Rapid detection of Escherichia coli O157:H7 by a fluorescent microsphere-based immunochromatographic assay and immunomagnetic separation

Accepted Manuscript Rapid detection of Escherichia coli O157:H7 by a fluorescent microsphere-based immunochromatographic assay and immunomagnetic separation Qianru Li, Yuexi Yang, Fei Hu, Yanxue Cai, Xiaoyun Liu, Xiaowei He PII:

S0003-2697(18)30898-4

DOI:

10.1016/j.ab.2018.10.009

Reference:

YABIO 13153

To appear in:

Analytical Biochemistry

Received Date: 25 August 2018 Revised Date:

30 September 2018

Accepted Date: 7 October 2018

Please cite this article as: Q. Li, Y. Yang, F. Hu, Y. Cai, X. Liu, X. He, Rapid detection of Escherichia coli O157:H7 by a fluorescent microsphere-based immunochromatographic assay and immunomagnetic separation, Analytical Biochemistry (2018), doi: https://doi.org/10.1016/j.ab.2018.10.009. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

Rapid detection of Escherichia coli O157:H7 by a fluorescent microsphere-based immunochromatographic assay and immunomagnetic separation

School of Food Science and Engineering, South China University of Technology,

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Qianru Li a, c, Yuexi Yang b, Fei Hu b, Yanxue Cai a, Xiaoyun Liu b, Xiaowei He a,*

b

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Guangzhou 510640, China

National & Local United Engineering Lab of Rapid Diagnostic Test, Guangzhou Wondfo Biotech Co., Ltd., Guangzhou 5l0663, China

College of Fisheries, Guangdong Ocean University, Zhanjiang 524088, China

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* Corresponding author.

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E-mail address: [email protected]

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Abstract

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To ensure food safety and avoid infections by Escherichia coli O157:H7 (E. coli

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O157:H7),

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immunochromatography assay (FM-ICA). FMs were conjugated to anti-E. coli O157:H7

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monoclonal antibody (MAb) as an ICA probe, Immunomagnetic beads (IM-beads) were

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prepared by conjugating functionalized magnetic microspheres with the antibody for

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enrichment and separation of pathogenic bacteria from complex food matrices. Under selected

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conditions, a standard curve of FM-ICA measurement of E. coli O157:H7 was developed with

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a linear range from 3 × 105 to 6 × 107 colony-forming units (CFU)/mL in PBS buffer. The

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recoveries of intra- and inter assay ranged from 101.64% to 107.03% and 95.62% to 110.2%

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respectively, with CV below 10%. The FM-ICA showed good sensitivity, accuracy and

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precision. When IM-beads separation plus FM-ICA (IMS-FICA) were used to assay raw food

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samples, sensitivity was 3 × 103 CFU/mL, a 33-fold improvement compared with FM-ICA

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only. Moreover, this method had high specificity for E. coli O157:H7 and can be used to

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assay E. coli O157:H7 in beef, milk and water samples. This assay can be completed within 2

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h and has great potential for on-site quantitation of E. coli O157:H7 with simplicity, rapidity,

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sensitivity, and cost-effectiveness.

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Keywords Fluorescent microsphere; immunochromatographic assay; immunomagnetic

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separation; Escherichia coli O157:H7; rapid detection

developed

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novel

fluorescent

microsphere

(FM)-based

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1. Introduction

Escherichia coli O157:H7 （E. coli O157:H7）is an important serotype of Shiga

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toxin-producing Escherichia coli (STEC), which can infect people and cause diarrhea,

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hemorrhagic enteritis, thrombotic thrombocytopenic purpura, and hemolytic uremic syndrome

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（HUS） [1]. Outbreaks have been reported in China, USA, Canada, Australia, Japan and

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many European countries [2]. From 2008 to 2014, there were 5955 cases of STEC-infection

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in European Union, of which 46.3% of the cases were caused by serotype O157 [3].

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According to the US Centers for Disease Control and Prevention (CDC), outbreaks of

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STEC-O157:H7 infection linked to soynut butter products have been reported from January to

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April 2017, which caused 32 people ill, with 9 HUS [4]. Recently, another outbreak linked to

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romaine lettuce in May 2018 had been reported by the US CDC, which caused 197

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individuals infected, 89 people hospitalized, with 26 HUS, and 5 deaths [5]. Usually, the main

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reservoirs of E. coli O157:H7 are ruminants and it is transmitted to people mainly via

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ingestion of inadequately processed contaminated food (25%) or water (9%) [6, 7].

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Consumption of infected sprouts, spinach, beef, lettuce, salami, unpasteurized milk, fresh

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strawberries and juice may result in E. coli O157:H7 infection [8-11]. Therefore, food

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screening is very important for consumer protection.

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Various methods have been developed for detecting E. coli O157:H7 in food samples,

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such as cultures and colony counting, multiplex polymerase chain reaction (PCR) [12],

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loop-mediated

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immunosorbent assay (ELISA) [14], and electrochemical immunosensors [15]. These 4

isothermal

amplification

methods

(LAMP)

[13],

enzyme-linked

ACCEPTED MANUSCRIPT methods are selective and reliable but most are time-consuming and laborious due to the

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multiple steps of bacterial enrichment and growth. Moreover, most approaches require

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expertise, so they are implausible for on-site detection. Immunochromatography assay (ICA)

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is simple, rapid, and cost-effective as well as highly sensitive and specific. However, the

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detection limit reported by other researchers was 105-106 CFU/mL [16, 17]. A lower detection

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limit was required for detection of this pathogen because the infectious dose of E. coli is very

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low. Furthermore, ICA can be affected by different matrices and background microflora

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causing false positives. Therefore, an improved ICA was needed.

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ICA has been used widely in the field of food safety to detect viral antigens [18], toxins

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[19, 20], heavy metals [21, 22], pesticides [23], and pathogens [24-26]. Colloidal gold is

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commonly used as a probe for ICA [20, 22] because it is inexpensive and rapid. However,

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colloidal gold-based ICA is limited when high sensitivity and accuracy is required. Recently

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advancements have been achieved in ICA [27], such as the use of novel labels [28], coupling

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ICA with other methods [29-31], and establishing signal amplification systems [32] to meet

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the needs of foodborne pathogen detection. In these developed methods, dye-doped

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fluorescent microsphere (FM) as probes for ICA had been used in lincomycin [33], aflatoxin

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B-1[34], T-2 toxin [35] and 4(5)-methylimidazole [36] residue detection because it was

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brighter and more photostable than fluorescent dye molecules. Moreover, FM-ICA is

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confirmed to have greater sensitivity and accuracy, and a lower detection limit than colloidal

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gold-based ICA [37, 38].

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Immunomagnetic beads (IM-beads) are often used to concentrate and isolate target

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bacteria from food matrices [39, 40]. With this sample preparation method, target bacteria are 5

ACCEPTED MANUSCRIPT concentrated from a large sample volume, and inhibitory agents are removed, significantly

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reducing background bacteria. IM-bead separation plus FM-ICA (IMS-FICA) for foodborne

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pathogen detection may significantly improve sensitivity and decrease detection times [30].

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Therefore, we created anti-E. coli O157:H7 IM-beads to separate pathogens from inoculated

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beef and milk matrices. Novel label FMs were used as probes for ICA. To our knowledge,

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this is the first report of IMS-FICA to quantify E. coli O157:H7.

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2. Material and methods

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2.1. Materials and reagents

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Anti- E. coli O157:H7 MAb A1, A2 were purchased from Aideed Biotechnology Co.,

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Ltd. (Zhuhai, China). Goat anti-mouse IgG and mouse IgG were purchased from Beijing

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Zhongshan Golden Bridge Biotechnology Co., Ltd. (Beijing, China). Fluorescent

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microspheres (418 nm diameter; λex 470 nm; λem 525 nm) were purchased from Merck

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(Darmstadt,

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1-(3-dimethyla-minopropyl)-3-ethylcarbodiimide hydrochloride (EDC), 2-(N-morpholino)

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ethanesulfonic acid (MES), and N-hydroxy-sulfo-succinimide (sulfo-NHS) were purchased

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from Sigma Aldrich (St. Louis, MO, USA). Nitrocellulose membrane, sample pads, and

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absorbent pads were obtained from Millipore (Bedford, MA, USA). Ultra-pure water was

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prepared using Elix-3 and Milli-QA (Molsheim, France). Magnetic beads were purchased

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from Xi'an Golden Magnetic Nano-Biotechnology Co. Ltd (Xian, China). All of the other

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reagents are analytical grade and purchased from Sinopharm Chemical Corp. (Shanghai,

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China).

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2.2. Bacterial strains and culture conditions

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Bovine

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albumin

(BSA),

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Germany).

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ACCEPTED MANUSCRIPT Four E. coli O157:H7 standard strains (NCTC 12900, ATCC 43888, CMCC 44828,

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ATCC 35150) and two wild E. coli O157:H7 strains (GIM1, GIM2) were obtained from the

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American Type Culture Collection (ATCC), National Collection of Type Culture (NCTC) and

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Guangdong Provincial Institute of Microbiology (GIM). Five non-E. coli O157 strains (SSl

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82000, CVCC 1490, ATCC 35401, ATCC 25922 and ATCC 12807) were obtained from

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Guangdong Culture Collection Center (GCCC) and ATCC. Ten other bacterial strains,

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namely, Staphylococcus aurous (CMCC 26003), Shigella spp. (ATCC 12022), Salmonella

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typhimurium (ATCC 14028), Salmonella paratyphi A (CMCC 50093), Salmonella paratyphi

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B (CMCC 50094), Bacillus thuringiensis (ATCC 10792), Sarcina (CMCC 28001), Listeria

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monocytogenes (ATCC 19115), and Vibrio parahaemolyticus (ATCC 17802, ATCC 33847)

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were procured from Guangdong Ocean University (GDOU), China Microbiological Culture

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Collection (CMCC) and Guangdong Institute of Microbiology(GIM). All strains were

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cultured in Luria-Bertani medium (LB, Oxoid, Basingstoke, UK) at 37 °C for 18 h, except

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Vibrio parahaemolyticus were cultivated with 3% NaCl alkaline peptone water at 37 °C for

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18 h. Viable cell counts of E. coli were measured by plating on MacConkey agar through

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serial dilutions in saline solution.

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2.3. Particle size of magnetic beads

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A laser particle size analyzer (BI-200SM, Brookhaven, USA) was used to evaluate the

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particle size of magnetic beads. Magnetic beads suspension (2 mL) was washed twice with

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ultrapure water to remove impurities, and then resuspended in ultrapure water for test at

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25 °C.

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2.4. IM-beads preparation 7

ACCEPTED MANUSCRIPT Conjugates of carboxylated magnetic beads and anti- E. coli O157:H7 MAb A1 were

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prepared using an EDC/NHS coupling procedure. EDC (200 µL) and sulfo-NHS (200 µL)

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solution (5 mg/mL in 10 mM MES buffer, pH 6.0) were mixed with magnetic beads (2 mg) to

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activate the carboxylic groups on the bead surfaces. Then, anti- E. coli O157:H7 MAb was

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immediately added into activated carboxyl-bead solution with gentle stirring for 2 h to form

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conjugates. Finally, the sites on the magnetic beads that were not bound were blocked with 1%

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BSA, and stored in refrigerator at 4 °C after adjusting the concentration to 2 mg/mL with

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borate-borax buffer with 0.2 mg/mL NaN3 and 1% BSA.

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2.5. IM-beads optimization

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2.5.1. Antibody concentration

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The optimum amount of MAb was estimated by monitoring the coupling rate of MAb

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and magnetic beads at different proportions of 1:1.1, 1:3.3, 1:5.5, 1:11, 1:22 (mg/mg). The

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coupled MAb was calculated through detecting the amount of antibody added and remained

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in the supernatant. Trace bicinchoninic acid (BCA) test kit (Nanodrop 2000, Thermo

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Scientific Co. Ltd., Wilmington, DE, USA) was used to test the amount of MAb before and

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after immunoreactions. Coupling rates were calculated as described in our published method

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[41].

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2.5.2. Working concentrations

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IM-beads (3.3 mg/mL) of 40, 60, 80, and 100 µL were separately added into four 1.5-mL

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centrifuge tubes which contain 1 mL E. coli O157:H7 diluted cultures (3 × 105 CFU/mL), and

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incubated at 37 °C for 30 min. The supernatant was separated from the conjugates of

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IM-beads and bacterium, then 50 µL volume was coated onto the MacConkey agar at 37 °C 8

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for 18 h for recording colonies number. Three parallels were set up for each experiment and

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blank bead coupled to BSA as negative control.

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2.5.3. Enrichment time IM-beads of 60 µL was added into four 1.5-mL centrifuge tubes which contain 1 mL

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diluted bacteria cultures of E. coli O157:H7 (3 × 105 CFU/mL), then incubated at 37 °C for 10,

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15, 30, 45 and 60 min. After magnetic separation, the supernatant of 50 µL was coated onto

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the MacConkey agar for recording colonies number. Three parallels were set for each sample,

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and a blank magnetic bead coupled with BSA was used as a negative control to calculate the

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capture efficiency.

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2.5.4. Sensitivity

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E. coli O157:H7 culture was diluted with sterile saline to a concentration of 102, 103, 104

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and 105 CFU/mL, which 1 mL were taken out from each dilution and mixed thoroughly with

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60 µL IM-beads. The number of colonies and capture efficiency of IM-beads were calculated

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as mentioned above.

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2.6. FM-ICA preparation

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2.6.1 Conjugates of FM labeled MAb and FM labeled rabbit IgG

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Conjugates of FM and anti- E. coli O157:H7 MAb A1 (FM-MAb A1), FM and rabbit

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IgG (FM-Rabbit IgG) were prepared as follows: first, carboxylated FM (0.5 mL, 1% solid

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content) were washed with 1 mL MEST buffer (50 mM MES, pH 6.0, 0.01% Triton X-100).

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Next, 50 µL EDC (50 mg/ml) and 50 µL sulfo-NHS (50 mg/ml) were added to form an

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amine-reactive sulfo-NHS ester at room temperature for 30 min on a rotary mixer. Afterward,

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the mixture was washed three times with 1 mL MEST buffer and excess EDC and sulfo-NHS 9

ACCEPTED MANUSCRIPT were removed. Finally, the precipitate was resuspended in 0.5 mL MES buffer (50 mM, pH

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6.0) which antibody concentration was 0.2, 0.3, 0.5 mg/mL separately for anti-E. coli

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O157:H7 MAb A1, 0.5 mg/mL for rabbit IgG. The mixture was incubated with gentle stirring

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for 2.5 h in the dark, and then centrifuged at 12,000 rpm for 10 min. After centrifugation,

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conjugates were washed three times with 1 mL tris buffered saline (TBS) (25 mM Tris-Cl,

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130 mM NaCl, 2.7 mM KCl, pH 8, 0.01% Triton X-100) and then suspended in 500 µL

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phosphate buffer saline (PBS) containing 1% BSA, 5% sucrose and 0.05% Tween 20, stored

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at 4 °C for later experiments.

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2.6.2. Fabrication of FM-ICA strips

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The ICA strip consists of a sample pad, a nitrocellulose (NC) membrane, and an

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absorbent pad. The sample pad was treated with PBS (10 mM, pH 7.2, 0.05% Tween-20, 2%

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BSA, 3% sucrose, and 0.05% sodium azide) for blocking and then dried at 37 °C for 24 h

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before use. Goat anti-rabbit (GAR) MAb and mouse anti- E. coli O157:H7 MAb A2 were

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dissolved in coating buffer separately and applied to the control line (C line) and the test line

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(T line) of the NC membrane using a Biostrip Dispenser (Biodot, Irvine, California) with a 4

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µL/cm jetting rate. This created two lines with a 4-mm interval. Then the NC membrane was

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dried at 50 °C for 48 h. After these preparation steps, the sample pad, the NC membrane and

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the absorbent pad were assembled on the PVC baking card with a 1-2 mm overlap (Fig. 1

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IIIB). Then the card was resized to 4.03 mm wide using an HGS 201 cutter (Autokun

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Technology Co., Ltd, Hangzhou, China) before being assembled to a plastic cartridge

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designed to fit the holder on the optical reader.

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ACCEPTED MANUSCRIPT For FM-ICA optimization, different amount of anti- E. coli O157:H7 MAb A2 (0.1, 0.2

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and 0.4 mg/mL) was sprayed on the T line (2 µL/cm), and GAR MAb of 0.2, 0.3, 0.5 and 1

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mg/mL was sprayed on the C line (2 µL/cm). FM-MAb conjugates (1% solid content) were

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diluted to 50, 100, and 200-fold and FM-Rabbit IgG conjugates were diluted to 500, 1000,

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and 2,000-fold for sample analysis. All experiments were conducted in triplicate.

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2.6.3. FM-ICA test procedure

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The diluted conjugates of FM-MAb A1 and FM-rabbit IgG were mixed with sample

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solution for 1 min, and then an 80 µL aliquot of mixture was pipetted into the sample hole for

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15 min. The fluorescent intensity of the test line (FT) and control line (FC) were recorded with

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a strip reader (λex 470 nm; λem 525 nm) supplied by Guangzhou Wondfo Biotechnology Co.

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Ltd. (Guangzhou, China). The ratio of FT/FC was used to offset the inherent heterogeneity

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from the test strips and the sample matrix.

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2.6.4. FM-ICA evaluation.

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The sensitivity, specificity and recovery of FM-ICA were investigated. For sensitivity

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analysis, a pure culture of E. coli O157:H7 was diluted with PBS buffer (0.01 M, pH 7.4) to

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form a final concentration of 6 × 104, 1 × 105, 3 × 105, 1 × 106, 6 × 106, 1 × 107, 6 × 107 and 1

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× 108 CFU/mL. This was mixed with FM-MAb in application buffer for test. Then, a

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dose-response curve was established by plotting FT/FC values against the log E. coli O157:H7

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concentration (LN (CFU/mL)). The limit of detection (LOD) was calculated by adding the

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blank sample’s FT/FC value plus three-fold standard deviations (SD). The limit of

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quantification (LOQ) was calculated by adding the blank sample’s FT/FC value plus ten-fold

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SD. For specificity analysis, 21 strains were chosen for cross-reactivity testing (Table. S2).

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ACCEPTED MANUSCRIPT The specificity of FM-ICA was evaluated by comparing FT/FC values with the LOD. The

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accuracy and precision of FM-ICA were evaluated by analyzing recoveries and the coefficient

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of variation (CV). E. coli O157:H7 concentration of 3 × 105 CFU/mL, 3 × 106 CFU/mL, 1 ×

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107 CFU/mL, 3 × 107 CFU/mL was tested 10 times separately using different batches of

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FM-ICA strip.

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2.7. IMS-FICA detection

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Beef, romaine lettuce, milk, and water (E. coli O157:H7 negative) were purchased from

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a local supermarket in Guangzhou (China). Beef matrices were spiked with diluted E. coli

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O157:H7 pure culture at a ratio of 1:20 (w: v) before homogenized, milk was mixed with

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diluted pure culture at a ratio of 1:10 (v: v), and water without any treatment after mixing with

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the culture. The three mixtures had the final bacteria concentration of 3 × 102, 3 × 103, 3 × 104,

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3 × 105 CFU/mL. Then, 100 µL bacteria solution was taken out for FM-ICA, and 10 mL for

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IMS+FICA test. 60 µL IM-beads was added into the solution with gently shaken at 37 °C for

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30 min. After magnet separation with a magnetic frame and washing three times with PBS,

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the conjugates of bacteria and IM-beads were resuspended with 100 µL PBS and eluted at

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75 °C for 5 min. The supernatant was assayed using an FM-ICA as previously described in

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test procedure. The detection process was shown in Figure 1. All tests were performed in

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triplicate.

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3. Results and discussion

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3.1. IM-beads optimization

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Magnetic beads about 1.59 µm (Fig. S1) were used for IM-beads preparation. Conditions

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that affected the capture rate of IM-beads were optimized. Results as shown in Fig. 2, an 12

ACCEPTED MANUSCRIPT amount of 1 mg antibody needed 3.3 mg magnetic beads for conjugates (Fig. 2A), a 60 µL

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IM-bead solution was used as a working concentration when the concentration of bacteria was

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equal to or less than 3 × 105 CFU/mL (Fig. 2B), 30 min was selected for enrichment (Fig. 2C).

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At these optimized conditions, the IM-beads had good sensitivity and the capture efficiency

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was higher than 80% (Fig. 2D).

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Fig. 1. The schematic illustration of (I) Preparation of immunomagnetic beads; (II) Process of

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immunomagnetic separation; (III) Detection process of fluorescence microsphere based

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ACCEPTED MANUSCRIPT immunochromatography assay, (A) Pathogen bacterium in sample combine with FM labeled

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antibody, (B) Immunoassay mechanism of ICA strip and (C) Scanning & analysis.

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Fig. 2. Various conditions for IM-beads separation, (A) Ratio of MAb to IM-beads for

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coupling; (B) Amount of IM-beads added; (C) Enrichment time; (D) Sensitivity of IM-beads

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to different concentration of bacteria.

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3.2. FM-ICA development

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Conjugates of FM-MAb and FM-rabbit IgG were used as probes for ICA. During the

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detection process, FM-MAb and FM-rabbit IgG were mixed with the sample, and if target

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strains were within the sample, they would be captured by the FM-MAb conjugates. Then, the

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mixture was added into the sample hole on the strip and this migrated smoothly to the T and C 14

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lines to form a sandwich format. The fluorescent intensities of T and C lines were recorded

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using the strip reader. Antibody was a key parameter in this detection process. So antibody and antigen

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interactions were optimized. Results as shown in Fig. 3, antibody concentration at 0.2, 0.3 and

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0.5 mg/mL for label had little effect on the fluorescence intensity and FT/FC value (Fig. 3A).

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However, when the concentration of MAb was 0.2 mg/mL, the least amount of MAb was

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required. Fold-dilutions of FM-MAb in the sample had obvious influence on fluorescence

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intensity, however, 50 and 100-fold dilutions made little difference to the FT/FC values (Fig.

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3B). At the same conditions, FT/FC value decreased with the increase of rabbit IgG

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concentration for label (Fig. 3C). Dilution of FM-rabbit IgG affected FT/FC values, and a good

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linear relationship was observed at a 1,000-fold dilution (Fig. 3D). The MAb sprayed on the C

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and T lines influenced FT/FC values; a hook effect was observed when the bacterial

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concentration exceeded 6 × 107 CFU/mL (Fig. 3C, E). By increasing the MAb coating

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concentration at the T line to exceed 0.5 mg/mL, a hook effect at 1 × 108 CFU/mL was no

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longer observed (Fig. S3), however, the detection limit remained the same. Therefore, the

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optimal conditions for FM-ICA were, 0.2 mg/mL MAb A1 for FM-MAb conjugates and 0.5

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mg/mL rabbit IgG for FM-rabbit IgG conjugates, FM-MAb diluted 100-fold and FM-rabbit

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IgG diluted 1,000-fold in the sample complex as a capture antibody, 0.5 mg/mL goat

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anti-rabbit MAb and 0.1 mg/mL anti- E. coli O157:H7 MAb A2 which were jetted onto the C

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and T line as detection lines.

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Fig. 3. Conditions for FM-ICA detection, (A) Antibody concentration for conjugation of

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FM-MAb A1; (B) Dilution fold of FM-MAb A1 conjugates; (C) Dilution fold of FM-rabbit

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IgG conjugates; (D) GAR MAb concentration sprayed on the C line; (E) Anti-E. coli

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O157:H7 MAb A2 concentration sprayed on the T line.

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3.3. FM-ICA evaluation 16

ACCEPTED MANUSCRIPT FM-ICA sensitivity was assessed by using different concentrations of E. coli O157:H7 in

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PBS buffer (1 × 105 - 3 × 108 CFU/mL) under optimized conditions. The LOD and LOQ data

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for FM-ICA appear in Table S1. Fluorescence intensity increased with increasing E. coli

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O157:H7 (Fig. 4A). A standard curve was established using the log bacterial concentration

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(LN (CFU/ml)) and its FT/FC value (Fig. 4B), and the linear detection ranged from 3 × 105 to

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6 × 107 CFU/mL. We observed that the FM-ICA had greater sensitivity (1 × 105 CFU/mL)

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than the colloidal gold-based ICA method (106 CFU/mL) [17]. Studies by Xie’s group

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confirmed that an FM-based immunochromatographic lateral flow assay was better than a

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colloidal gold immunochromatographic lateral flow assay with regard to sensitivity,

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coefficient of variation (CV), and antibody needed [37]. However, when this method was

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used to measure Campylobacter jejuni, the sensitivity was 106 CFU/mL [38], a finding that is

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inconsistent with our results. This may be explained by the use of different antibodies and

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reaction conditions, which could affect the sensitivity.

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25.00

B

y = 0.0003e 0.6258x R² = 0.9949

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Ratio of HT/HC

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LN(CFU/mL)

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Fig. 4. The fluorescence intensity on the NC membrane at E. coli O157:H7 concentration

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from 3 × 105 to 6 × 107 CFU/mL (A) and the standard curve (B).

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ACCEPTED MANUSCRIPT Accuracy of the developed FM-ICA for E. coli O157:H7 detection was evaluated by

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recovery of the spiked experiments. The precision was evaluated by CV of intra-assay and

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inter-assay. Data appeared in Table 1. The average recoveries of intra-assay were ranged from

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101% to 106%, with CV lower than 10%. The inter-assay recoveries were ranged from 95.62%

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to 110.2%, with CV lower than 10%. The results suggested that the FM-ICA for E. coli

286

O157:H7 quantitation had higher reproducibility.

287

Table 1

288

Accuracy and precision of FM-ICA.

(CFU/mL)

Recovery

(n=10)

c

CV (%)

(%) 103.80

8.08

3×106

106.15

2.40

7

107.03

1.68

3×107

101.64

Recovery

TE D

3×105

1×10

SC Inter-assay b

(n=10)

Spiked

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Intra-assay a

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281

1.94

(%)

c

CV (%)

104.32

9.56

96.50

4.26

95.62

3.56

110.2

6.45

a

Intra-assay was completed using ten strips from same batch.

290

b

Inter-assay was completed using ten strips randomly selected from different batches.

291

c

Recovery = (detection concentration/spiked concentration) * 100%.

292

CV=SD/mean * 100%.

293

Recoveries were affected by various factors, such as sample pad, NC membrane,

294

absorbent pad, chromatography time and so on. When non-specific adsorption occurred, more

295

FMs were conjugated to the detection line, and stronger fluorescence intensities were

296

recorded by the strip reader. Thus, the recoveries calculated showed high than 100%. In

297

addition, when the fluorescent intensity of C-line was lower than normal, higher HT/HC ratios

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ACCEPTED MANUSCRIPT 298

were obtained, which also result in higher recoveries. Although recovery was closer to 100%,

299

a more accurate detection was achieved. However, recoveries of about 80% to 120% were

300

deemed acceptable [42-44]. In addition, the stability of this present method was studied by accelerated test. Results

302

appeared in Fig. S2. The ICA strips remained stable at 50 °C for 35 days. The test strip

303

identified four standard E. coli O157:H7 strains and two wild E. coli O157:H7 strains with

304

strong signals, but did not identify fifteen non-target bacterial strains. The FT/FC values less

305

than the LOD produced no signal (Table S2).

306

3.4. IMS-FICA detection.

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To remove interference of different food matrices and lower detection limits, IM-beads

308

combined with FM-ICA (IMS-FICA) were prepared to detect E. coli O157:H7. Artificially

309

contaminated samples were prepared by adding E. coli O157:H7 to beef, romaine lettuce,

310

milk and water. Under optimized conditions, the detection limit was lowered from 1×105 to

311

3×103 CFU/mL, 33-fold lower than an FM- ICA method alone (Data as shown in Table 2).

312

The sensitivity of this combined method was higher than a personal glucose meter (PGM)

313

based-ICA assay (the detection limit was 6.2 × 104 CFU/mL) [45] and a little lower than Eu

314

(III)-doped polystyrene nanoparticle-linker ICA assay (9.54 × 102 CFU/mL) [43]. This

315

present method has the same sensitivity as a LAMP (103 CFU/mL) and PCR (103 CFU/mL)

316

without pre-enrichment [13]. However, recoveries for this IMS-FICA method were slightly

317

lower than for FM-ICA due to capture efficiency of IM-beads and elution step. Although

318

other researchers have shown that the capture efficiency of IM-beads for E. coli O157:H7 in

319

ground beef and milk samples were 94.4% and 99.8%, respectively [40]. Which was similar

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ACCEPTED MANUSCRIPT to our conjugated IM-beads, yet IM-beads and bacteria were separated at 70 °C for 5 min, the

321

epitopes of the antigen-antibody binding sites can be damaged and cause a lower test

322

concentration than what is theoretical. More work is required to optimize the elution method

323

to increase detection sensitivity. Furthermore, recoveries obtained from beef were

324

significantly lower than from other matrices, indicating that the application conditions of

325

IM-beads are different. More work is required to test the utility of IMS-FICA under field

326

conditions. Our method can be completed within 2 h (15 min for test strip and 1 h for

327

IM-beads separation), including the time required to enrich the bacteria and elute it from the

328

IM-beads, which is much faster than conventional biochemical methods.

329

4. Conclusions

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A novel approach was developed to detect foodborne pathogen E. coli O157:H7 with

331

IM-beads to enrich bacteria from food matrices followed by an FM-ICA for bacterial

332

quantification. This combined method has a sensitivity of 3×103 CFU/mL in raw materials

333

and requires an assay time of 2 h or less. IMS-FICA method is technically simple and requires

334

no special equipment. It may hold promise for a rapid, simple and highly sensitive detection

335

of foodborne pathogens used in emergent disease detection and monitoring.

336

Acknowledgments

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This research was supported by the National Science and Technology Support Program

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of China (Nos. 2012BAK08B00 and 2012BAK08B07), Science and Technology Plan Project

339

of Guangdong Province (2017A020208014). The authors thank Guangzhou Wondfo Co., Ltd.,

340

China in Guangzhou for providing assistance. We thank Accdon (www.accdon.com) for its

341

linguistic assistance during the preparation of this manuscript. 20

ACCEPTED MANUSCRIPT 342

Table 2

343

Changes of FM-ICA and IMS-FICA in E. coli O157:H7 detection in spiked beef, romaine

344

lettuce, milk and water samples.

Sample

Spiked

matrices

(CFU/mL)

HT/HC

Result

Beef

3 × 105

0.87 ± 0.05

+

3 × 104

0.69 ± 0.01

-

3 × 10

3

0.67 ± 0.02

3 × 10

2

Milk

Water

Recovery

113.06

10.43 ± 0.25

+

60.22

—

2.61 ± 0.28

+

58.70

-

—

0.72 ± 0.01

+

—

0.68 ± 0.02

-

—

0.69 ± 0.01

-

—

3 × 105

0.83±0.03

+

104.81

12.05±0.16

+

75.83

3 × 104

0.67±0.02

3 × 10

3

0.68±0.02

3 × 10

2

0.67±0.03

3 × 10

5

0.86 ± 0.10

3 × 10

4

0.66 ± 0.04

3 × 10

3

0.68 ± 0.03

3 × 10

2

0.67 ± 0.02

3 × 10

5

3 × 10

4

3 × 10

3

3 × 10

2

(%)

SC

Result

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lettuce

Recovery

IMS + FICA (n = 3)

HT/HC

(%)

-

—

2.90±0.14

+

77.68

-

—

0.70±0.02

+

—

-

—

0.66±0.02

-

—

+

112.05

10.93 ± 0.15

+

66.01

-

—

2.54 ± 0.08

+

75.60

-

—

0.72 ± 0.02

+

—

-

—

0.68 ± 0.01

-

—

0.88 ± 0.09

+

116.8

12.18 ± 0.13

+

77.14

0.68 ± 0.02

-

—

2.87 ± 0.07

+

76.42

0.68 ± 0.01

-

—

0.72 ± 0.02

+

—

0.67 ± 0.02

-

—

0.67 ± 0.01

-

—

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Romaine

b

FM-ICA (n = 3)

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Negative result (-) and positive result (+) are judged according to LOD value.

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Test concentration (CFU/mL) was obtained according to the standard curve within the linear

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detection range.

348

—: not in the linear detection range.

349

a

The samples were not treated with IM-beads.

350

b

The samples (10 mL) were treated with 60 µL IM-beads and concentrate to 100 µL for FICA.

351

testing.

21

ACCEPTED MANUSCRIPT 352

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coli

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using

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meter

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on

ACCEPTED MANUSCRIPT

Highlights Immunomagnetic beads were prepared for separating and concentrating bacteria Fluorescence microspheres as label for immunochromatography assay

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IMS-FICA detection has the sensitivity of 3×103 CFU/mL and can be completed

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within 2 h.