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Single-Step Recombinase Polymerase Amplification Assay Based on a Paper Chip for Simultaneous Detection of Multiple Foodborne Pathogens Heeseop Ahn, Bhagwan Sahebrao Batule, Youngung Seok, and Min-Gon Kim Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b01309 • Publication Date (Web): 03 Aug 2018 Downloaded from http://pubs.acs.org on August 5, 2018

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Analytical Chemistry

Single-Step Recombinase Polymerase Amplification Assay Based on a Paper Chip for Simultaneous Detection of Multiple Foodborne Pathogens

Heeseop Ahn†, Bhagwan Sahebrao Batule†, Youngung Seok, and Min-Gon Kim* Department of Chemistry, School of Physics and Chemistry, Gwangju Institute of Science and Technology, 261 Cheomdan-gwagiro, Gwangju 500-712, Republic of Korea

* Corresponding author. M. G. Kim: Tel: +82-62-715-3330; Fax: +82-62-715-3419; E-mail address: [email protected] †

Heeseop Ahn and Bhagwan Sahebrao Batule contributed equally.

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ABSTRACT A paper chip device-based recombinase polymerase amplification (RPA) method was developed for highly sensitive and selective single-step detection of foodborne pathogens. A paper chip was manufactured by simply stacking functional papers. RPA reagents and fluorescent probe were dried on the reaction zone of a patterned polyethersulfone membrane. The RPA reaction was initiated by adding pathogen DNAs into an injection hole. Paper chipbased analysis of pathogens showed optimal performance at 37°C for 20 min and the results were comparable to those obtained with solution-based RPA reactions. Based on the paper chip-based fluorescence signal, Escherichia coli, Staphylococcus aureus, and Salmonella typhimurium were simultaneously detected with detection limits of 102 cfu/ml. The diagnostic utility of the device was demonstrated by the reliable detection of E. coli and S. aureus present in spiked milk. This ready-to-use device could be integrated with simple nucleic acid extraction for food pathogen detection in resource-limited settings.

Keywords:

Paper chip;

Polyethersulfone; Recombinase

Foodborne pathogen; Fluorescence detection


polymerase

amplification;

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INTRODUCTION Research on the development of paper-based diagnostic technologies had rapidly evolved in recent years owing to the potential for successful application to point-of-care testing (POCT).1–4 Paper materials have several advantages as a detection platform in POCT devices including

simplicity

of

operation,

portability,

biocompatibility,

cost

efficiency,

biodegradability, simple fabrication, use of dry chemistry, and natural fluid flow by capillary action due to porosity.5,6 In addition, hydrophilic channels can be built on the paper platform by simple patterning techniques with a hydrophobic material, which enables the fabrication of multi-functional diagnostic tools.7,8 Paper materials have been used for nucleic acid testing (NAT) based on isothermal amplification methods.9–11 In resource-limited areas, these technologies have enabled more sensitive detection of target pathogens compared to conventional diagnostic methods.12,13 However, previously reported paper-based NAT methods have certain limitations such as difficulty in detecting the end point,14,15 inadequate limit of detection (LOD) and reproducibility,16 complex operation,17 and singleplex detection.18,19 As such, there is broad scope for improving paper-based detection platforms for disease diagnosis. There have been only a few reports on paper-based recombinase polymerase amplification (RPA) despite its low and wide range of reaction temperatures, short reaction time, and robustness.17,20,21 One study used a RPA-based paper and plastic device to detect human immunodeficiency virus DNA, which yielded a LOD comparable to a solution-based RPA reaction.22 However, operating this platform is complex and reactions require freshly prepared reagents. A reverse transcription RPA paper-based device in which RPA reagents were freeze-dried on a paper membrane was proposed for the multiplexed analysis of Ebola virus (with single-stranded RNA as the target) in the resource-limited environments.23 However, this method requires improvement in terms of sensitivity. Therefore, simple, cost-effective, real-time, sensitive,



and selective paper-based disease diagnosis devices that can be used in resource-limited settings are needed. We recently reported a simple paper-based loop-mediated isothermal amplification (LAMP) technique for simultaneous detection of multiple DNA targets in real time.24 In this platform, all LAMP reaction components were dried on the reaction membrane without lyophilization. However, there was an evaporation problem due to relatively high reaction temperature and long reaction time (60 min at 60 – 65°C) of LAMP. To address these issues, in the present study we developed a paper chip that allows multiple RPA reactions simultaneously and fluorescence-based detection of major foodborne pathogens such as Shiga toxin-producing E. coli, S. aureus, and S. typhimurium in a single-step operation, thereby reducing the time, labor, and cost of detection.25 On-field detection of foodborne pathogens requires a rapid, portable, cost-effective, user-friendly, selective, and sensitive multiplex bioanalytical assay.26–29 We designed a paper chip for RPA by stacking two functional layers that were held together with sealing tape. The RPA reagents and fluorescent probe were ovendried on the paper pads at 37°C, and reaction proceeded at 37°C for 20 min upon sample injection. This paper-based RPA showed sensitivity and selectivity that were comparable to a solution-based RPA reaction in actual spiked samples.

EXPERIMENTAL SECTION Materials and chemicals. The Twistamp Basic and Exo kits were purchased from TwistDX (Cambridge, UK). Glass fiber (Grade 8964) and absorbent cotton pad (Grade 222) were from Ahlstrom (Stockholm, Sweden), asymmetric polysulfone membrane (vivid plasma separation membrane-GF) were from Pall Corporation (Ann Arbor, MI, USA). The nitrocellulose (HiFlow Plus 180 membrane) membrane was from Millipore (Billerica, MA, USA). Modified glass fiber (Fusion 5) were from Whatman (Maidstone, UK). The polyethersulfone (PES)


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membrane filter was from Sterlitech (Kent, WA, USA). Enzyme-linked immunosorbent assay (ELISA) sealing tape was from Excel Scientific (Victorville, CA, USA). Oligonucleotide primers were from Genotech (Daejeon, South Korea) and RPA exo probes were from LGC Biosearch Technologies (Petaluma, CA, USA); their sequences are shown in Table S1. DNA/DNAase-free water was from Sigma-Aldrich (St. Louis, MO, USA). Preparation of bacterial culture samples. Escherichia coli O157:H7 (ATCC 35150), Staphylococcus aureus (ATCC 25923), and Salmonella enterica typhimurium (ATCC 19585) were employed as the model target stain for the evaluation of paper chip-based RPA. All bacterial strains were purchased from the American Type Culture Collection (ATCC) and grown in tryptic soy broth (Difco Laboratories, Franklin Lakes, NJ, USA) under gentle shaking at 37 °C with 150 rpm shake for 12 h. Then, the concentrations of pathogenic bacteria were calculated by cell counting on tryptic soy agar plates (Difco Laboratories, Franklin Lakes, NJ, USA).30 Lastly, the bacterial cell cultures were re-suspended in PBS to obtain bacterial suspensions containing 100–106 colony forming units per microliter (cfu mL−1) and stored at −20 °C before use.31 The bacterial genomic DNAs were extracted with the QIAamp DNA mini kit (Qiagen, Inc., CA, USA) and DNA copy numbers were calculated using a dsDNA copy number calculator.24,32 RPA exo assay on paper membrane for reaction material testing. The glass fiber, asymmetric polysulfone membrane, nitrocellulose membrane, modified glass fiber, polyethersulfone membrane filter, and absorbent cotton pad were cut into 5 × 5 mm squares. Drying solution was prepared by adding 45.2 µl water, 4.2 µl of 10 µM primer sets, and 0.6 µl of 10 µM Twistamp exo probe to lyophilized RPA proteins. The mixture was loaded directly onto the membrane by pipetting. Loading volume was determined according to the intrinsic absorption capacity of each paper, which was measured by dipping the material in a beaker of water on an electronic scale and calculating the reduction in water weight. Reagent-



treated membranes were prepared by drying the solution-loaded membranes for 30 min in a 37°C drying oven. The sample solution composed of 2.5 µl magnesium acetate and 29.5 µl of reaction buffer of unknown concentration provided by the manufacturer, 1.2 µl template DNA, and 16.8 µl water were pipetted onto the reagent-treated membrane and heated for 20 min at 37°C. Fluorescence data were obtained using a Chemi Doc XRS+ imaging system (Bio-Rad Laboratories, Hercules, CA, USA) with a blue light source and 605/50 filters. Reaction efficiency was evaluated based on the ratio of change in fluorescence intensity between the control and test pad.

Primer screening. To obtain primer pairs with optimal amplification performance, a set of primer candidates were evaluated in a screening test. Target regions of template DNAs were first selected; for multiplex detection, this was done in such a way as to avoid overlap with target regions of other pathogen DNAs. Within a selected target region, two sets of staggered, opposing oligonucleotides were randomly selected as forward and reverse primers. Nine primers were selected per direction, and by screening all reverse primers against a single forward primer, we selected the best among the nine candidate reverse primers and used it to screen the nine forward primers. Amplification performance of primer candidates was evaluated with the Twistamp basic kit (with detection by gel electrophoresis) and the Twistamp exo kit (with detection and fluorescence read-out). Optimized primer sequences are listed in Table S1.

Paper chip fabrication. The asymmetric polysulfone membrane was cut into 14 × 14-mm squares using a cutter. Four 5-mm reaction compartments were formed on the 8.0-µm PES filter using a wax printer (ColorQube 8570; Xerox Corporation, Norwalk, CT, USA).7 The drying solution (11.5 µl) (see above) was pipetted onto each zone and dried for 30 min at


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37°C in a drying oven. The paper chip was fabricated as follows. Four holes were punched into the ELISA sealing tape, which was attached to the PES membrane while aligning the holes with the reaction zones. The diameter of the holes was about 6 mm. A sample injection hole (diameter: 1 mm) was punched in center of this structure, which was stacked on an asymmetric polysulfone membrane. The large-pore side of this membrane made contact with the patterned PES membrane. This paper chip was used in a multiplex RPA-exo assay.

RPA analysis on the paper device. The fabricated paper device was placed in a Petri dish and the reaction was initiated with a single injection of the sample solution (80 µl) into the sample injection hole, followed by incubation at 37°C. Fluorescence data were obtained using a blue light source and 605/50 filters on a Chemi Doc XRS+ imaging system. Results of the RPA analysis are presented as the ratio of change in fluorescence intensity between the control and test. (∆S/∆N), taking into consideration the high background signal of paper materials or paper chip.

Detection of foodborne pathogens in the spiked milk sample. The milk sample was purchased from a local supermarket in the city of Gwangju, Korea and directly employed without dilution. The milk samples with different bacterial copies (such as 102, 103, 104 and 105 cfu/ml) of foodborne pathogens such as E. coli (Gram negative strain) and S. aureus (Gram positive strain) were prepared by serial dilution. The genomic DNAs were isolated from each spiked milk samples with QIAamp DNA mini kit (Qiagen, CA, USA) and directly utilized to the paper chip-based RPA device without further purification.

RESULTS AND DISCUSSIONS



Basic concept of the paper chip-based RPA device. The design and functioning of the paper chip-based RPA device are shown in Figure 1. The paper chip had a stacked structure composed of a transfer pad, wax-printed PES reaction membrane, and sealing tape. The reaction membrane was divided into four compartmentalized reaction zones, each of which contained dried RPA reaction reagents including pathogen-specific primers, RPA-exo probe, and proteins for amplifying specific target DNA. The four compartments were labeled as C (Control) and P1–P3 (Probes 1–3). P1, P2, and P3 contained primers and probes for E. coli, S. aureus, and S. typhimurium, respectively. C contained RPA reagents without primers and probes. Each reaction zone was independently operated and the primer sets and probes in each reaction zone allowed target-specific RPA reactions (Fig. 1a). When the sample solution (containing DNA) was loaded into the sample injection hole, it soaked the transfer pad and then flowed to the PES reaction membrane where it mixed with dried RPA reagent in the reaction membrane (Fig. 1b). For active and uniform transfer of the sample solution to the reaction pads, we used an asymmetric transfer pad membrane with the large-pore side of the membrane in contact with the reaction pad. We designed the device with the sample injection hole and reaction membrane on the same side, which is advantageous for adding instruments for heating and fluorescence analysis—e.g., a heat block under the bottom layer and an optical instrument above the device. In addition, the device can be fabricated by simply stacking the functional layers sequentially, yielding a small and compact structure.

RPA on different membranes. To obtain a maximum signal-to-noise ratio, we examined the fluorescence signal of the RPA-exo reaction on the various paper materials and found detectable fluorescence in the glass fiber and PES (Fig. 2). It was previously shown that papers with a large pore size such as glass fiber and polyester can capture and store reagents in a stable and dry format at ambient temperature.6 In addition, a large pore size provides


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more spaces that allow mixing and diffusion of reagent components. Especially, glass fiber was reported to the matrix that is most compatible with RPA.22 In our experiments, PES as well as glass fiber showed strong fluorescence, providing an effective platform for RPA. We therefore selected PES as the reaction material since wax printing directly on the PES membrane is possible unlike for glass fiber, thereby simplifying the fabrication process. In the paper-RPA experiment, the good performance of the PES membrane may have been due to the large pore size, hydrophilicity, and biocompatibility with RPA reagents. PES membrane-based detection of three different targets showed that the maximum fluorescence change occurred upon addition of the target DNA (Fig. 3). In these experiments, we employed a mixture of target pathogens and obtained specific fluorescence signal with respective target-specific primer set. We did not find any non-specific amplification. We therefore conclude that the PES membrane is a useful material for the paper chip for simultaneous detection of multiple pathogenic DNAs.

Optimization of paper chip structure. We confirmed that that RPA reaction was detectable on both bare and patterned PES membranes (Fig. S1a). The membrane pores of the transfer pad are asymmetric, and the side with the larger pores was in contact with the reaction membrane, allowing uniform transfer of the sample solution to the reaction zones (Fig. S1b). ELISA sealing tape was used to ensure tight contact between paper membranes and active flow of injected sample solution and to delay reagent evaporation during the RPA reaction. However, the area of contact between the sealing tape and reaction zone required a hole to allow the reaction on the paper chip to proceed (Fig. S1c). Pressure caused by the sealing tape inhibited the flow of sample solution to the reaction zone and the mixing of the solution with dried reagents. By creating holes in the sealing tape, the maximum injection capacity increased by over 50%.



Multiplex detection of foodborne pathogens. We next detected target template DNAs of three pathogen—i.e., E. coli, S. aureus, and S. typhimurium—on the paper chip device. Target-specific primer sets and probe were deposited in specific reaction zones of the patterned PES membrane. The RPA paper chip detected the three pathogen DNAs in multiplex reactions—specifically, E. coli (Fig. S2a); E. coli plus S. aureus (Fig. S2b); and E. coli, S. aureus, and S. typhimurium (Fig. S2c). There was no cross-reactivity or non-specific amplification of the three target pathogens. Under optimized conditions, we evaluated the sensitivity of paper chip-based RPA by detecting E. coli, S. aureus, and S. typhimurium. The ∆S/∆N value continuously increased with pathogens concentration. The detection range of E. coli, S. aureus, and S. typhimurium was 102–105 cfu/ml with an LOD of 102 cfu/ml (Fig. 4). By comparison, the LOD of the solution-based RPA assay was 102 cfu/ml and the detection range was 102–105 cfu/ml (Fig. S3). Thus, the two approaches had similar sensitivity. Further, we achieved over 90 % stability of dried RPA reaction mixture on membrane more than 4 weeks at room-temperature (Fig. S4). The dried RPA reagents present on the membrane were activated by simple addition of sample or buffer. The long-term storage stability widen its potential application in resource-limited settings. It indicates the robustness of RPA-paper chip developed in this manuscript. We have avoided contamination of RPA product by conducting RPA paper-chip reaction in the Petri plates. In addition, the closed structure of paper-chip with sealing tape avoided contamination of RPA reaction products. A comparison with previously reported methods showed that our paper chip-based RPA assay has adequate analytical performance with a suitable LOD, short detection time, and low-temperature detection (Table 1). We assured that our paper-chip RPA could further utilized in the detection DNA and RNA target with simple and rapid nucleic acid extraction device.


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Detection of foodborne pathogens in spiked milk sample. The practical applicability of the our assay system was verified by detecting pathogens such as E. coli and S. aureus spiked in milk sample, which typically have biological interference from proteins, hormones, glucose, ions, and other molecules.36-37 Milk samples spiked with different concentrations (such as 102, 103, 103, and 104 cfu/ml) of foodborne pathogens (such as E. coli and S. aureus) were analyzed by proposed paper-chip-based RPA method. Recoveries of E. coli in the range (97.80±1.62 to 101.56±1.73) and S. aureus in the range of (97.56±0.07 to 102.40±2.01) were obtained (Table 2). We did not find any inhibitory effect on the RPA reaction with genomic DNAs extracted from spiked milk samples. But, the direct use of milk sample spiked with genomic DNA of pathogen inhibits the fluorescence of RPA reaction. Therefore, we believe that our developed RPA-paper-chip could be utilized with simple genomic DNA extraction methods. We successfully tested the sensitivity and reproducibility of the proposed system with gram-positive and gram-negative pathogens. These results demonstrated that paper chipbased RPA can be used to reliably quantify food pathogens from complex biological samples.

CONCLUSIONS We successfully designed and tested a paper chip-based RPA assay for sensitive and selective multiplex detection of foodborne pathogens. The reaction was performed at 37°C for 20 min. The performance of our assay was comparable to that of RPA reaction in solution, but has the advantages of cost effectiveness, simple fabrication, easy manipulation with single sample loading, tolerance to sample impurities, low reaction temperature, rapidity, and multiplex detection. Our paper chip-based RPA can be integrated with simple nucleic acid extraction and used for detection of foodborne pathogens in resource-limited settings.



AUTHOR INFORMATION Corresponding Author * E-mail: [email protected] (Min-Gon Kim) ORCID Min-Gon Kim: 0000-0002-3525-0048 Author Contributions †

Heeseop Ahn and Bhagwan Sahebrao Batule contributed equally.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT The authors would like to acknowledge financial support from GIST (Gwangju Institute of Science and Technology), Korea, under the Practical Research and Development support program supervised by the GTI (GIST Technology Institute) and the Mid-career Researcher Program (NRF-2017R1A2B3010816) through a National Research Foundation grant funded by the Ministry of Science, ICT, and Future Planning.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Optimization of paper chip structure, Specificity test of paper chip-based multiplexed detection of three pathogen DNAs. Sensitivity analysis of pathogenic DNA based on RPA reaction in the solution phase, Effect of storage time on single-step RPA assay


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Figure captions

Figure 1. Schematic illustration of paper chip-based RPA reaction. (a) Fabrication and (b) operation of RPA paper chip.

Figure 2. Selection of paper substrate for the RPA reaction. ∆S/∆N in the fluorescence intensity analysis of the RPA reaction on various reaction paper substrates. ∆S, change in fluorescence intensity of the reaction pad from before to after the RPA reaction; ∆N, change in fluorescence intensity of the control pad from before to after the RPA reaction without target DNA. Data represent average values ± standard deviation of three replicates.

Figure 3. Feasibility study of PES membrane-based RPA with mixture of different target pathogens. E. coli (target 1), S. aureus (target 2), and S. typhimurium (target 3). ∆Fluorescence intensity, change in fluorescence intensity of the reaction pad from before to after RPA reactions on PES membranes. Data represent average values ± standard deviation of three replicates.

Figure 4. One-step quantitative detection of three pathogen DNAs on the RPA paper chip. E. coli (target 1), S. aureus (target 2), and S. typhimurium (target 3). ∆S, change in fluorescence intensity of the reaction pad after the RPA reaction; ∆N, change in fluorescence intensity of the control pad after the RPA reaction without primer and probe. Data represent average values ± standard deviation of three replicates.


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Figure 1.



Figure 2.


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Figure 3.



Figure 4.


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Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

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Table 1. Comparison of isothermal amplification methods using paper substrate Detection strategy

Pathogen

Paper substrate

Ebola virus

Cellulose

Limit of

Detection time

detection

and temperature

107 copies/µl

Operation steps

Ref.

20 min and 40°C

one

23

90 min and 37°C

More than five

33

Reverse transcription and recombinase polymerase amplification (RT-RPA) Nucleic acid sequencebased amplification

Zika virus

Cellulose

(NASBA) Loop-mediated isothermal

Streptococcus

amplification (LAMP)

pneumoniae

RPA Helicase-dependent amplification (HDA) HDA LAMP

1.7 × 106 copies/ml

Glass fiber

102 copies/ml

70 min and 65°C

One

24

Plasmodium

Cellulose

5 copies/µl

35 min and 37°C

More than five

12

Salmonella typhimurium

Glass fiber

102 cfu/ml

60 min and 65°C

Four

19

Cellulose

100 copies

~45 min at 65°C

More than five

34

Cellulose

100 copies

~60 min at 63°C

More than five

35

Polyethersulfone

102 cfu/ml

20 min at 37°C

One

This study

Mycobacterium tuberculosis DNA Listeria monocytogens Escherichia coli,

This work

Staphylococcus aureus, and Salmonella typhimurium


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Table 2. Detection of foodborne pathogens in the spiked milk samples. aMean of three

Food

Added pathogen (cfu/mL)

S. aureus

Recovery (%) a

(cfu/mL)a

pathogens

E. coli

Found pathogen

0

0

1.0 x 102

(0.978 ± 0.016) x 102

97.80 ± 1.62

1.0 x 103

(1.016 ± 0.017) x 103

101.56 ± 1.73

1.0 x 104

(0.963 ± 0.021) x 104

96.26 ± 2.11

1.0 x 105

(0.982 ± 0.011) x 105

98.2 ± 1.13

0

0

1.0 x 102

(0.980 ± 0.018) x 102

98.03 ± 1.81

1.0 x 103

(0.995 ± 0.022) x 103

99.46 ± 2.20

1.0 x 104

(0.972 ± 0.007) x 104

97.56 ± 0.07

1.0 x 105

(1.024 ± 0.020) x 105

102.40 ± 2.01

measurements.



For TOC only


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Single-Step Recombinase Polymerase Amplification Assay Based on

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