = 3) (n = 5) 8.45 × 103 (84.5%) – 9.05 × 104 (90.5%) 8.81 × 104 (88.1%) 5 8.82 × 10 (88.2%) 7.79 × 105 (77.9%) 1.014 × 107 8.46 × 106 (84.6%) (101.4%) 9.03 × 103 (90.3%) – 1.03 × 105 (103%) 8.93 × 104 (89.3%) 9.16 × 105(91.6%) 7.94 × 105(79.4%) 8.72 × 106(87.2%) 9.95 × 106 (99.5%)
Neither the entire manuscript nor any part of its content has been published or accepted elsewhere. All authors have approved this submission. The authors have no conflicts of interest to declare. Our institutions have expressly agreed to the publication of this work in Food Control. All authors are jointly responsible for this publication. We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of, the manuscript entitled “Lanthanide-labeled fluorescent-nanoparticle immunochromatographic strips allow the rapid and quantitative detection of Escherichia coli O157:H7 in food samples “.
time. In our previous study, we used magnetic beads to conjugate antiE. coli O157:H7 Abs (which were also used in the present study) to form immunomagnetic beads whose capture efficiency was increased to > 70.24% for a range of 100–105 CFU/mL E. coli O157:H7 (Long et al., 2018). Coupling immunomagnetic separation with the EuNP-ICT strip might improve applicability for on-site detection; therefore, this application will be tested in a future study. We performed a recovery experiment to evaluate the accuracy and precision of the EuNP-ICT strips by using animal-derived food samples spiked with E. coli O157:H7. Ground pork was used as a sample matrix, and an initial concentration of E. coli O157:H7 was inoculated and then enumerated by colony counting in triplicate. The fortified samples with different concentrations of two strains of E. coli O157:H7 were simultaneously tested with EuNP-ICT and sandwich ELISA. The recoveries of the strips ranged from 84.5% to 103% (Table 2), thus indicating that the EuNP-ICT strips satisfied the requirements for quantitative detection of E. coli O157:H7. Therefore, this comparison demonstrated the accuracy and higher sensitivity of our EuNP-ICT strips for detecting E. coli O157:H7. Furthermore, 50 beef samples and 50 pork samples with unknown
Acknowledgments This study was financially supported by the National Key Research and Development Programs of China (no. 2016YFD0501101, 2017YFC1200201), and the Shanghai Science and Technology Commission Research Project (nos. 18140900700 and 17140900400). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.foodcont.2019.106894. References Blais, B. W., Leggate, J., Bosley, J., & Martinez-Perez, A. (2004). Comparison of fluorogenic and chromogenic assay systems in the detection of Escherichia coli O157 by a novel polymyxin-based ELISA. Letters in Applied Microbiology, 39, 516–522.
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real-time PCR. Animal Husbandry & Veterinary Medicine, 50, 68–76 (in Chinese). MacDonald, K. L., O'Leary, M. J., Cohen, M. L., Norris, P., Wells, J. G., Noll, E., et al. (1988). Escherichia coli O157:H7, an emerging gastrointestinal pathogen. Results of a one-year, prospective, population-based study. Journal of the American Medical Association, 259, 3567–3570. Malorny, B., Tassios, P. T., Rådström, P., Cook, N., Wagner, M., & Hoorfa, J. (2003). Standardization of diagnostic PCR for the detection of foodborne pathogens. International Journal of Food Microbiology, 83, 39–48. Omidfar, K., Khorsand, F., & Darziani Azizi, M. (2013). New analytical applications of gold nanoparticles as label in antibody based sensors. Biosensors and Bioelectronics, 43, 336–347. Pang, B., Zhao, C., Li, L., Song, X., Xu, K., Wang, J., et al. (2018). Development of a lowcost paper-based ELISA method for rapid Escherichia coli O157:H7 detection. Analytical Biochemistry, 542, 58–62. Saeedi, P., Yazdanparast, M., Behzadi, E., Salmanian, A. H., Mousavi, S. L., Nazarian, S., et al. (2017). A review on strategies for decreasing E. coli O157:H7 risk in animals. Microbial Pathogenesis, 103, 186–195. Song, C., Liu, J., Li, J., & Liu, Q. (2016). Dual FITC lateral flow immunoassay for sensitive detection of Escherichia coli O157:H7 in food samples. Biosensors and Bioelectronics, 85, 734–739. Wang, J., Katani, R., Li, L., Hegde, N., Roberts, E. L., Kapur, V., et al. (2016). Rapid detection of Escherichia coli O157 and shiga toxins by lateral flow immunoassays. Toxins (Basel), 25, 92. Wang, Q., Liu, C., Wang, M., Chen, Y., & Jiang, W. (2018). A multiplex immunochromatographic test using gold nanoparticles for the rapid and simultaneous detection of four nitrofuran metabolites in fish samples. Analytical and Bioanalytical Chemistry, 410, 223–233. Wang, C., Peng, J., Liu, D. F., Xing, K. Y., Zhang, G. G., Huang, Z., et al. (2018). Lateral flow immunoassay integrated with competitive and sandwich models for the detection of aflatoxin M1 and Escherichia coli O157:H7 in milk. Journal of Dairy Science, 101, 8767–8777. Westerman, R. B., He, Y., Keen, J. E., Littledike, E. T., & Kwang, J. (1997). Production and characterization of monoclonal antibodies specific for the lipopolysaccharide of Escherichia coli O157. Journal of Clinical Microbiology, 3, 679–684. Yang, S. C., Lin, C. H., Aljuffali, I. A., & Fang, J. Y. (2017). Current pathogenic Escherichia coli foodborne outbreak cases and therapy development. Rrchives of Microbiology, 1998, 811–825. Zhang, Y., Yan, C., Yang, H., Yu, J., & Wei, H. (2017). Rapid and selective detection of E. coli O157:H7 combining phagomagnetic separation with enzymatic colorimetry. Food Chemistry, 234, 332–338. Zhang, F., Zou, M. Q., Chen, Y., Li, J. F., Wang, Y. F., Qi, X. H., et al. (2014). Lanthanidelabeled immunochromatographic strips for the rapid detection of Pantoea stewartii subsp. Stewartii. Biosensors & Bioelectronics, 51, 29–35.
Chen, M., Yu, Z., Liu, D., Peng, T., Liu, K., Wang, S., et al. (2015). Dual gold nanoparticle lateral flow immunoassay for sensitive detection of Escherichia coli O157:H7. Analytica Chimica Acta, 876, 71–76. Cho, I. H., Bhunia, A., & Irudayaraj, J. (2015). Rapid pathogen detection by lateral-flow immunochromatographic assay with gold nanoparticle-assisted enzyme signal amplification. International Journal of Food Microbiology, 206, 60–66. Donhauser, S. C., Niessner, R., & Seidel, M. (2011). Sensitive quantification of Escherichia coli O157:H7, Salmonella enterica, and Campylobacter jejuni by combining stopped polymerase chain reaction with chemiluminescence flow-through DNA microarray analysis. Analytical Chemistry, 83, 3153–3160. Gordillo, R., Rodríguez, A., Werning, M. L., Bermúdez, E., & Rodríguez, M. (2014). Quantification of viable Escherichia coli O157:H7 in meat products by duplex realtime PCR assays. Meat Science, 96, 964–970. Guo, Q., Han, J. J., Shan, S., Liu, D. F., Wu, S. S., Xiong, Y. H., et al. (2016). DNA-based hybridization chain reaction and biotin-streptavidin signal amplification for sensitive detection of Escherichia coli O157:H7 through ELISA. Biosensors and Bioelectronics, 86, 990–995. Huang, X. L., Aguilar, Z. P., Xu, H. Y., Lai, W. H., & Xiong, Y. H. (2016). Membrane-based lateral flow immunochromatographic strip with nanoparticles as reporters for detection: A review. Biosensors and Bioelectronics, 75, 166–180. Huang, H., Liu, M., Wang, X., Zhang, W., Yang, D. P., Cui, L., et al. (2016). Label-free 3D Ag nanoflower-based electrochemical immunosensor for the detection of Escherichia coli O157:H7 pathogens. Nanoscale Research Letters, 11, 507. Hu, J., Huang, R., Wang, Y., Wei, X., Wang, Z., Geng, Y., et al. (2018). Development of duplex PCR-ELISA for simultaneous detection of Salmonella spp. and Escherichia coli O157: H7in food. Journal of Microbiological Methods, 154, 127–133. Jiang, W., Liu, Y. C., Chen, Y. J., Yang, Q. F., Chun, P., K, L., et al. (2015). A novel dynamic flow immunochromatographic test (DFICT) using gold nanoparticles for the serological detection of Toxoplasma gondii infection in dogs and cats. Biosensors and Bioelectronics, 72, 133–139. Lai, X. H., Liang, R. L., Liu, T. C., Dong, Z. N., Wu, Y. S., & Li, L. H. (2016). A Fluorescence immunochromatographic assay using europium (III) chelate microparticles for rapid, quantitative and sensitive detection of creatine kinase MB. Journal of Fluorescence, 26, 987–996. Liang, R. L., Xu, X. P., Liu, T. C., Zhou, J. W., Wang, X. G., Ren, Z. Q., et al. (2015). Rapid and sensitive lateral flow immunoassay method for determining alpha fetoprotein in serum using europium (III) chelate microparticles-based lateral flow test strips. Analytica Chimica Acta, 891, 277–283. Lin, D., Pillai, R. G., Lee, W. E., & Jemere, A. B. (2019). An impedimetric biosensor for E. coli O157:H7 based on the use of self-assembled gold nanoparticles and protein G. Mikrochimica Acta, 86, 169. Long, M. Y., Jiang, W., Chen, Y. J., Xiong, T., Chen, Y. J., Han, Y. R., et al. (2018). Rapid detection of Escherichia coli O157 in meat products by immunomagnetic beads and
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Lanthanide-labeled fluorescent-nanoparticle immunochromatographic strips enable rapid and quantitative detection of Escherichia coli O157:H7 in food samples
T
Quan Wanga, MengYao Longa, CaiYun Lvb, SiPei Xina, XianGan Hana, Wei Jianga,∗ a b
Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, 518 Ziyue Road, Minhang, Shanghai, 200241, PR China School of Life and Environmental Sciences, Huangshan University, 44 Daizheng Road, Tunxi, Huangshan, 245041, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Lanthanide-labeled fluorescent nanoparticles Fluorescence immunochromatographic strips Escherichia coli O157:H7 Quantitative detection
A simple, rapid, highly sensitive, and quantitative fluorescence-based immunochromatographic test (ICT) was successfully developed for the determination of Escherichia coli O157:H7 in food samples. Europium (III) chelated nanoparticles (EuNPs) conjugated to monoclonal antibodies specific to the O-specific polysaccharide fractions of E. coli O157:H7 were used as the fluorescence reporter. The fluorescence was visually observable within 15 min under dark conditions with an ultraviolet light source, and a test-strip reader was used for quantitative analysis. Under optimal conditions, the visual limit of detection (LOD) of the strip was 104 CFU/mL, and the LOD for quantitative detection was as low as 5 × 102 CFU/mL with the test-strip reader. No crossreaction was observed in six E. coli strains with different somatic (O) antigens and five other major foodborne pathogenic strains, thus indicating the good specificity of this method. Additionally, the detection sensitivity of E. coli O157:H7 was improved to 1 CFU/mL of the original bacterial content by pre-incubation for 8 h in meat (beef, pork, and chicken) and 6 h in milk samples. The average recovery of E. coli O157:H7 spiked in pork was 84.5–103%, thus indicating the accuracy and reliability of the novel test strips. The developed EuNP-ICT strip assay is highly sensitive, specific, and replicable. This detection system provides a tool that can potentially be used to detect E. coli O157:H7 and other pathogenic bacteria in food samples.
1. Introduction Food contaminated by pathogenic bacteria causes foodborne illnesses, which pose a serious threat to human health and are therefore a major public health concern (Yang, Lin, Aljuffali, & Fang, 2017). Escherichia coli O157:H7 is listed by the United States Centers for Disease Control and Prevention (CDC) as one of the top five pathogens resulting in hospitalization. Infection with E. coli O157:H7 often leads to hemolytic uremic syndrome, bloody diarrhea, and possibly death; as many as 100 deaths are caused by E. coli O157:H7 annually in the United States alone (Cho, Bhunia, & Irudayaraj, 2015; Song, Liu, Li, & Liu, 2016; Zhang, Yan, Yang, Yu, & Wei, 2017). This pathogen enters the host via ingestion of contaminated food, such as meat, milk, and water, and the dose necessary for E. coli O157:H7 infection is extremely low (10–100 CFU of viable bacteria) (MacDonald et al., 1988; Saeedi et al., 2017). Therefore, sensitive and selective detection of E. coli O157:H7 in food is important to promote food quality and safety. Traditional culturing and colony counting methods are gold-
∗
standard approaches for detecting E. coli O157:H7; however, these methods are inconvenient, because they require considerable time and highly trained personnel (Cho et al., 2015). Other detection methods, such as polymerase chain reaction (PCR), quantitative real-time PCR (qPCR), enzyme-linked immuno-sorbent assay (ELISA), PCR-ELISA, and electrochemical immunosensors (Blais, Leggate, Bosley, & MartinezPerez, 2004; Donhauser, Niessne, & Seidel, 2011; Gordillo, Rodríguez, Werning, Bermúdez, & Rodríguez, 2014; Hu et al., 2018; Huang et al., 2016; Malorny et al., 2003), are less time consuming but require expensive equipment and involve complex procedures and relatively extended analysis times, thus restricting their application in on-site detection of E. coli O157:H7. Therefore, methods enabling faster detection with greater sensitivity should be developed. Immunochromatographic tests (ICTs) are useful tools because of their simplicity, rapidity, and low cost, as well as their efficacy in detecting single or multiple analytes ((C. Wang et al., 2018; Q. Wang, Liu, Wang, Chen, & Jiang, 2018)). The most widely used format involving strip sensors uses gold nanoparticles (AuNPs) as reporters for
Corresponding author. E-mail addresses: [email protected], [email protected] (W. Jiang).
https://doi.org/10.1016/j.foodcont.2019.106894 Received 5 July 2019; Received in revised form 11 September 2019; Accepted 13 September 2019 Available online 14 September 2019 0956-7135/ © 2019 Elsevier Ltd. All rights reserved.
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2.2. Preparation of monoclonal antibodies (McAbs) against E. coli O157:H7
colorimetric detection (Jiang et al., 2015; Lin, Pillai, Lee, & Jemere, 2019). However, AuNP-based strips have severe limitations in high sensitivity quantification, mainly because of the insufficient signal intensity of ICTs with 20–30 nm AuNPs (Huang, Aguilar, Xu, Lai, & Xiong, 2016; Omidfar, Khorsand, & Darziani, 2013). Moreover, traditional AuNP-based ICTs provide only qualitative or semi-quantitative results (yes/no or positive/negative), and visual interpretation of bands on a test strip with the naked eye is prone to human error. Recently, fluorescent nanoparticles labeled with multiple lanthanide chelates have attracted increasing research attention because of their excellent fluorescence properties, including long fluorescence lifetimes, long emission wavelengths, narrow emission spectra, large Stokes shifts, and excellent photostability (Zhang et al., 2014). These properties make lanthanide-labeled nanoparticles promising for improving the sensitivity and multiplexing capabilities of bioassays and biosensors. Europium (III)-chelated nanoparticles (EuNPs) bearing modified carboxylic acid groups perform effectively in these assays, because they can covalently conjugate with proteins, increase the stability of the fluorescent label, and decrease interference (Liang et al., 2015). Additionally, EuNPs contain thousands of fluorescent chelates within a single polystyrene shell, thereby providing a high emission fluorescence intensity and enhanced labeling efficiency (Lai et al., 2016). Here, we successfully prepared a sandwich format fluorescence strip using an EuNP as a reporter for detecting E. coli O157:H7. Use of this method together with a portable strip reader provided both qualitative and quantitative results. We evaluated the performance of the EuNPbased ICT in terms of sensitivity, linearity, specificity, and reproducibility. We also compared the accuracy of the fabricated strips with an ELISA method for E. coli O157:H7 detection.
For identification of the O157 antigen essential for the detection of E. coli O157:H7, McAbs specific to E. coli O157:H7 lipopolysaccharide (LPS) were produced as previously described (Westerman, He, Keen, Littledike, & Kwang, 1997), with modifications. Briefly, LPS was extracted from E. coli through the hot phenol-water method, and O-specific polysaccharide (O-SP) fractions were prepared by acid hydrolysis from the phenol phase of the LPS fraction, collected, and lyophilized (concentration: 40 μg/mL). Female BALB/c mice (8–10 weeks old; Slack Shanghai Laboratory Animal Co., Ltd., Shanghai, China) were injected with inactivated E. coli O157:H7 emulsified in Montanide ISA 50 V (SEPPIC, Paris, France). When the PcAb titer reached 1:10,000, the mice were sacrificed, and the spleens were collected and fused with Sp2/0 cells for hybridoma screening. E. coli O157:H7 and O-SPs were used as the coating antigens to screen positive putative hybrids by indirect-ELISA (i-ELISA), and several non-O157:H7 E. coli or other Gramnegative strains were used as negative controls. Positive hybridomas were subcloned more than three times to ensure stable antibody production. The ascitic fluid was collected 7–14 days after injection of the hybridoma cells into the BALB/c mice, and McAbs were purified with a HiTrap Protein G purification column (GE Healthcare, Pittsburgh, PA, USA). The titers of the purified McAbs were determined separately by iELISA, and concentrations were determined with a BCA protein assay kit (Thermo Fisher Scientific). 2.3. Preparation of EuNPs coupled with anti-E. coli O157:H7 McAbs EuNP and anti-E. coli O157:H7 McAb conjugates were prepared as follows. Activation of EuNPs (2 mg) was performed in 1 mL of 25 mM MES solution (pH 7.2) supplemented with EDC (0.2 mg/mL) and sulfoNHS (0.4 mg/mL) for 30 min at room temperature with gentle shaking; this was followed by centrifugation at 12,000g for 15 min at 4 °C. After removal of the supernatant, the activated EuNPs were resuspended in 200 μL of 50 mM boric acid buffer by sonication. Subsequently, 0.1 mg of the purified anti-E. coli O157:H7 McAb was mixed with the activated EuNPs, and the reaction mixture was agitated for 24 h at 4 °C. Uncoupled Ab was removed by centrifugation at 12,000g for 20 min at 4 °C. After two washes with 0.01 M phosphate-buffered saline (PBST), McAb-linked EuNPs were blocked by the addition of an equal volume of blocking solution (50 mM Tris-HCl buffer containing 0.5% BSA and 0.2% sodium azide [pH 8.0]) for 6 h at 4 °C with shaking. The conjugates were then centrifuged, dissolved in 300 μL of dilution buffer (50 mM Tris-HCl buffer containing 0.5% BSA, 0.2% sodium azide, and 0.5% polyvinyl pyrrolidone [pH 8.0]), and stored at 4 °C until use. Conjugated and unconjugated EuNPs were characterized by transmission electron microscopy (TEM).
2. Materials and methods 2.1. Bacterial strains and reagents The microorganisms used in this study were E. coli O157:H7 (ATCC35150, ATCC43889), E. coli O6 (ATCC25922), Yersinia enterocolitica (ATCC23715), Staphylococcus aureus (ATCC6538), Listeria monocytogenes (ATCC19115), and Salmonella Typhimurium (ATCC13311), which were obtained from the American Type Culture Collection Center. E. coli O1 (CVCC249), E. coli O2 (CVCC1565), E. coli O78 (CVCC1490), E. coli O26 (CVCC1540), and E. coli O111 (CVCC1450) were purchased from the China Veterinary Culture Collection Center. Streptococcus suis (HA9801) was provided by Dr. Xiangan Han (Chinese Academy of Agricultural Sciences, Shanghai Veterinary Research Institute). E. coli O157:H7 strain 038 is a clinical strain isolated from pigs; the E. coli O157 strains C017, C029, C041, and C047 were isolated from pork or pig fecal samples, and were provided by Professor Zhiyong Ma and Dr. Shaohui Wang (Chinese Academy of Agricultural Sciences, Shanghai Veterinary Research Institute). The rabbit polyclonal antibody (PcAb) against E. coli O157:H7 was produced in our laboratory. Nitrocellulose (NC) membranes, glass fibers, absorbent papers, and polyvinyl chloride membranes were purchased from Shanghai Kinbio Co., Ltd. (Shanghai, China); 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) was purchased from Thermo Fisher Scientific (Waltham, MA, USA); and N-hydroxy succinyl amine (NHS) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Glycine, goat anti-mouse IgG, 2-(N-morpholino)ethanesulfonic acid (MES), and albumin from bovine serum (BSA) were purchased from Sigma-Aldrich. EuNPs were purchased from Chengdu Micro-Rui Biotechnology Co., Ltd. (Chengdu, China). All solvents and other chemicals were of analytical reagent grade, and all solutions used in this study were prepared with ultrapure water (> 18 MΩ).
2.4. Preparation of the EuNP-based test strips The lanthanide-labeled fluorescent-NP-based lateral-flow test strips comprised four components. The sample pad, NC membrane, and absorbent pad were assembled sequentially onto a plastic backing sheet. Images of the ready-to-use test strip and the opened cassette are presented in Fig. 1a. The sample pad (20 × 200 mm) made from glass fiber was impregnated with the optimal sample-pad buffer solution (0.2% BSA and 0.2% Tween-20 in 0.01 M PBST) and dried for 24 h at 37 °C. The NC membrane (25 × 300 mm) was spotted with an XYZ biostrip dispenser (Bio-Dot, Irvine, CA, USA), and anti-E. coli O157:H7 PcAb and goat anti-mouse IgG served as the test and control lines, respectively. The absorbent pad, which was made from 100% pure cellulose fiber, was cut into strips (20 × 300 mm), and all three components were assembled on a backing plate in the correct order, with the ends of the components overlapped to achieve continuous flow from the sample pad to the absorbent pad by capillary action. The assembled product 2
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Fig. 1. (a) Components of the EuNP-based strip test and the opened cassette. (b) The fluorescence strip reader integrated with the strip shown in (a).
7.2) containing 1% BSA and tested them with the strips. The visual limit of detection (LOD) of the strip was defined as the minimum target concentration producing a fluorescence intensity at the test line that differed significantly from that produced by a blank-control strip in the absence of target bacteria. The fluorescence intensity of the test line (FIT) was recorded by the strip reader to quantitatively determine E. coli O157:H7. A standard calibration curve was established by plotting the FIT from the standard bacterial solution against the logarithmic concentrations, and the LOD for quantitative detection by the strip reader was defined as the minimum concentration of E. coli O157:H7 in the sample solution capable of producing a FIT ≥2.1 × the FIT of the blank control (solution without E. coli O157:H7). The specificity of the test strip was determined by individually testing E. coli strains with different somatic (O) antigens, including O1, O2, O6, O26, O78, O111, and O157. Additionally, we tested other bacterial non-E. coli strains, including L. monocytogenes, S. Typhimurium, Y. enterocolitica, S. suis, and S. aureus, which were prepared in sterile PBST at a high concentration of 108 CFU/mL to avoid false-negative results. E. coli O157:H7 (106 CFU/mL) was used as a positive control, and sterile PBS solution was used as the blank control. 2.7. Simulated sample detection and comparison with ELISA To estimate the applicability of the EuNP-based test strips, we first used PCR methods to validate the absence of E. coli O157:H7 in milk, beef, pork, and chicken samples purchased from local food markets. Then meat slices from the beef, pork, and chicken samples were homogenized in a blender, and all four samples were artificially contaminated with 1–10 CFU/mL E. coli O157:H7, then analyzed by ELISA and the test strips after incubation in enrichment broth. Aliquots (5 mL) from each bottle at various incubation times (0, 2, 4, 6, 8, 10, and 12 h) were collected and centrifuged at 10,000g for 5 min at 4 °C, and the pellets were re-suspended in 1 mL of sterile PBST solution. Pork samples spiked with E. coli O157:H7 at different concentrations were quantitatively analyzed with ELISA and the test strips. Additionally, strain ATCC35150 and the clinical strain 038 were added to the homogenized pork samples at a ratio of 1:20 (w/v), and 5 mL of each mixed solution was centrifuged. The pellets were re-suspended in 1 mL of sterile PBST, tested with the EuNP-ICT strips, and quantified with the strip reader. Fifty beef samples and 50 pork samples with unknown target bacteria concentrations were sourced from retail outlets in Shanghai, China. All collected samples were assayed with the EuNP-ICT strips and ELISA. Sandwich ELISA was performed as follows. First, high-binding 96well microtiter plates (Shanghai GenoMintel Medical Instrument Co., Ltd., Shanghai, China) were coated with 100 μL of anti-E. coli O157:H7 PcAb diluted in carbonate buffer (pH 9.6) and incubated overnight at 4 °C. After the wells were washed three times and blocked with 1% gelatin, standard bacterial solutions or sample solutions were added to the microtiter plates, incubated for 45 min at 37 °C, and then washed;
Fig. 2. TEM images of EuNPs solution (a) and EuNP-McAb conjugate solution (b).
was cut into 4-mm wide strips with a CM4000 cutter (Bio-Dot) and maintained as strip cassettes with silica desiccant gel, which were stored under dry conditions at room temperature until use. 2.5. Analytical procedures The EuNP-based test strips were used by transferring 100 μL of the sample solution and 50 μL of the McAb-EuNP conjugates into the sample well. After incubation for 15 min, the strip results were visualized in a dark room under an ultraviolet light source with a 365-nm filter, and the fluorescence intensity of the test line was determined with a test-strip reader (Micro-Rui, Ltd., Chengdu, China) for quantitative analysis (Fig. 1b). The reader was a portable, single-channel fluorescence lateral flow instrument, and a fixed excitation wavelength of 420 nm and an emission wavelength of 615 nm were used for measuring the EuNPs. 2.6. Evaluation of the sensitivity and specificity of the EuNP-ICT strips To confirm the sensitivity of the EuNP-ICT strips, we diluted E. coli O157:H7 samples from 107 CFU/mL to 101 CFU/mL in 0.01 M PBST (pH 3
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Fig. 3. Schematic diagram of process of detecting E. coli O157:H7 using the strip sensor. (a) Schematic illustration of the assay procedure. (b) Fluorescence detection by the strip reader. Positive: peaks appear in the control and test regions; negative: a single peak appears in the control zone.
subsequently, 100 μL of horseradish peroxidase-labeled anti-E. coli O157 McAb was added to each well and incubated at 37 °C for 45 min. After the plates were washed, reactions were detected with 100 μL of 3,3′,5,5′-tetra-methylbenzidine for 15 min, and the enzyme reaction was stopped by addition of 50 μL of 1 M H2SO4. Absorbance was measured with a microplate reader at an optical density of 450 nm (OD450), and a standard curve was obtained by plotting the OD450 values from the standard bacterial solution against the logarithmic concentrations. The visual LOD of the sandwich ELISA was defined as the minimum bacterial concentration necessary to produce an OD450 > 2.1 × that of
the blank control.
3. Results and discussion 3.1. Preparation of the McAbs and EuNP-McAb conjugates We used i-ELISA to detect hybridoma McAb production on day 8 after cell fusion. Hybridomas producing Abs capable of recognizing both E. coli O157:H7 and O-SPs were subcloned three times through the limiting-dilution method, and three hybridoma cells displaying a high 4
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Fig. 4. Optimization of immunosensor parameters. (a) Effects of PcAb concentration and dilution time of EuNP-McAb conjugates on FITs/FITb. (b) Effect of immunoreaction time on fluorescence-peak heights (left) and FITs/FITb (right).
antigen–antibody reaction, thus forming an EuNP-McAb-E. coli O157:H7-PcAb complex and resulting in visible fluorescence in the test zone (marked “T”) under an ultraviolet light source. The density of the fluorescent band is proportional to the concentration of E. coli O157:H7 in the sample. Additionally, excess EuNP–McAb conjugates flow over the test line and bind purified goat anti-mouse antibodies on the control line (marked “C”), thus forming another fluorescent band at the control line of the strip. The results can be assessed with the naked eye within 15 min under an ultraviolet light source (Fig. 3b; right): a positive result is indicated by the appearance of two red lines in the test and control regions, and a negative result is indicated by a single line in the control region. The “C” line should always be present; if no line is observed at the control position, the test should be considered invalid. For quantitative detection, the strip is subjected to fluorescence detection with the strip reader, and positive and negative results are differentiated according to the appearance of peaks in the test regions. The higher the concentration of E. coli O157:H7 in the sample solution, the more bacteria are subsequently captured by the EuNP-McAbs and the anti-E. coli O157:H7 PcAbs, thus leading to increased complex binding on the “T” line and an increased fluorescence signal, which is inversely proportional to target-bacteria concentrations in the sample (Fig. 3b; left). Therefore, the E. coli O157:H7 concentration is directly proportional to the ratios of the heights of the fluorescence peaks of the test lines produced by the sample solution and the blank control (the FITs/FITb
antibody titer (1F1, 8D3, and 6H12) were found to stably secrete McAbs. One of these hybridoma cell lines (1F1) was used for McAb preparation, yielding 11.8 mg McAb per mL. E. coli O157:H7 at 1 × 107 CFU/well resulted in highly sensitive detection of the 1F1 McAb at a titer of 1:1.6 × 105, according to i-ELISA. TEM images revealed that the EuNPs were round with a homogeneous size distribution, with an average size of 103 nm ± 1.4 nm (Fig. 2a). The carboxylic acid on the EuNPs was activated with EDC and NHS to produce an active ester, which then reacted with the amine groups on the antibody and formed an amide bond, thereby conjugating the antibodies to the EuNPs. After conjugation, the EuNP size increased, and the hydrodynamic diameter of the EuNP-McAb reached 114 nm ± 1.7 nm (Fig. 2b), thus indicating successful preparation of the EuNP-McAb conjugates.
3.2. Detection procedure and principles of EuNP-ICT Use of the designed strip sensor based on a sandwich-immunoassay format is outlined in Fig. 3a. Detection was initially performed by placing an aliquot (100 μL) of sample solution and 50 μL of anti-E. coli O157: H7 McAb-EuNP conjugate reagents onto the sample pad; the liquid moved along the NC membrane via capillary action. When E. coli O157:H7 is present in the sample, the target bacteria are captured by the anti-E. coli O157:H7 PcAb coated on the test line via an 5
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Fig. 5. Sensitivity of the EuNP-ICT strip sensor. (a) Image of the test strip (membrane area) in the presence of E. coli O157:H7 samples diluted from 107 to 101 CFU/ mL and the blank control (solution without E. coli O157:H7). (b) EuNP-ICT-detection curve for E. coli O157:H7 according to the fluorescence-strip reader. The curve illustrates the linear relationship between relative fluorescence intensity (FITs/FITb) and different concentrations of E. coli O157:H7.
sample in parallel. Each value was based on quintuplicate measurements, and the error was represented by the standard deviation. The kinetic reaction curve showed that the FITs and FITb sharply increased within the first 15 min, then equilibrated after 21 min. Similarly, the FITs/FITb ratio increased rapidly 12 min before equilibration and plateaued at 15 min, after which the ratio stabilized. Therefore, we selected 15 min as the optimum immunoreaction time for this system. These results indicate that the FITs/FITb ratio is a better parameter for quantifying E. coli O157:H7 concentration than FITs alone, on the basis of its ability to eliminate the effects of differences in immunoreaction kinetics and to shorten turnaround time.
ratio), as well as the FITs. However, the intrinsic heterogeneity of the lateral-flow test strip might adversely affect assay reliability and reproducibility when FITs alone is used for signal quantification. On the basis of this process, we used the FITs/FITb ratio for measurement. 3.3. Optimization of experimental parameters Because strip sensitivity is dependent on the fluorescence intensity of the test line, strip performance is primarily affected by the concentrations of the EuNP-McAb conjugates and the PcAbs used for coating. To increase sensitivity, we tested different dilution times for the fluorescence conjugates (from 25 to 100-fold) and different PcAb concentrations (from 0.5 to 2.5 mg/mL) and optimized strip performance according to the checkerboard-titration method, using a 106 CFU/mL E. coli O157:H7 sample and a negative sample in parallel. These parameters were selected on the basis of the ratio of the fluorescence intensity (FITs/FITb) of the E. coli O157:H7 standard sample (106 CFU/mL) to that of a blank sample without E. coli O157:H7. The FITs/FITb ratio peaked at a 35-fold dilution of EuNP-McAb conjugates and PcAb at 1.0 mg/mL (Fig. 4a); therefore, this dilution was used to coat the test line, and a 35-fold dilution of the conjugates was used in subsequent experiments. We then optimized the immunoreaction time to maximize the fluorescence signal. As shown in Fig. 4b, immunoreaction time was evaluated by monitoring FITs, FITb, and FITs/FITb over a 3- to 42- min incubation with a 106 CFU/mL E. coli O157:H7 sample and a negative
3.4. Sensitivity of the EuNP-ICT strips We evaluated the sensitivity of the EuNP-ICT strips by analyzing a series of diluted E. coli O157:H7 solutions in quintuplicate (Fig. 5a). We observed that the fluorescence intensity of the test lines increased with increasing E. coli O157:H7 concentration. Additionally, the fluorescence intensity of the test line at 104 CFU/mL of E. coli O157:H7 differed significantly from that of the blank control; therefore, we established 104 CFU/mL as the visual LOD for the EuNP-ICT strip sensor. The calibration curve obtained by plotting the relative fluorescence intensity (FITs/FITb) against the logarithm of the E. coli O157:H7 concentration (101–107 CFU/mL) suggested a good linear range for the fluorescence strips between 104 and 107 CFU/mL, according to the equation: y = 17.73x − 61.01, R2 = 0.9954 (Fig. 5b). The LOD for 6
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Fig. 6. Specificity of the EuNP-ICT strip sensor. (a) Image of fluorescence strips (membrane area) prepared using 12 standard strains:1. E.coli O157; 2. E.coli O1; 3. E.coli O2; 4. E.coli O78; 5. E.coli O6; 6. E.coli O26; 7. E.coli O111; 8. L.monocytogenes;9. S.typhimurium;10. Y.enterocolitica;11. S. suis;12. S. aureus; 13. Blank control (PBST solution). (b) Relative fluorescence intensity (FITs/FITb) of the immunosensor in the presence of 12 standard strains and blank control.
effective up to 48 weeks under proper storage.
quantitative detection was defined as the concentration of E. coli O157:H7 capable of inducing a fluorescence signal 2.1-fold higher than that of the blank (negative) control. We established the LOD for quantitative detection at 5 × 102 CFU/mL, thus suggesting that the EuNP-ICT strips had higher sensitivity than colloidal gold-based or traditional ELISA methods (Chen et al., 2015; Pang et al., 2018; Wang et al., 2016).
3.6. Sample analysis and comparison with ELISA The sensitivities of the EuNP strips and sandwich ELISA were compared with the same pair of antibodies. A standard curve was observed for the sandwich ELISA, and the LOD was found to be 105 CFU/ mL (Fig. S2), which was 200 times higher than that of the EuNP-ICT strips. Bacterial-microbiological assays are often preceded by culture enrichment to allow viable cells to grow, thereby enabling the detection of very low levels of bacteria. To verify the applicability of the EuNPICT strips, we detected simulated samples spiked with E. coli O157:H7 after different incubation periods. Generally, we observed a good correlation between the techniques. As shown in Table 1, beef, milk, pork, and chicken samples showed positive results after incubation for 8 h, 6 h, 8 h, and 8 h, respectively, and the detection sensitivity of the EuNPICT strips increased to 1 CFU/mL. The test results from three non-spiked samples incubated > 12 h were all negative. The enrichment times required to achieve positive results were 2 h longer for ELISA than the EuNP-ICT strips. Culture enrichment is time consuming and labor intensive. Recently, simple and time-saving methods, such as immunomagnetic separation, have been applied to achieve efficient and simple isolation or concentration of target bacteria from complex samples (such as food matrices), thereby decreasing total experimental
3.5. Specificity of the EuNP-ICT strips We confirmed the specificity of EuNP-ICT strips with 106 CFU/mL E. coli O157:H7 and 11 non-E. coli O157: H7 strains (108 CFU/mL). Only E. coli O157:H7 strains resulted in fluorescence in the test region, whereas no fluorescence was observed for the other non-E. coli O157:H7 strains or the blank control (PBS) (Fig. 6a). Moreover, the relative fluorescence intensity ratio (FITs/FITb) of E. coli O157:H7 was significantly higher than those of the 11 non-E. coli O157:H7 strains, which were near that of the blank control (Fig. 6b). Furthermore, six other standard and food/animal isolates of E. coli O157 were also tested with the EuNP-ICT strips and yielded a positive signal at the test line (Fig. S1). These results indicated that the EuNP-ICT strips are highly specific for detecting E. coli O157 without cross-reactivity with other strains, even at high concentrations. We investigated the strips via periodic testing of control samples and found that the strips remained 7
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concentrations of target bacteria were analyzed with the EuNP-ICT strips and the ELISA method. We obtained no positive results through either method, thus indicating that the data obtained with these two methods correlated well. ELISA is widely considered an effective tool for detecting foodborne pathogenic bacteria, such as E. coli O157:H7, in regulatory and industrial laboratories (Guo et al., 2016; Long et al., 2018; Pang et al., 2018). However, it requires a laboratory, skilled technicians, and special instruments. Moreover, measurements take ~2 h to complete, thus making rapid on-site detection difficult. Compared with ELISA, the EuNP-ICT strips described here had higher specificity and sensitivity without a need for special equipment, and the results were obtained within 15 min with a test-strip reader.
Table 1 Sample detection for E. coli O157:H7 over different incubation periods by EuNP-ICT strips and ELISA. Samples
Time
beef
0h 2h 4h 6h 8h 10 h 12 h 0h 2h 4h 6h 8h 10 h 12 h 0h 2h 4h 6h 8h 10 h 12 h 0h 2h 4h 6h 8h 10 h 12 h
milk
Pork
chicken
Method EuNP-ICT strip (n = 3) – – – – + + + – – – + + + + – – – – + + + – – – – + + +
ELISA (n = 3) – – – – – + + – – – – + + + – – – – – + + – – – – – + +
4. Conclusions Lateral-flow immunoassays are an accepted point-of-care testing technique for pathogen detection that is based on generating colorimetric signals from AuNP tracers that can be perceived by the naked eye. However, these methods often have the drawbacks of poor quantitative discrimination and low analytical sensitivity. To address these limitations, we used EuNPs as fluorescent labels conjugated to an McAb specific for the O-SP of E. coli O157:H7. Using a strip reader as a quantitative test system, we detected concentrations as low as 5 × 102 CFU/mL E. coli O157:H7 within 15 min, representing a 200fold increase in the LOD, obtained with a 2 h shorter processing time, as compared with ELISA. Additionally, the EuNP method was successfully used to detect E. coli O157:H7 in beef, milk, pork, and chicken samples, and it can potentially be used for other foods. These results suggest the efficacy of the EuNP-based ICT strips for quantitative determination of foodborne pathogens. The method might also be useful for applications such as point-of-care clinical analysis and food and agricultural field operations.
Table 2 Sample detection for different concentrations of E. coli O157:H7 by EuNP-ICT strips and ELISA. Spiked E. coli O157:H7 stains
Spiked concentration (CFU/mL) 4
ATCC35150
1.0 × 10 1.0 × 105 1.0 × 106 1.0 × 107
038
1.0 × 104 1.0 × 105 1.0 × 106 1.0 × 107
Conflicts of interest
Numbers of E. coli O157:H7 detected by the two methods (CFU/mL) EuNP-ICT strip ELISA (n = 3) (n = 5) 8.45 × 103 (84.5%) – 9.05 × 104 (90.5%) 8.81 × 104 (88.1%) 5 8.82 × 10 (88.2%) 7.79 × 105 (77.9%) 1.014 × 107 8.46 × 106 (84.6%) (101.4%) 9.03 × 103 (90.3%) – 1.03 × 105 (103%) 8.93 × 104 (89.3%) 9.16 × 105(91.6%) 7.94 × 105(79.4%) 8.72 × 106(87.2%) 9.95 × 106 (99.5%)
Neither the entire manuscript nor any part of its content has been published or accepted elsewhere. All authors have approved this submission. The authors have no conflicts of interest to declare. Our institutions have expressly agreed to the publication of this work in Food Control. All authors are jointly responsible for this publication. We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of, the manuscript entitled “Lanthanide-labeled fluorescent-nanoparticle immunochromatographic strips allow the rapid and quantitative detection of Escherichia coli O157:H7 in food samples “.
time. In our previous study, we used magnetic beads to conjugate antiE. coli O157:H7 Abs (which were also used in the present study) to form immunomagnetic beads whose capture efficiency was increased to > 70.24% for a range of 100–105 CFU/mL E. coli O157:H7 (Long et al., 2018). Coupling immunomagnetic separation with the EuNP-ICT strip might improve applicability for on-site detection; therefore, this application will be tested in a future study. We performed a recovery experiment to evaluate the accuracy and precision of the EuNP-ICT strips by using animal-derived food samples spiked with E. coli O157:H7. Ground pork was used as a sample matrix, and an initial concentration of E. coli O157:H7 was inoculated and then enumerated by colony counting in triplicate. The fortified samples with different concentrations of two strains of E. coli O157:H7 were simultaneously tested with EuNP-ICT and sandwich ELISA. The recoveries of the strips ranged from 84.5% to 103% (Table 2), thus indicating that the EuNP-ICT strips satisfied the requirements for quantitative detection of E. coli O157:H7. Therefore, this comparison demonstrated the accuracy and higher sensitivity of our EuNP-ICT strips for detecting E. coli O157:H7. Furthermore, 50 beef samples and 50 pork samples with unknown
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