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Food Sensing: Aptamer-Based Trapping of B. cereus Spores with Specific Detection via Real Time PCR in Milk Christin Fischer, Tim Hünniger, Jan-Hinnerk Jarck, Esther Gesine Frohnmeyer, Constanze Kallinich, Ilka Haase, Uli Hahn, and Markus Fischer J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b03738 • Publication Date (Web): 26 Aug 2015 Downloaded from http://pubs.acs.org on August 26, 2015

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Journal of Agricultural and Food Chemistry

Food Sensing: Aptamer-Based Trapping of B. cereus Spores with Specific Detection via Real Time PCR in Milk

Christin Fischer1a, Tim Hünniger1a, Jan-Hinnerk Jarck1a, Esther Frohnmeyer1, Constanze Kallinich1, Ilka Haase1, Ulrich Hahn2, Markus Fischer1*

1

HAMBURG SCHOOL OF FOOD SCIENCE; Institute of Food Chemistry, University of Hamburg,

Grindelallee 117, 20146 Hamburg, Germany, *Corresponding author: Tel.: +49-40428384357; Fax: +49-40-428384342; E-Mail: [email protected] 2

Institute of Biochemistry and Molecular Biology, University of Hamburg, Martin-Luther-

King-Platz 6, 20146 Hamburg, Germany; E-Mail: [email protected]

a

These authors contributed equally

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Abstract

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Aerobic spores pose serious problems for both food product manufactures and consumers.

3

Milk is particularly at risk and thus an important issue of preventive consumer protection

4

and quality assurance. The spore-former Bacillus cereus is a food poisoning gram-positive

5

pathogen which mainly produces two different types of toxins, the diarrhea inducing and the

6

emetic toxins. Reliable and rapid analytical assays for the detection of B. cereus spores are

7

required, which could be achieved by combining in vitro generated aptamers with highly

8

specific molecular biological techniques.

9

For the development of routine bioanalytical approaches, already existing aptamers with

10

high affinity to B. cereus spores have been characterized by surface plasmon resonance (SPR)

11

spectroscopy and fluorescence microscopy in terms of their dissociation constants and

12

selectivity. Dissociation constants in the low nanomolar range (from 5.23 to 52.37 nM) were

13

determined. Subsequently, the characterized aptamers were utilized for the establishment

14

and validation of an aptamer-based trapping technique in both milk simulating buffer and

15

milk with fat contents between 0.3 and 3.5 %. Thereby, enrichment factors of up to 6-fold

16

could be achieved. It could be observed that trapping protocol and characterized aptamers

17

were fully adaptable to the application in milk. Due to the fact that aptamer selectivity is

18

limited, a highly specific real time PCR assay was utilized following trapping to gain a higher

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degree of selectivity.

20 21 22

Keywords

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spore trapping, milk, real time PCR, SPR, food poisoning, spores, B. cereus

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Introduction

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Aptamers are short single-stranded oligonucleotides (DNA or RNA) which are capable of

27

interacting highly specific and affine with various targets ranging from ions to whole cells,

28

due to their distinct three-dimensional structures.1-3 Aptamers have a wide scope of

29

therapeutic and diagnostic applications as well as in biological and bacterial detection

30

systems.4-11 In comparison to antibodies the chemical and physical stability of aptamers is an

31

advantage besides the in vitro production which is not limited by the natural disadvantages

32

and restrictions of animals or eukaryotic cells.12-15

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Bacillus cereus is a common food poisoning agent and closely related to B. anthracis, the

34

bacterium responsible for causing the lethal disease anthrax, and B. thuringiensis, a

35

bacterium toxic to many insect larvae.16-21 B. cereus is able to propagate under aerobic and

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anaerobic conditions. In reduced oxygen environment it is able to survive for many years in

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form of spores.22,

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concentrations up to 7 %, between 4 - 50 °C with an optimum of 28 - 35 °C, and at a pH

39

ranging from 2.8 to 10.0.24-29 B. cereus causes serious food poisoning through two different

40

groups of toxins, differentiated into diarrhea inducing and emetic toxins.30, 31 Symptoms of

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diarrhea inducing toxins include diarrhea and profuse abdominal cramping; an emetic

42

intoxication is characterized by vomiting and nausea after a shorter incubation time of about

43

0.5 to 5 h.32, 33

44

Aerobic spores are undesirable microorganisms in food; they especially occur in milk and

45

dairy products. B. cereus is known as a pathogen and spoilage microorganism for its ability to

46

produce toxins and extracellular enzymes by its metabolic activity. Thus, it is very important

47

to prevent especially warm-up foods or milk from B. cereus contaminations under aspects of

48

consumer protection and quality assurance.

23

The mesophilic and thermoduric strains of B. cereus grow at salt



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Concerning the pathogenicity (toxin producers) various legal regulations are available.

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According to article 3 section 1 in conjunction with annex 1 chapter 2 process hygiene

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criteria (chapter 2.2 milk and dairy products) of Commission Regulation (EC) No. 2073/2005

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on microbiological criteria for foodstuffs, B. cereus and the corresponding toxins are strictly

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regulated so that rapid and reliable analytical assays are mandatory.34

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The detection of B. cereus spores in milk could be fulfilled by the utilization of aptamers in

55

combination with various techniques (e. g. sensor chip technologies) to establish a rapid and

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cost-efficient trapping and detection method for routine analysis in the food industry

57

without the necessity to perform a time-consuming microbiological enrichment. Aptamers

58

with high affinity to B. cereus spores were characterized and utilized for a novel aptamer-

59

based trapping technique of B. cereus spores in milk. In order to validate the trapping

60

technique a specific optimized real time PCR method for detection of B. cereus was applied.

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35

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1.5 and 3.5 % fat) for verification of the suitability of the technique under real conditions.

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The aptamer-based trapping technique was developed to replace the necessary time-

64

consuming microbiological enrichment of B. cereus spores in routine analysis.

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The novelty of the presented work is given by the application of aptamers for spore

66

enrichment in different milk samples. The combination of an aptamer-based enrichment by

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magnetic separation and a specific real-time PCR represents a new and rapid spore detection

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system. Additionally, due to the fact that spore forming germs are important for many

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industrial food processes this enrichment technique is easily adaptable to further

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investigations. In particular the presented system could be adapted for aptamer-based

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enrichment of e.g. Bacillus spp. from soups, spices, and fruits or Clostridium spp. in chilled

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foods, cheese, and meat products.36, 37

The trapping was realized in milk simulating buffer and in sterilized milk (containing 0.3,


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Materials and methods

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Aptamers with an affinity to B. cereus spores

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The aptamers used in this study have been generated via the SELEX process (systematic

76

evolution of ligands by exponential enrichment) (unpublished data). Aptamer sequences

77

(Table 1) were purchased from Invitrogen Life Technologies GmbH (Darmstadt, Germany) for

78

further characterization.

79

Determination of dissociation constants (KD) of aptamers using SPR

80

Spore lysis

81

To generate a spore lysate of 106 CFU/mL, different B. cereus strains (Table 2) were

82

diluted in 400 µL 10 mM sodium acetate (pH 5.0) and 250 mg glass beads (∅=0.5 mm) were

83

added. The spore solution was lysed by mechanical treatment (5 min at 30 Hz, twice) using a

84

TissueLyser (Qiagen GmbH, Hilden, Germany). The supernatant with fragmented spores was

85

subsequently used for immobilization on a SPR sensor chip.

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SPR chip preparation

87

A SPR-2 biosensor system (Sierra Sensors GmbH, Hamburg, Germany) was used for SPR

88

spectroscopy. One channel of this two-channel-system, was used as active spot and

89

contained the immobilized spore lysate, the other channel with immobilized bovine serum

90

albumin (BSA) was used as a reference to detect unspecific binders. The temperature control

91

together with an integrated autosampler allowed automated measurements at a

92

temperature of 25 °C. All SPR experiments were carried out with a flow rate of 25 µL/min

93

using degassed PBS buffer containing 0.05 % Tween 20 as running buffer.

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SPR-AS-AM sensor chips (Sierra Sensors GmbH, Hamburg, Germany) with carboxylated

95

surface were used for immobilization of fragmented spores. Each sensor chip was initially 5 ACS Paragon Plus Environment


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activated for 7 min by injecting a mixture (1/1, v/v) of 100 mM N-Hydroxysuccinimide (NHS)

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and

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Subsequently, fragmented spores diluted in 10 mM sodium acetate (pH 5.0) were injected

99

for 7 min, followed by an injection of 3 min using 50 µg/mL bovine serum albumine (BSA)

100

diluted in 10 mM sodium acetate (pH 5.0). The remaining surface areas were blocked by

101

injecting 1 M ethanolamine (pH 8.5) for 7 min.

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SPR assay design

400 mM

N-ethyl-N-(dimethyl-aminopropyl)

carbodiimide

hydrochloride

(EDC).

103

The aptamers were diluted (2.5 mM, 5.0 mM, 7.5 mM and 10.0 mM) in milk simulating

104

buffer (55 mM NaCl, 20 mM MgCl2, 67 mM CaCl2, 80 mM KCl, 40 mM Tris-HCl, pH 6,6). For

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each assay, aptamers were injected onto the chip surface for 6 min in increasing

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concentrations. A dissociation time of 3 min was set to observe the binding behavior after

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completed injection. Bound aptamers were removed by injecting a mixture (1/1, v/v) of

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10 mM sodium hydroxide and 1 M sodium chloride for 30 s. Four different concentrations of

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each aptamer were measured. Response units (RU) of the spot containing spore fragments

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were subtracted by RU of the reference spot containing BSA and RU of milk simulating buffer

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injections for 6 min to obtain double-referenced SPR data. The evaluation was performed

112

using AnalyserR2 (Sierra Sensors GmbH, Hamburg, Germany) and Scrubber (BioLogic

113

Software Pty Ltd, Campbell, Australia) software. The mean values of resulting dissociation

114

constants were calculated and presented in nM (Table 2).

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Determination of aptamer selectivity via SPR

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SPR chips were prepared essentially as previously described with the following

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modifications: A SPR-4 four-channel biosensor system (Sierra Sensors GmbH, Hamburg, 6 ACS Paragon Plus Environment

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Germany) was used for SPR spectroscopy. Three spots were used as active spots and

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contained immobilized streptavidin and the three biotinylated aptamers that were used for

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subsequent aptamer-based trapping of B. cereus spores. The fourth spot was used as a

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reference with immobilized streptavidin without aptamer. The sensor chips SPR-AS-AM were

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used for immobilization of streptavidin to bind biotinylated aptamers on the sensor chip

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surface. Initially, the sensor chip was activated as mentioned and streptavidin, diluted in

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10 mM sodium acetate (pH 5.0) to a concentration of 0.9 µM, was injected for 7 min,

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followed by blocking of the remaining surface areas as mentioned. Afterwards, biotinylated

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aptamers were diluted in milk simulating buffer (10 µM) and injected for 6 min, each on one

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channel to determine the selectivity of each aptamer individually.

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SPR assay design

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For determination of aptamer selectivity, various Bacillus spp. spores (Table 2) were

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diluted in milk simulating buffer (final concentration 107 CFU/mL) and lysed as mentioned

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above. B. cereus strain MHI M1, which was used among other B. cereus strains as a selection

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target, was measured as a reference. For the assay, the spore lysates were injected for 1 min

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onto the prepared sensor chip surface. A dissociation time of 5 min was set and afterwards

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the bound spores were removed by injecting a mixture (1/1, v/v) of 10 mM sodium

136

hydroxide and 1 mM sodium chloride for 30 s. The resulted RU signals were put into relation

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to the obtained RU signal for the reference strain MHI M1 in order to classify the aptamer

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selectivity of each strain. Data analysis was performed using the AnalyserR2 (Sierra Sensors

139

GmbH, Hamburg, Germany) software.

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Fluorescence microscopy



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Fluorescence microscopy was carried out using a fluorescence correlation spectroscope

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ConfoCor2 (Carl Zeiss GmbH, Jena, Germany) to visualize the aptamer target interactions.

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Therefore, aptamers were labeled at the 5’-terminus by PCR with fluorescein modified

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primers as described as follows: After an initial denaturation at 95 °C for 5 min, 25 cycles

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(95 °C for 30 s, 58 °C for 30 s, and 72 °C for 30 s each) were carried out. For a final

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elongation, reaction mixtures were incubated for 7 min at 72 °C. The PCR mix consisted of

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the following: 1x DreamTaq buffer (10x, Fisher Scientific-Germany GmbH, Schwerte,

148

Germany), 0.8 mM dNTPs (10 mM, Bioline GmbH, Luckenwalde, Germany), 0.05 units

149

DreamTaq polymerase (5.0 U/µL, Fisher Scientific-Germany GmbH, Schwerte, Germany), 1.0

150

µM of each primer (Invitrogen Life Technologies GmbH, Darmstadt, Germany), 25 µL of the

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DNA solution, and ddH2O filled up to 50 µL. For subsequent strand separation 5´-

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biotinylated aptamer were used. Strand separation was based on binding of biotin to

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streptavidin-coated magnetic beads (DynaBeads® M-280 Streptavidin, Invitrogen Life

154

Technologies GmbH, Darmstadt, Germany) to separate ssDNA for fluorescence microscopy.

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5 nM of fluorescent labeled aptamers were incubated with B. cereus spores (106 CFU/mL) in

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350 µL milk simulating buffer for 1 h at 25 °C. After incubation, spore suspensions were

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washed by centrifugation (5 min at 10500x g) and spore pellets were resuspended in 350 µL

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milk simulating buffer for fluorescence microscopy. The analyses were carried out under

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following conditions: argon laser (5 mW), 25 % output, transmission: 15 %, wavelength:

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488 nm, 40x and 100x lens.

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Aptamer-based trapping of B. cereus spores in milk simulating buffer and milk

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Preparation of aptamer-linked magnetic beads


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Before the aptamers could be used for trapping, they had to be linked to magnetic

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SiMAG-Carboxyl Beads (chemicell GmbH, Berlin, Germany). Therefore, 200 µL SiMAG beads

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were washed twice with 1 mL MES-buffer (0.1 M 2-(N-morpholino)ethanesulfonic acid, pH

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5.0),

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dimethylaminopropyl)carbodiimide hydrochloride) and incubated under agitation for

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10 min. Afterwards, 50 µL of aminated aptamers were added and the mix was incubated for

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2 h. After incubation, magnetic beads were washed three times with 1 mL PBS buffer and

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finally redissolved in 1 mL Blocking/Storage-buffer (PBS buffer containing 0.1 % BSA, 0.05 %

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sodium azide). Before each trapping experiment the required volume of magnetic beads was

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transferred to a new reaction tube, washed and taken up in the initial volume of ddH2O. For

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the subsequent aptamer-based trapping a mixture of aptamer-linked magnetic beads

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(BacApt3, BacApt4 and BacApt5) was used to cover a wide range of potential binding sites.

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Aptamer-based trapping using magnetic separation

suspended

in

250 µL

MES-buffer

containing

10 mg

EDC

(1-ethyl-3-(3-

176

Before each experiment, the mixture of aptamer-linked magnetic beads was washed once

177

with the same volume milk simulating buffer and refolded for 5 minutes at 95 °C with

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subsequent cooling to 4 °C to ensure correct aptamer folding. Then 10 µL of aptamer-linked

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magnetic bead mixture were diluted with 940 µL (i) milk simulating buffer, (ii) milk with

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0.3 % fat, (iii) milk with 1.5 % fat, and (iv) milk with 3.5 % fat. These solutions were spiked

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each with 50 µL B. cereus spores (107 CFU/mL, used strains MHI M1 and two wild types) and

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incubated for 30 min under agitation. Thereafter, the solution was centrifuged (2 min,

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10000xg) and the supernatant was removed. The resulting bead pellets were washed three

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times with 1 mL ddH2O and finally taken up into 100 µL ddH2O. Aptamer-bound spores were

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then heat eluted for 5 min at 96 °C and the hot supernatant containing enriched spores was

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transferred to a new reaction tube. The samples were stored at -20 °C until further use. 9 ACS Paragon Plus Environment


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Real time PCR detection of B. cereus spores

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Real time PCR was performed to demonstrate the success of aptamer-based trapping.

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Therefore, the solutions containing enriched spores were diluted 1:10 with ddH2O, lysed as

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mentioned above and real time PCR assay was performed using a iCycler iQ5 (BioRad

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Laboratories, Inc.; Hercules, CA). The used real time assay and conditions have been

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described in detail elsewhere.35 The primers used in this assay target the hbID gene of

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B. cereus. Each reaction mix consisted of following components: 1x DreamTaq buffer (10x,

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Fisher Scientific-Germany GmbH, Schwerte, Germany), 0.8 mM dNTPs (10 mM, Bioline

195

GmbH, Luckenwalde, Germany) 0.25 units DreamTaq polymerase (5.0 U/µL, Fisher Scientific-

196

Germany GmbH, Schwerte, Germany), 0.25 µM of each primer (mp3L1R1for and

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mp3L1R1rev, Invitrogen Life Technologies GmbH, Darmstadt, Germany), 0.3125x SYBR Green

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I nucleic acid gel stain (10000x, Invitrogen GmbH, Karlsruhe, Germany), 3 µL enriched and

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lysed spore solution and ddH2O filled up to 20 µL. After an initial denaturation at 95 °C for

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10 min, 35 cycles (95 °C for 15 s, 60 °C for 10 s, and 72 °C for 10 s each) were carried out. For

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final elongation, the reaction mixtures were incubated for 10 min at 72 °C. For quantitation

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and comparison, an external calibration with B. cereus (wild type, 103 to 107 CFU/mL) was

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applied. A milk simulating buffer/milk sample spiked at a concentration equal to pre-

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enrichment was used as control. The assay was additionally performed using B. subtilis and

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B. thuringiensis spores as template to demonstrate the specificity of the protocol.

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Results and discussion

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The presented work includes the characterization of aptamers with an affinity to B. cereus

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spores by SPR and fluorescence microscopy and subsequent development of an aptamer-

210

based trapping technique in milk. The trapping was performed in both milk simulating buffer

211

and milk containing different fat contents. Moreover its effectiveness was demonstrated by

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real time PCR.35

213

Determination of dissociation constants (KD) of aptamers using SPR

214

Suitable SPR sensor chips were prepared to determine the dissociation constants (KD) of

215

the aptamers to B. cereus spores. Immobilization of spores fragmented using glass beads and

216

a TissueLyser enabled the preparation of suitable sensor chips for the determination of

217

dissociation constants (KD).

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Spore lysis

219

The degree of spore fragmentation proofed to be a crucial parameter for successful

220

sensor chip preparation. Thus, several treatments (e.g. ultrasonic disruption), pH-values, and

221

spore fragmentation methods were investigated and assessed (data not shown). The

222

developed mechanical treatment with glass beads is applicable for fragmentation of spores

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and supplies the ideal degree of spore fragmentation for subsequent immobilization on

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carboxylated SPR sensor chips.

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During immobilization of fragmented spores on activated SPR sensor chip surfaces

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approximately 400 - 800 RU could be observed. According to Karlsson one RU corresponds to

228

approximately 1 pg immobilized fragmented compounds per mm2 38. Thus, on a sensor chip 11 ACS Paragon Plus Environment


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surface of approximately 1.2 mm2, 440 - 960 pg fragmented spores can be immobilized. The

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high amount of immobilized fragments allows the conclusion that a sufficient amount of

231

spore coat fragments were immobilized and therefore the developed SPR immobilization

232

method is suitable for investigations concerning interaction of the aptamers and spore

233

surfaces.

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SPR assay

235

Regeneration conditions were obtained using sodium hydroxide (10 mM) and sodium

236

chloride (1 M) (1/1, v/v) and a contact time of 30 s. The stability and the activity of the chip

237

surface were further monitored by multiple injections of an aptamer solution (10 mM) and

238

subsequent regeneration. Due to the obtained response signals of 214 RU (first aptamer

239

injection) and 191 RU (50th aptamer injection) a decrease of approximately 10.75 % in

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surface activity was determined which is less than the recommended cut-off value of 20 %.39,

241

40

242

requirements.

Therefore the developed SPR chip surface regeneration procedure fulfilled the compulsory

243

SPR analyses were carried out using milk simulating buffer, because the selected

244

aptamers will be used for applications in milk. Milk itself was not suitable for SPR

245

measurements because of its fat content and the included milk sugar. Therefore, the

246

selection conditions were chosen as far as possible similar to the conditions of milk by using

247

a simulating buffer. Dissociation constants (KD) of aptamers were measured by

248

immobilization of fragmented spores on chip surface and injection of several aptamer

249

concentrations (2.5 mM, 5.0 mM, 7.5 mM, and 10.0 mM). Exemplary overlays of aptamer

250

injections are shown in Figure 1 and Figure S1. RU of spore fragment containing spots were

251

subtracted by RU of reference spots containing BSA and injections of milk simulating buffer

252

for 6 min to obtain double referenced SPR data. The obtained curves were fitted in 12 ACS Paragon Plus Environment

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accordance with the Langmuir 1:1 model including a mass transport limitation factor to

254

determinate the dissociation (Figure 1). Although an appropriate multivalent binding kinetic

255

could be more precise for performed experiments, it is not yet available, so that the

256

Langmuir binding model was chosen. SPR data were normally distributed according to David

257

(confidence intervals of 90 %) regarding the calculation of dissociation constants.41

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Furthermore, no outlier according to Dixon (confidence intervals of 95 %) was identified.42 A

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summary of calculated KD values is shown in Table 1.

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In summary, the dissociation constants of aptamers with an affinity to B. cereus spores could

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be detected in a low-nanomolar range. Hence, the selected aptamers were considered

262

suitable for the subsequent development of aptamer-based trapping technique.

263

Determination of aptamer selectivity via SPR

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Aptamer selectivity was determined to investigate the specificity of our trapping protocol

265

and the aptamers used in this study. For that, the three aptamers applied for capturing were

266

immobilized onto a SPR chip surface and binding interaction differences were observed

267

between aptamers and B. cereus spores as well as various other Bacillus spp. spores (Figure

268

S2). The spores were chosen for their degree of relationship to B. cereus and included both

269

closely related (B. thuringiensis and B. subtilis) and more distant spores (e.g. B.

270

weihenstephanensis and B. coagulans). The selectivity results were calculated in relation to

271

the strain B. cereus MHI M1, which had been one of the targets for aptamer selection. MHI

272

M1 was chosen as a reference because it was expected that aptamers have the highest

273

affinity to their selection target.

274

Principally, all aptamers exhibited a higher affinity to B. cereus spores in comparison to

275

other Bacillus spores (Figure 2). However, the selectivity was not equal for all B. cereus

276

strains used in this assay. This could be due to small differences in surface composition and 13 ACS Paragon Plus Environment


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spore lysates preparation. For instance, the affinity of BacApt3 to B. cereus AH 187 is much

278

higher than to the reference strain MHI M1.

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Figure 3 graphically describes the affinity of the three used aptamers to all Bacillus spores

280

in comparison. It can be clearly seen that, unsurprisingly, the selectivity towards B. cereus

281

spores is highest, followed by closely related spores, i.e. B. subtilis and B. thuringiensis

282

spores. Considerably lower interactions between the selected aptamers and more distantly

283

related spores could be observed. In conclusion, the use of the selected aptamers for highly

284

selective trapping of targets is limited due to structural similarities of Bacillus spp. spores.

285

However, high selectivity can be achieved by performing a highly selective real time PCR

286

following trapping which excludes closely related spores.43 Real time PCR results will be

287

discussed later.

288

Fluorescence microscopy

289

Fluorescence microscopy was carried out to visualize the interactions of aptamers with

290

B. cereus spores and therefore to verify the aptamer affinities. Spores without prior

291

incubation were used as a blank sample to define the optimal settings for fluorescence

292

microscopy. Adjustment of the blank sample settings was necessary to obtain low intrinsic

293

fluorescence of spores (shown in Figure 3, 1A to 1C). The aptamer spore complexes were

294

investigated by microscopy after incubation of fluorescent-labeled aptamers with spores and

295

corresponding washing steps. Furthermore, to generate representative fluorescence images,

296

a mixture of all nine aptamers was used for fluorescence microscopy (shown in Figure 3, 2A

297

to 2C, 3A to 3C).


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The visualization of aptamer spore interactions by fluorescence microscopy confirmed the

299

affinity of selected aptamers to B. cereus spores and underlined the obtained SPR results,

300

respectively.

301

Aptamer-based trapping of B. cereus spores in milk

302

Aptamer based trapping of B. cereus spores using real time PCR detection

303

To verify the suitability and specificity of the used real time PCR assay, the PCR was

304

performed using B. cereus, B. subtilis, and B. thuringiensis spores (each 103 CFU/mL). An

305

external calibration was used (Figure 4 A and B) for which a regression coefficient of 0.99

306

was obtained. The results demonstrate that B. cereus spores were amplified while B. subtilis

307

and B. thuringiensis spores show no amplification in obtained threshold cycle number (data

308

not shown). The real time PCR assay thus proofed to be a highly specific tool for detection

309

and quantitation of B. cereus spores after trapping.

310

Trapping was performed in three different matrices and milk simulating buffer. Milk

311

simulating buffer was included as a trapping matrix since aptamers are expected to achieve

312

highest target affinities in their selection buffer. This is due to conformational changes

313

caused by e.g. ion concentration and pH value of the medium. Milk simulating buffer was

314

chosen for aptamer selection as it on one hand simulates milk in ion concentration and pH

315

value and on the other hand does not contain substances like sugars and fat which could

316

disturb the SELEX process. Milk with three different fat contents was selected to

317

demonstrate the applicability of the trapping technique under real conditions. This was done

318

to evaluate a possible interference of fat and sugar with aptamer confirmation.

319

Overall enrichment factors between 3.1 and 7.5 (Tab. 3) could be achieved. As expected, the

320

highest enrichment factor was observed in milk simulating buffer (6.0 ± 1.0 on average). 15 ACS Paragon Plus Environment


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Trapping was also successful in milk, however at a lower efficiency in comparison to milk

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simulating buffer (on average 3.5 ± 1.0 to 5.4 ± 1.9). No significant differences were

323

observed between different fat contents. Thus, it can be assumed that fat content is not a

324

relevant parameter for trapping. The successful enrichment in milk also allows the

325

assumption that milk sugar does not influence the aptamer-target interaction because

326

aptamers shows high affinity despite milk samples containing milk sugar.

327

The ion strength of milk (or milk simulating buffer) has been shown to be a disruptive factor

328

for subsequent detection and quantitation via real time PCR. Therefore it was necessary to

329

include several washing steps of aptamer-linked magnetic beads with ddH2O following

330

trapping in order to achieve successful real time PCR quantitation. Furthermore, PCR results

331

were improved by diluting the samples prior to measurement to minimize the effect of

332

residual ions (data not shown).

333 334

In this study, we demonstrate the successful aptamer-based entrapment of B. cereus spores

335

in milk simulating buffer as well as in milk with different fat contents. The presented work

336

includes an extensive characterization of DNA aptamers with an affinity to B. cereus spores.

337

The detection of vegetative cells was not considered relevant for the presented study

338

because pasteurization, which is typically performed during the production of milk,

339

inactivates vegetative B. cereus cells and only spores survive heat treatment. The aptamers

340

were characterized by SPR and fluorescence microscopy to gain dissociation constants and

341

to determine the selectivity of the aptamers used for trapping. Aptamer affinities in a low-

342

nanomolar range could be obtained. However, more detailed binding properties in terms of

343

kinetics and binding sites are to be determined by further research. Thus, selecting only one

344

aptamer for developed detection methods seems not advisable. Due to the presumption of 16 ACS Paragon Plus Environment

Page 17 of 32


345

multivalent binding kinetics, utilization of an aptamer mix for aptamer-based trapping was

346

chosen. It was shown that trapping was successful in buffer and different milk samples,

347

whereby as expected the best enrichment factor was achieved by using milk simulating

348

buffer. Moreover, it was shown that the usage of aptamers to perform a high selective

349

trapping is limited. We recommend to perform a B. cereus specific real time PCR following

350

trapping to ensure a reliable detection.44

351

In general, it was shown that aptamers are suitable for applications in trapping and

352

detection tools. Further research could amplify the range of aptamer-based application e. g.

353

in Apta-PCR, which allows an indirect detection of targets at low concentrations.45,

354

Moreover the development of aptamer-based lateral flow dipsticks/devices (LFDs) could

355

fulfill many expectations for manufacturing industry, because they enable a specific, rapid,

356

and simple optical target detection with less expenditure of instrumentation due to

357

interactions with specific aptamers.47, 48

46

358



Page 18 of 32

359

Abbreviations

360

SELEX, systematic evolution of ligands by exponential enrichment; ssDNA, single stranded

361

DNA; PCR, polymerase chain reaction; ATP, adenosine triphosphate; FACS, fluorescence-

362

activated cell sorting; SPR, surface plasmon resonance; NHS, N-Hydroxysuccinimide; EDC, N-

363

ethyl-N-(dimethyl-aminopropyl) carbodiimide hydrochloride; BSA, bovine serum albumin;

364

LFD, lateral flow dipstick;

365

Acknowledgements

366

We acknowledge Sven Malik from Sierra Sensors GmbH, Hamburg, for the support with the

367

SPR evaluations, and Hauke Wessels from the HAMBURG SCHOOL OF FOOD SCIENCE;

368

Institute of Food Chemistry, University of Hamburg for proof reading. Furthermore, we

369

acknowledge our project partners at the Ludwig-Maximilians-Universitaet Muenchen,

370

Lehrstuhl fuer Hygiene und Technologie der Milch (Oberschleißheim, Germany) for support.

371

Funding

372

This research project was supported by the German Ministry of Economics and Technology

373

(via AiF) and the FEI (Forschungskreis der Ernaehrungsindustrie e. V., Bonn, Germany);

374

Project AiF 331 ZN.

375

Associated Content

376

Supporting information: Overlays of several BacApt injection sensorgrams for the KD

377

determination.

378 379


Page 19 of 32


380

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Page 24 of 32

Figure Captions Figure 1: Exemplary overlay of fitted raw data (BacApt2) for evaluation of dissociation constants (KD). The raw data were fitted using the Langmuir 1:1 model including mass transport limitation. The data was analyzed using AnalyserR2 (Sierra Sensors GmbH, Hamburg, Germany) and Scrubber (BioLogic Software Pty Ltd, Campbell, Australia) software. Figure 2: Aptamer selectivity of BacApt3, BacApt4 and BacApt 5 determined by SPR measurements with spores of different Bacillus strains. As a reference, the affinity of B. cereus MHI M1 was set to 1. The selectivity of other spores is assigned as relative affinity compared to the reference. Figure 3: Fluorescence microscopy recordings of B. cereus spores in milk simulating buffer as blank sample (1A to 1C with 40 x lens). Exemplary images of spores after incubation with a fluorescently labeled mix of obtained aptamers (2A to 2C and 3A to 3C with 40 x lens). (A) fluorescence images; (B) visual images; (C) overlays of the fluorescence and visual images. Figure 4: (A) Example of amplification curves during real time PCR with different B. cereus MHI M1 spore concentrations (from left to right): 107 CFU/mL, 106 CFU/mL, 105 CFU/mL, 104 CFU/mL, 103 CFU/mL and 0 CFU/mL. (B) Resulting calibration line during real time PCR with R2 = 0.99. Spore concentration (from left to right): 0 CFU/mL, 103 CFU/mL, 104 CFU/mL, 105 CFU/mL, 106 CFU/mL, 107 CFU/mL.


Page 25 of 32


Figure graphics Figure 1



Page 26 of 32

Figure 2


Page 27 of 32


Figure 3

B.



Page 28 of 32

Figure 4


Page 29 of 32


Tables Table 1: Sequences of the used aptamers and calculated dissociation constants for B. cereus

spores. Primer regions of aptamers were marked as italic. Aptamer

Aptamer sequence (5’-3’)

KD [nM]

BacApt1

CATCCGTCACACCTGCTCGGTGCAGACCCATAGGGGGGGCGT GCGGATGTGCGGAGTAGGGTGTTGGCTCCCGTATC

5.73

BacApt2

CATCCGTCACACCTGCTCCCCAATGAAGCGAGATGGACGCCTA GCACCCCCCGCGTCCGGTGTTGGCTCCCGTATC

6.84

BacApt3

CATCCGTCACACCTGCTCGGTGCAGACCCATAGGGGGGGCGT GCGGATGTAGGAGTAGGGTGTTGGCTCCCGTATC

35.52

BacApt4

CATCCGTCACACCTGCTCCCAGCGTGCGTCGACCCGGACCCCT GTCAGCCCCCTCGCGGGTGTTGGCTCCCGTATC

44.57

BacApt5

CATCCGTCACACCTGCTCCAGGTGGGGGGGCGTATTACTGAG GCAGAGTAGTTGGCCGGGTGTTGGCTCCCGTATC

19.12

BacApt6

CATCCGTCACACCTGCTCCATTGACGTTGTCAGGTAATGGTTTG GGAGGTCGTGGTGTGGTGTTGGCTCCCGTATC

22.16

BacApt7

CATCCGTCACACCTGCTCGCCGGGAGAACGGTACTGGTGGGG GATGACAGCTCGGGGGGGTGTTGGCTCCCGTATC

23.54

BacApt8

CATCCGTCACACCTGCTCCCGCCAGGCAATGCCTGCCGCGTCTC 31.56 GAACACGTACGGTCGGTGTTGGCTCCCGTATC

BacApt9

CATCCGTCACACCTGCTCGCACGGGTGGTTGGTCACGCCTAGT CTCCAATTGCGTTGCGGTGTTGGCTCCCGTATC

52.57



Page 30 of 32

Table 2: Bacillus spp. strains used in this study for determination of aptamer selectivity. Bacillus spp.

strain

B. cereus

MHI M1*

B. cereus

AH 187

4x B. cereus

wild types

B. weihenstephanensis

wild type

B. thuringiensis

ATCC 10792

B. subtilis

DSM2109

B. subtilis

ATCC 6633

B. licheniformis

DSM 13

B. coagulans

ATCC 7050

B. circulans

ATCC9966

B. sphaericus

ATCC 245

Paenibacillus polymixa

ATCC10401

*

This strain was used for aptamer selection.


Page 31 of 32


Table 3: Obtained enrichment factors for aptamer-based trapping in milk simulating buffer

and milk with different fat contents. The maximal trapping factor was determined mathematically to 10. matrix

milk simulating

milk

milk

milk

strain

buffer

0.3 % fat

1.5 % fat

3.5 % fat

B. cereus wild type

5.2

3.5

4.3

4.0

B. cereus wild type

5.7

3.1

6.7

4.8

B. cereus MHI M1

7.1

3.9

3.2

7.5

mean value

6.0 ± 1.0

3.5 ± 0.4

4.7 ± 1.8

mean value 2

5.4 ± 1.9

4.91 ± 1.07



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