Accepted Manuscript Title: Multiplexed detection of foodborne pathogens based on magnetic particles Author: Delfina Brand˜ao Susana Li´ebana Mar´ıa Isabel Pividori PII: DOI: Reference:

S1871-6784(15)00053-9 http://dx.doi.org/doi:10.1016/j.nbt.2015.03.011 NBT 776

To appear in: Received date: Revised date: Accepted date:

2-10-2014 16-3-2015 22-3-2015

Please cite this article as: Brand˜ao, D., Li´ebana, S., Pividori, M.I.,Multiplexed detection of foodborne pathogens based on magnetic particles, New Biotechnology (2015), http://dx.doi.org/10.1016/j.nbt.2015.03.011 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.

1 2

Multiplexed detection of foodborne pathogens based on magnetic particles

3 4

ip t

5

1

7

Grup de Sensors i Biosensors, Departament de Química, Universitat Autònoma de

Applied Enzyme Technology Ltd., Gwent Group Ltd., Monmouth House, Mamhilad Park, Pontypool, NP4 OHZ, UK.

an

2

us

Barcelona, 08193 Cerdanyola del Vallès (Bellaterra), Spain

8 9 10

cr

Delfina Brandão1, Susana Liébana2 and María Isabel Pividori1*

6

M

11

* Tel: +34 93 581 4937, Fax: +34 93 581 2473. E-mail address:

13

*Authors to whom correspondence should be sent: [email protected]

pt Ac ce

14

ed

12

1

Page 1 of 34

Abstract

15

This paper addresses the novel approaches for the multiplex detection of food poisoning

16

bacteria, paying closer attention to three of the most common pathogens involved in

17

food outbreaks: Salmonella enterica, Escherichia coli O157:H7 and Listeria

18

monocytogenes. End-point and real-time PCR, classical immunological techniques,

19

biosensors, microarrays and microfluidic platforms, as well as commercial kits for

20

multiplex detection of food pathogens will be reviewed, with special focus on the role

21

of magnetic particles in these approaches. Although the immunomagnetic separation for

22

capturing single bacteria from contaminating microflora and interfering food

23

components has demonstrated to improve the performance on these approaches, the

24

integration of magnetic particles for multiplex detection of bacteria is still in a

25

preliminary stage and requires further studies.

26

Keywords: Food safety, simultaneous detection, magnetic particle, electrochemical

27

biosensor, multiplex PCR, food pathogens.

cr

us

an

M

ed

pt Ac ce

28

ip t

14

2

Page 2 of 34

Introduction

29

Food safety in Europe

30

Food contamination caused by pathogens is a significant public health concern for

31

consumers worldwide. Therefore, identification and detection of microorganisms in

32

food processing play an important role at control programs provided by Food regulatory

33

agencies, in order to prevent food outbreaks [1, 2]. In 2013, it was reported by the Rapid

34

Alert System for Food and Feed (RASFF) that pathogenic microorganisms, mycotoxins

35

and pesticide residues were the main cause of notification in Europe, especially for meat

36

products, as well as for fruits and vegetables [3]. For instance, the notifications of

37

pathogens in meat products are shown in Figure 1, being Salmonella, E. coli and L.

38

monocytogenes the most common reported pathogens.

M

an

us

cr

ip t

28

39 40

ed

Preferred position for Figure 1

41

Hence, food safety standards, such as RASFF were introduced in Europe in order to

43

prevent foodborne outbreaks and to ensure that marketed products are safe to the

44

consumers.

45

Preventive approaches like Hazard Analysis and Critical Control Point (HACCP) and

46

the Codex Alimentarius were also successfully implemented, which can considerably

47

reduce the survival of pathogens during the process of handling, preparation and storage

48

of food. But there is still a need for the development of new tools and technologies to

49

prevent problems related to food safety, including rapid approaches for the

50

identification and quantification of foodborne pathogens [8, 9].

Ac ce

pt

42

51

3

Page 3 of 34

Traditional methods for the detection of foodborne pathogens

53

Threshold limits for the presence of certain microorganisms had been set down for some

54

food products. If the food product contains an amount above the required legislation, it

55

must be rejected from the market. Therefore, in this section, the traditional methods

56

commonly used for the single detection of foodborne pathogens will be discussed, with

57

special focus on their advantages and disadvantages.

ip t

52

cr

58

Conventional microbiological culture techniques

60

Conventional microbiological culture techniques are currently the gold standard for

61

isolation, detection, and identification of microorganisms. These methods are very

62

simple, consisting in the following steps: pre-enrichment, selective enrichment,

63

selective plating, biochemical screening and serological confirmation. Although they

64

are considered to be reliable, they are also time-consuming, laborious and might

65

introduce sampling and enumeration errors, due to the low concentration of pathogenic

66

bacteria in food samples [9, 10].

an

M

ed

pt

67

us

59

Immunological assays

69

Immunological assays (IAs) relies on the specificity of the antigen-antibody

70

recognition, being suitable for the detection of whole bacterial cells or specific cellular

71

components as lipopolysaccharides or other biomolecules present on the bacterial outer

72

membrane. Enzyme Linked ImmunoSorbent Assays (ELISAs), such as sandwich with

73

direct and indirect labelling are the most common formats used for the detection of

74

pathogens. ELISA methods have been approved by regulatory agencies, being

75

commercially available. The limit of detection (LOD) for pathogens are normally in the

76

range of 104 and 105 CFU mL-1 and the assay time can take around 48 h, since a pre4

Ac ce

68

Page 4 of 34

enrichment step is commonly required in order to achieve the threshold limits for the

78

presence of the bacteria on food samples [8- 10].

79

Immunomagnetic separation (IMS) is also an example of an immunoassay commonly

80

used for bacteria detection. In this technique, superparamagnetic particles modified with

81

a variety of molecular groups are coated with antibodies specific to the target bacteria.

82

Therefore, bacteria will be captured and concentrated either from a culture medium or

83

from complex food matrices. The use of magnetic particles provides several advantages

84

to IAs such as (i) pre-concentration of the target bacteria into smaller volumes for

85

further testing (ii) reducing and simplifying the pre-enrichment step (iii) eliminating the

86

matrix effect of the food components [11].

87

Magnetic particles can also be modified with bacteriophages for capturing and pre-

88

concentration of bacteria, in an ELISA-like format, named as phagomagnetic

89

immunoassay, decreasing significantly the LOD of a classic immunoassay, up to 19

90

CFU mL−1 in 2.5 h without any pre-enrichment in milk samples [12].

91

Therefore, IAs are advantageous for decreasing the assay reaction time in comparison

92

with microbiological culturing techniques, providing also the possibility of being easily

93

integrated in automated equipments, which consists an important advantage for

94

industrial applications. Nevertheless, the efficiency of an immunoassay is strongly

95

dependent on the antibodies affinity and specificity towards the target pathogen. The

96

risk of antibody cross-reactions consists of a disadvantage of immunological assays by

97

increasing the possibility of false positive results or high background signals [8- 11].

Ac ce

pt

ed

M

an

us

cr

ip t

77

98 99

Nucleic acid amplification methods

100

Nucleic acid amplification methods include end-point polymerase chain reaction (PCR)

101

and real-time PCR (qPCR) for single or multiplex detection of bacteria. PCR allows the 5

Page 5 of 34

production of multiple copies of DNA from the amplification of a single copy or a few

103

copies of a DNA template. Due to its high sensitivity, nucleic acid amplification has

104

been widely used for the identification and detection of pathogens in food samples,

105

being considered as an alternative to conventional microbiological culture techniques.

106

As occurred in the immunological assays, PCR methodologies require an enrichment

107

step, being able to detect, for instance in the case of Salmonella, few CFUs in 25 g of

108

food product. The fact that this methodology does not discriminate between live of dead

109

cells are pointed out as the main limitations [8- 13].

110

Hence, it was shown that traditional methodologies can be sensitive for food

111

microbiological control. However, the stricter and increased legislations and controls to

112

implement public food safety lead mostly to a need for the development of rapid

113

methodologies. In this context, the development of new methodologies with

114

multiplexing capabilities becomes an important advantage presenting a cost effective

115

and time saving strategy.

cr us

an

M

ed

116

ip t

102

Immunosensors, DNA biosensors and phagosensors

118

Over the recent years, a lot of effort has been directed into the study and development of

119

rapid methods for foodborne pathogens as an alternative methodology to

120

microbiological culturing, IAs and PCR approaches [14]. Biosensors technology has

121

been revolutionised research conducted in food safety. These devices are available in a

122

wide range of readout platforms, such as surface plasmon resonance [15],

123

electrochemical [16] and various kinds of optical biosensors [17]. Most of the current

124

developed biosensors for pathogenic bacteria are based on the specific antigen –

125

antibody binding reactions, where the antibody is immobilised on the sensor platform to

126

capture the bacteria that are of interest. Then, the bacteria detection is measured through

Ac ce

pt

117

6

Page 6 of 34

electrochemical,

optical,

or

piezoelectric

readouts.

[18]

Moreover,

genetic

128

biorecognition is also widely used in biosensing, as well as the biorecognition through

129

bacteriophages. The integration of magnetic particles in immunosensors, genosensors

130

and phagosensors was reported, for instance, in electrochemical based approaches,

131

improving the analytical performance in terms of LODs. A magneto immunosensor

132

with electrochemical readout was reported for the detection of Salmonella in milk

133

(Figure 2, A). In this approach, the bacteria were captured and pre-concentrated from

134

milk samples with magnetic micro or nanoparticles through an immunological reaction.

135

A second polyclonal antibody labelled with peroxidase was used for the electrochemical

136

detection based on a magneto-electrode [16, 19]. This strategy was able to detect 1x104

137

CFU mL-1 in 1h. If the sample is pre-enriched for 8 h, as low as 2.7×CFU in 25 g of

138

milk were detected accordingly with the legislation. Another approach involves the lysis

139

of the captured bacteria after immuno or phagomagnetic separation, followed by

140

amplification of the genetic material by PCR with a double-tagging set of primers

141

(Figure 2, B). Then, the double-tagged amplicon was immobilised on streptavidin-

142

modified magnetic beads based on a high affinity interaction though the biotin tagging

143

the 5`end DNA of the amplicon, while the digoxigenin label was used for the enzymatic

144

reaction. The electrochemical detection was finally achieved by an enzyme marker,

145

such as anti-digoxigenin horseradish peroxidase (HRP). A LOD of 1 CFU mL-1 was

146

obtained in 3.5 h without any pre-treatment. If the milk is pre-enriched for 6 h, the

147

method is able to feasibly detect as low as 1 CFU in 25 g of milk [20, 21]. The

148

integration of the magnetic particles improved the analytical performance of these

149

approaches, providing the pre-concentration of the bacteria during the IMS, reducing

150

the time required for the pre-enrichment step and the LODs, eliminating the matrix

Ac ce

pt

ed

M

an

us

cr

ip t

127

7

Page 7 of 34

151

effect of the food components and PCR inhibitors, and serving also as a platform for the

152

electrochemical readout based on magneto-actuated electrodes.

153 154 155

ip t

Preferred position for Figure 2

Simultaneous detection of pathogenic bacteria

157

In this section, different methods for the simultaneous detection of pathogenic bacteria

158

based on multiplex PCR, DNA microarrays and biosensors will be fully reviewed and

159

summarised in the Tables 1 and 2.

an

us

cr

156

160

Multiplex PCR

162

End-point multiplex PCR (mPCR) has been widely used to detect simultaneously

163

multiple targets in the same amplification reaction. The efficiency of this methodology

164

is strongly dependent on different factors, such as primers specificity, buffer,

165

magnesium chloride and Taq DNA polymerase concentration, thermal cycling

166

conditions and the amount of DNA template. [22] When applied to the detection of

167

foodborne pathogenic bacteria, a pre-enrichment step is required in order to enhance

168

detection of pathogens in samples. Recently, mPCR for the detection of five different

169

pathogens in artificially contaminated pork samples was reported [23- 24]. In Table 1,

170

selected studies published in the literature are shown, summarising and highlighting the

171

most important parameters in terms of the analytical performance, including the food

172

matrix, the time required for the pre-enrichment step, the total assay time and the LODs.

173

For instance, the simultaneous detection of E. coli, Salmonella spp. and L.

174

monocytogenes by end-point multiplex PCR showed a LOD of 10 CFU in 25 g of

Ac ce

pt

ed

M

161

8

Page 8 of 34

sample within 15 h of pre-enrichment [25]. The detection by end-point mPCR usually

176

requires the analysis of the PCR final products on an agarose gel or on capillary

177

electrophoresis-based DNA sequencer, being for this reason more laborious and time

178

consuming than Real Time PCR (qPCR), which provides the detection and

179

quantification during the amplification process in each cycle. In this methodology, the

180

fluorescence intensity of the amplicons is measured by either intercalation of

181

fluorescent dyes in the double-stranded DNA or with dual-labelled fluorescent

182

oligonucleotide probes, among others readout strategies [26]. The application of qPCR

183

for simultaneous detection has been studied for several years and it requires the use

184

oligonucleotides tagged with different fluorophores specific for the different

185

microorganisms to be detected. Moreover, in some cases an internal amplification

186

control (IAC) is recommended to be added into the PCR mixture [26, 27], which

187

consists on a non-target DNA sequence, in order to avoid false-negative results caused

188

by inhibitors, such as phenolic compounds, fats and glycogen, that may affect different

189

steps of the qPCR method. qPCR procedures are in general faster than the related

190

conventional methodology, with similar sensitivity. Nevertheless, expensive bench top

191

equipment and high technical requirements are the main limitation of this methodology.

192

Recently, it has been reported in the literature the combination of magnetic particles

193

with PCR based methodologies. As an example, a rapid and simultaneous detection of

194

Salmonella, Shigella, and Staphylococcus aureus in fresh pork was reported, being able

195

to detect all pathogens at 10 CFU g-1, within 6 h [28, 29]. In this example, the use of

196

magnetic particles provided a significant decrease of the total assay time, including the

197

pre-enrichment step. This strategy is already commercially available for single

198

pathogens detection, proving the potential application of magnetic particles in food

199

industry.

Ac ce

pt

ed

M

an

us

cr

ip t

175

9

Page 9 of 34

200

Microfluidic approaches

202

Microfluidic systems provide several advantages for the simultaneous determination of

203

multiple foodborne bacterial pathogens, such as portability, lower reagent consumption,

204

rapidity and possibility for automation. Usually, these systems are combined with

205

agarose gel electrophoresis for DNA analysis, but they can also be coupled with other

206

platforms [30]. The risk of cross-contamination in the process of sample loading is

207

pointed out as one of the main inconvenients of these systems [31]. In the Table 1, some

208

approaches for the simultaneous detection of pathogens using PCR based microfluidic

209

systems are presented. For instance, the simultaneous detection of S. enterica, E. coli

210

and L. monocytogenes was reported within 35 min, with LODs of 399, 314, and 626

211

DNA copies per μL, respectively [32].

M

an

us

cr

ip t

201

212 213 214

ed

Preferred position for Table 1

DNA microarrays

216

DNA microarrays were reported for the detection and identification of several

217

pathogens. In these arrays, a DNA probe or oligonucleotide is immobilised at fixed

218

positions on a substrate and used to capture the target molecule through hybridisation of

219

the amplified DNA, offering in this way higher capacity for multiplexing, as well as the

220

possibility of miniaturisation and automation [33]. Several examples of DNA

221

microarrays for the simultaneous detection of foodborne pathogens are summarised on

222

the Table 1. In this context, it is emphasised that DNA microarrays provide an

223

important advantage of bacteria screening in a high number of food samples. The

224

detection is achieved by measuring the fluorescence intensity, which presents the

Ac ce

pt

215

10

Page 10 of 34

limitation of being expensive and non-portable. Alternatively, colorimetric methods and

226

biochips combined with chemiluminescent labels can be used. Therefore, a DNA

227

microarray based on a colorimetric detection was reported using digoxigenin and biotin

228

labelled on the DNA. The simultaneous detection of Salmonella spp., Shigella spp., L.

229

monocytogenes, and E. coli was achieved with a LOD of 105 CFU mL-1, without any

230

enrichment step [34]. A pathogen detection microarray combined with PCR

231

methodologies with fluorescence readout was developed for the simultaneous detection

232

of E. coli, S. enterica, L. monocytogenes and Campylobacter jejuni being able to detect

233

as low as 103 CFU mL-1 of culture medium or food sample [35].

234

Combination of DNA microarrays with nanomaterials are also being explored as an

235

alternative to overcome problems related to photo bleaching caused by fluorescent the

236

organic dyes. In this context, the identification of twelve bacterial strains, using

237

quantum dots coated with streptavidin as fluorescent labels was achieved with a LOD of

238

10 CFU mL-1 in pure culture, without any enrichment step [36]. Furthermore, magnetic

239

particles can also be integrated on DNA microarrays opening the possibility of final

240

detection based on a digital camera or a light microscopy [37]. In this context, it was

241

reported the detection of E. coli, S. enterica, C. jejuni, presenting LODs of 136, 500,

242

and 1 CFU mL-1, respectively, without any enrichment procedures. [38]

243

Nevertheless, the microarray fabrication and also the hybridisation procedure can be

244

time consuming, presenting the main disadvantage of this methodology.

cr

us

an

M

ed

pt

Ac ce

245

ip t

225

246

Biosensors

247

Over the past years, a new challenge has been attracting researchers in this field, the

248

design of novel biosensors with multiplexing capabilities, where the integration of

249

nanomaterials plays an important role. These novel bionanomaterials, including 11

Page 11 of 34

nanostructured carbon materials, inorganic nanoparticles (i.e., semi-conducting, noble

251

metal and magnetic nanoparticles), among others, appears to be keys in bacteria

252

multiplex detection in biosensors, enhancing the biological reactions, providing high

253

selectivity and improving the LODs [39, 40, 41, 42].

254

In this section, different biosensor strategies will be discussed. These strategies are

255

summarised in the Table 2, with special focus on the assay type, detection technique,

256

food matrix, LODs and assay times, being classified according to the type of

257

nanomaterial integrated in each approach.

us

cr

ip t

250

258 259

an

Preferred position for Table 2

260

M

261

Biosensing based on metallic nanomaterials

263

Metallic nanomaterials, such as gold or silver nanoparticles, as well as gold films are

264

the most common selection for the immobilisation of biomolecules and/or signal

265

amplification. In this context, a biosensor for the detection of Salmonella and E. coli

266

based on Raman spectroscopy was reported, in which gold, silver and core–shell

267

nanoparticles were coated with a Raman reporter molecule to improve the LOD of the

268

assay [43]. This strategy was able to detect both bacteria with a LOD of 102 CFU mL-1

269

in 45 min. A multi-channel SPR biosensor for the simultaneous detection of S.

270

Typhimurium, L. monocytogenes, C. jejuni, and E. coli based on a sandwich

271

immunoassay was also reported, presenting LODs of from 3.4×103 to 1.2×105 CFU mL-

272

1

Ac ce

pt

ed

262

in 50 min [44].

273 274

Biosensing based on Quantum Dots 12

Page 12 of 34

Semiconductor particles as quantum dots (QDs) are also a common choice in the

276

strategies for multiplexing, either as a support for the target analyte or as a label to

277

enhance the optical or electrochemical readout. In this context, an electrochemical

278

immunosensor for the multiplex detection of E. coli, Campylobacter and Salmonella

279

based on a sandwich immunoassay with QDs modified with three antibodies specific for

280

each bacterium was reported with a LOD of 400 CFU mL−1 for Salmonella and

281

Campylobacter and 800 CFU mL−1 for E. coli in 1h [45]. Another strategy was reported

282

recently using aptamers specific for Vibrio parahaemolyticus and S. Typhimurium

283

immobilised on quantum dots. The quantification of bacteria was based on dual

284

fluorescence resonance energy transfer (FRET) between QDs and carbon nanoparticles,

285

being able to detect 25 and 35 CFU mL-1 of V. parahaemolyticus and S. Typhimurium

286

respectively within 2h 20 min. [46]

M

an

us

cr

ip t

275

287

Biosensing based on Magnetic Particles

289

Along this review, the integration of magnetic particles, either with micrometer or

290

nanometer sizes on the strategies for the detection of foodborne pathogenic bacteria has

291

been emphasised. The possibility of immobilisation of a variety of biomolecules, such

292

as enzymes, antibodies, oligonucleotides onto the magnetic particles surface, as well as

293

the possibility of being easily manipulated by an external magnetic field gradient,

294

provides a selective capture of the target bacteria, offering an attractive application

295

towards the development of new biosensors and microfluidic devices [47].

296

Magnetic particles have also been combined with several biosensors platforms,

297

especially biosensors with optical readout. In this context, some studies were reported

298

using both magnetic particles and quantum dots in a sandwich immunoassay. For one

299

side, IMS of bacteria was achieved with further fluorescence detection, using different

Ac ce

pt

ed

288

13

Page 13 of 34

quantum dots modified with antibodies specific for each bacterium [48, 49]. Recently,

301

an aptasensor with optical readout for the simultaneous detection of S. aureus, V.

302

parahemolyticus, and S. Typhimurium was reported, using magnetic particles and

303

multicolor upconversion nanoparticles (UCNPs), as luminescence labels (Figure 3). In

304

this strategy, multicolor UCNPs were conjugated with aptamers specific for each

305

bacterium and hybridised with the complementary DNA sequence, which was coupled

306

to magnetic particles, these last providing an important advantage of improving the

307

washing steps. These conjugates are capable of emitting strong visible luminescence

308

with the excitation of NIR light (typically 980 nm), using a 980 nm laser, giving three

309

independent peaks at different wavelengths for each of the three bacteria. Upon addition

310

of the bacteria, these signals are proportionally reduced, since the multicolor UCNPs

311

conjugated with the aptamers react with their specific bacterial target being then

312

eliminated as a supernatant after applying a magnetic field, as shown in Figure 3. The

313

remaining UCNPs-MNPs were then separated and washed three times, and the

314

luminescence was measured with a 980 nm excitation laser.

315

The concentration of the three bacteria was related to the corresponding emission peak

316

of the multicolor UCNPs. The basic principle of the strategy was that aptamers could

317

form a defined conformation when binding to the targets and were also able to hybridise

318

to the complementary DNA sequences attached to the MPs to form a duplex structure.

319

When the targets and the complementary oligonucleotides were introduced, the

320

aptamers preferentially bound to the targets, resulting in the specific recognition of the

321

targets. Therefore, < 25 CFU mL-1 of all pathogens were detected in approximately 1h

322

with this approach [50].

323

Ac ce

pt

ed

M

an

us

cr

ip t

300

Preferred position for Figure 3 14

Page 14 of 34

Electrochemical immuno and genosensors have been extensively explored for food

325

safety applications due to their high sensitivity, rapidity, low cost and possibility of

326

being a hand-held platform operated by batteries for field applications. However, the

327

use of electrochemical biosensors remains in a preliminary stage towards multiplex

328

detection of bacteria [1].

ip t

324

329

Commercial approaches

331

The commercialisation of novel devices for the detection of food microorganisms is

332

based on a set of specific standards in food microbiology, such as quality control of

333

culture media, preparation of test samples, uncertainty estimation, method validation

334

and proficiency testing [51].

335

Currently, there are an increasing number of kits commercially available for a rapid

336

simple and reliable detection of pathogenic bacteria in food samples. In Table 3,

337

selected kits, mostly for single bacteria detection are compared in terms of assay format,

338

target pathogens, pre-enrichment step time, total assay time and LODs. Commercial kits

339

are based on different assays formats either immunological assays or PCR

340

methodologies for the detection of several foodborne pathogens, being able to detect in

341

general 1 CFU in 25 g of food sample as required for the legislation, after a pre-

342

enrichment culturing step. Most recent kits make use of a fully automated system,

343

which reduces the assay time, increasing the number of samples per test until 300

344

samples in one assay (Atlas® System, Assurance® EIA or GeneQuence). Rapid nucleic

345

acid amplification and detection technologies have been increasingly applied to

346

pathogen detection in food industry. In DuPont™ BAX® System, a qPCR methodology

Ac ce

pt

ed

M

an

us

cr

330

15

Page 15 of 34

is used for the detection of several pathogens with LODs of one CFU per food sample

348

ranged from 25 g to 375 g.

349

The use of nano-microsized particles on bioassays has been successfully introduced into

350

food industry. Lateral Flow System has been commercialised by Dupont (DuPontTM

351

Lateral Flow System) and Biocontrol (VIP® Gold), where food samples are combined

352

with gold-colloid particles coated with antibodies specific for the target bacteria. The

353

results are produced in only 10 min, being for this reason a suitable tool for pathogens

354

screening.

355

Finally, magnetic particles modified with antibodies specific to the target bacteria have

356

been used for IMS as a pre-concentration tool from food samples, being able to reduce

357

substantially the time of enrichment step. IMS is usually combined with PCR

358

methodologies, as occurred in Assurance GDS® MPX and Pathatrix®. Currently, the

359

commercialisation of kits for the simultaneous detection of foodborne pathogens is

360

available in a reduced number, being mostly based on PCR methodology (Assurance

361

GDS® MPX).

us

an

M

ed

pt

362 363

Preferred position for Table 3

Ac ce

364

cr

ip t

347

365

Conclusions

366

A considerable progress regarding food safety in EU has been done in terms of rapid

367

and multiplexed approaches for detecting bacteria outbreaks. However, food

368

contamination caused by pathogenic bacteria is still a serious threat for the consumers.

369

The common strategies for the detection of pathogenic microorganisms are consisted of

370

the gold standard conventional microbiological culturing techniques, IAs and PCR 16

Page 16 of 34

methodologies. The development of novel strategies with multiplexing capabilities is

372

highlighted as a rapid and cost effective alternative for the detection of bacteria.

373

Therefore, in this review, it was shown that mPCR based methodologies could detect

374

below 10 CFU in 25 g of sample after a pre-enrichment step up to 30 h [25, 53]. The

375

integration of magnetic particles with PCR based methodologies leads to a LOD of less

376

than 10 CFU g-1 within 6 h [28]. DNA microarrays showed LODs below 500 CFU mL-1

377

when using magnetic particles, in approximately 3.5 h, without the need of a pre-

378

enrichment step [38]. Biosensors combined with nanomaterials have led to a significant

379

improvement of the assay time and LOD. In this context, no significant differences were

380

noticed between the different nanomaterials, however it was observed that the

381

integration of magnetic particles allows a significant decrease of the enrichment times,

382

reducing thus the total assay time [50, 59-60].

383

To summarise, in food safety applications the integration of magnetic carriers and IMS

384

step for capturing the bacteria through an immunological reaction from contaminating

385

microflora and interfering food components introduces advantages on the analytical

386

performance due to the pre-concentration upon magnetic actuation for further testing or

387

readout. For instance, the matrix effect is eliminated and the PCR inhibitors are avoided

388

which leads to a decrease of the background signals. The improvement of the washing

389

steps and the decrease of the time required for enrichment steps are also other

390

advantages. Finally, magnetic particles offer an attractive support to be incorporated in

391

magnetic actuated biosensors, microfluidic platforms or other devices.

392

Magnetic particles have also been successfully integrated in commercially available kits

393

for a rapid, simple and reliable detection of single pathogenic bacteria. However, there

394

are still only a few commercial kits available for simultaneous detection, with integrated

Ac ce

pt

ed

M

an

us

cr

ip t

371

17

Page 17 of 34

395

magnetic nanoparticle functionality. As a conclusion the integration of magnetic

396

particles for the multiplex detection of bacteria is still in a preliminary stage, requiring

397

further studies due to their promising features.

398

Acknowledgments

400

Financial support from BioMaX “Novel diagnostic bioassays based on magnetic

401

particles”, Marie Curie Initial Training Networks (FP7-PEOPLE-2010-ITN) and the

402

Ministry of Economy and Competitiveness (MINECO), Madrid (Project BIO2013-

403

41242-R) are acknowledged.

an

us

cr

ip t

399

M

404

FIGURE CAPTIONS

406

Figure 1. Notifications for pathogens found in meat samples in 2012 and 2013.

407

Figure 2. Integration of magnetic particles in immunosensors and genosensors. After an

408

immunomagnetic separation step, (A) a magneto immunosensor and (B) a magneto

409

genosensor with electrochemical readout were reported for the detection of Salmonella

410

in milk. Adapted from [45].

411

Table 1. Strategies for the simultaneous detection of pathogenic bacteria based on

412

Nucleic acid amplification methods, Multiplex PCR based microfluidic systems and

413

DNA microarrays.

414

Table 2. Biosensors platforms based on the integration of nanomaterials for the

415

simultaneous detection of bacteria.

Ac ce

pt

ed

405

18

Page 18 of 34

Figure 3. Simultaneous detection of S. aureus, V. parahemolyticus, and S.

417

Typhimurium, using magnetic particles (MNPs) and multicolor upconversion

418

nanoparticles (UCNPs), as luminescence labels. UCNPs were modified with aptamers

419

(Apt1-3) specific for the different bacterium. Complementary oligonucleotides (c1-3DNA-

420

MPs) were immobilised on the MNPs. Reprinted with permission from [50]. Copyright

421

(2014) American Chemical Society.

422

Table 3. Commercial kits for the detection of pathogenic bacteria.

cr us

423 424

an

425 426

M

427

Ac ce

pt

ed

428 429

ip t

416

19

Page 19 of 34

References

430

(1) Swaminathan B, Feng P. Rapid detection of food-borne pathogenic bacteria. Annu

431

Rev Microbiol 1994; 48:401-26.

432

(2) Tirado MC, Clarke R, Jaykus LA, McQuatters-Gollop A, Frank JM. Climate change

433

and food safety: A review. Food Res Int 2010; 43:1745–65.

434

(3) The Rapid Alert System for Food and Feed 2013, Annual report. European Union.

435

http://ec.europa.eu/food/safety/rasff/index_en.htm

436

(4) Hendriksen RS, Vieira AR, Karlsmose S, Danilo MA, Wong LF, Wegener HC, et al.

437

Global Monitoring of Salmonella Serovar Distribution from the World Health

438

Organization Global Foodborne Infections Network Country Data Bank: Results of

439

Quality Assured Laboratories from 2001 to 2007. Foodborne Pathog Dis 2011;8.

440

(5) Grad YH, Lipsitch M. Feldgarden M, Arachchi HM, Cerqueira GC, FitzGerald M, et

441

al. Genomic epidemiology of the Escherichia coli O104:H4 outbreaks in Europe 2011.

442

Proc Natl Acad Sci USA 2012;109:3065-70.

443

(6) Frank C, Milde-Busch A, Werber D. Results of surveillance for infections with

444

Shiga toxinproducing Escherichia coli (STEC) of serotype O104:H4 after the large

445

outbreak in Germany, July to December 2011. Euro Surveill 2014; 19(14):pii=20760.

446

Available online: http://www.eurosurveillance.org/ViewArticle.aspx?ArticleId=20760

447

(7) Jadhav S, Bhave M, Palombo EA. Methods used for the detection and subtyping of

448

Listeria monocytogenes. J Microbiol Methods 2012;88:327–41.

449

(8) Newell DG, Koopmans M, Verhoef L, Duizer E, Aidara-Kane A, Sprong H, et al.

450

Food-borne diseases — The challenges of 20 years ago still persist while new ones

451

continue to emerge. Int J Food Microbiol 2010;139:S3–S15.

Ac ce

pt

ed

M

an

us

cr

ip t

429

20

Page 20 of 34

(9) Velusamy V, Arshak K, Korostynska O, Oliwa K, Adley C. An overview of

453

foodborne pathogen detection: In the perspective of biosensors. Biotechnol Adv

454

2010;28:232–54.

455

(10) López- Campos G, Martínez-Suarez JV, Aguado-Urda M, López-Alonso V.

456

Microarray detection and characterization of bacterial foodborne pathogens. US:

457

Springer;2012, pp. 16-33.

458

(11) Yang H, Li H, Jiang X. Detection of foodborne pathogens using bioconjugated

459

nanomaterials. Microfluid Nanofluid 2008;5:571–83.

460

(12) Laube T, Cortés P, Llagostera M, Alegret S, Pividori MI. Phagomagnetic

461

immunoassay for the rapid detection of Salmonella. Appl Microbiol Biotechnol

462

2014;98:1795–805.

463

(13) Mandal PK, Biswas AK, Choi K, Pal UK. Methods for the rapid detection of

464

foodborne pathogens: An overview. Am J Food Technol 2011;6:87-102.

465

(14) Liébana S, Brandão D, Alegret S, Pividori MI. Electrochemical immunosensors,

466

genosensors and phagosensors for Salmonella detection. Anal Methods 2014; 6:8858-74.

467

(15) Knez K, Janssen KPF, Spasic D, Declerck P, Vanysacker L, Denis C. Spherical

468

Nucleic Acid Enhanced FO-SPR DNA Melting for Detection of Mutations in

469

Legionella pneumophila. Anal Chem 2013;85:1734−42.

470

(16) Liébana S, Lermo A, Campoy S, Barbé J, Alegret S, Pividori MI. Rapid detection

471

of Salmonella in milk by electrochemical magneto-immunosensing. Bios Bioelec

472

2009;25:510-13.

473

(17) Ohk SH, Koo OK, Sen T, Yamamoto CM, Bhunia AK. Antibody–aptamer

474

functionalized fibre-optic biosensor for specific detection of Listeria monocytogenes

475

from food. J Appl Microbiol 2010;109:808–17.

Ac ce

pt

ed

M

an

us

cr

ip t

452

21

Page 21 of 34

(18) Ivnitski D, Abdel-Hamid I, Atanasov P, Wilkins E. Biosensors for detection of

477

pathogenic bacteria. Bios Bioelec 1999;14:599–24.

478

(19) Brandão D, Liébana S, Campoy S, Cortés P, Alegret S, Pividori MI.

479

Electrochemical magneto-immunosensing of Salmonella based on nano and micro-sized

480

magnetic particles. J Phys Conf Ser 2013;413:012020.

481

(20) Liébana S, Lermo A, Campoy S, Barbé J, Alegret S, Pividori MI. Magneto

482

Immunoseparation of Pathogenic Bacteria and Electrochemical Magneto Genosensing

483

of the Double-Tagged Amplicon. Anal Chem 2009;81:5812–20.

484

(21) Liébana S, Spricigo DA, Cortés MP, Barbé J, Llagostera M, Alegret S, Pividori

485

MI. Phagomagnetic Separation and Electrochemical Magneto Genosensing of

486

Pathogenic Bacteria. Anal Chem 2013;85:3079−86.(22) Yanfang Y, Wentao X, Zhifang

487

Z, Hui S, Yunbo L, Zhuojun C, Kunlun H. Universal Primer-Multiplex PCR Approach

488

for Simultaneous Detection of Escherichia coli, Listeria monocytogenes, and

489

Salmonella spp. in Food Samples. J Food Sci 2009;74:446-52.

490

(23) Guan ZP, Jiang Y, Gao F, Zhang L, Zhou GH, Guan ZJ. Rapid and simultaneous

491

analysis of five foodborne pathogenic bacteria using multiplex PCR. Eur Food Res

492

Technol 2013;237:627–37.

493

(24) Chen J, Tang J, Liu J, Cai Z, Bai X. Development and evaluation of a multiplex

494

PCR for simultaneous detection of five foodborne pathogens. J Appl Microbiol

495

2012;112:823–30.

496

(25) Germini A, Masola A, Carnevali P, Marchelli R. Simultaneous detection of

497

Escherichia coli O175:H7, Salmonella spp., and Listeria Monocytogenes by multiplex

498

PCR. Food Control 2009;20:733–38.

Ac ce

pt

ed

M

an

us

cr

ip t

476

22

Page 22 of 34

(26) Omiccioli E, Amagliani G, Brandi G, Magnani M. A new platform for Real-Time

500

PCR detection of Salmonella spp., Listeria Monocytogenes and Escherichia coli O157

501

in milk. Food Microbiol 2009;26:615–22.

502

(27) Suo B, He Y, Tu S, Shi X. A Multiplex Real-Time Polymerase Chain Reaction for

503

Simultaneous Detection of Salmonella spp., Escherichia coli O157, and Listeria

504

Monocytogenes in Meat Products. Foodborne Pathog Dis 2010;7(6):619-28.

505

(28) Ma K, Deng Y, Bai Y, Xu D, Chen E, Wu H, et al. Rapid and simultaneous

506

detection of Salmonella, Shigella, and Staphylococcus aureus in fresh pork using a

507

multiplex real-time PCR assay based on immunomagnetic separation. Food Control

508

2014;42:87-93.

509

(29) Yang Y, Xu F, Xu H, Aguilar ZP, Niu R, Yuan Y, et al. Magnetic nano-beads

510

based separation combined with propidium monoazide treatment and multiplex PCR

511

assay for simultaneous detection of viable Salmonella typhimurium, Escherichia coli

512

O157:H7

513

Microbiol 2013;34(2):418-24.

514

(30) Zhang C, Wang H, Xing D. Multichannel oscillatory-flow multiplex PCR

515

microfluidics for high-throughput and fast detection of foodborne bacterial pathogens.

516

Biomed Microdevices 2011;13:885–97.

517

(31) Ramalingam N, Rui Z, Liu H, Dai C, Kaushik R, Ratnaharika B, et al. Real-time

518

PCR-based microfluidic array chip for simultaneous detection of multiple waterborne

519

pathogens. Sens Actuators B Chem B 2010;145:543–52.

520

(32) Wang H, Zhang C, Xing D. Simultaneous detection of Salmonella enterica,

521

Escherichia coli O157:H7 and Listeria Monocytogenes using oscillatory-flow multiplex

522

PCR. Microchim Acta 2011;173:503–12.

M

an

us

cr

ip t

499

monocytogenes

ed

Listeria

in

food

products.

Food

Ac ce

pt

and

23

Page 23 of 34

(33)

Kalantar-zadeh

K,

Fry

B.

Nanotechnology-Enabled

Sensor.

Springer

524

Science+Business Media; 2008, pp. 436-470.

525

(34) Kupradit C, Rodtong S, Ketudat-Cairns M. Development of a DNA macroarray for

526

simultaneous detection of multiple foodborne pathogenic bacteria in fresh chicken meat.

527

World J Microbiol Biotechnol 2013;29:2281–91.

528

(35) Suo B, He Y, Paoli G, Gehring A, Tu S, Shi X. Development of an

529

oligonucleotide-based microarray to detect multiple foodborne pathogens. Mol

530

Cell Probes 2010;24:77–86.

531

(36) Huang A, Qiu Z, Jin M, Shen Z, Chen Z, Wang X, Li J. High-throughput detection

532

of food-borne pathogenic bacteria using oligonucleotide microarray with quantum dots

533

as fluorescent labels. Int J Food Microbiol 2014;185:27–32.

534

(37) Sun H, Mo Q, Lin J, Yang Z, Tu C, Gu D, Shi L, Lu W. Rapid simultaneous

535

screening of seven clinically important enteric pathogens using a magnetic bead based

536

DNA microarray. World J Microbiol Biotechnol 2011; 27: 163–69.

537

(38) Donhauser SC, Niessner R, Seidel M. Sensitive Quantification of Escherichia coli

538

O157:H7, Salmonella enterica, and Campylobacter jejuni by Combining Stopped

539

Polymerase Chain Reaction with Chemiluminescence Flow-Through DNA Microarray

540

Analysis. Anal Chem 2011;83(8): 3153–60.

541

(39) Mannoor MS, Tao H, Clayton JD, Sengupta A, Kaplan DL, Naik RR, et al.

542

Graphene-based wireless bacteria detection on tooth enamel. Nat Commun 2012;3:763-

543

71.

544

(40) Mao X, Baloda M, Gurung AS, Lin Y, Liu G. Multiplex electrochemical

545

immunoassay using gold nanoparticle probes and immunochromatographic strips.

546

Electrochem commun 2008;10:1636–40.

Ac ce

pt

ed

M

an

us

cr

ip t

523

24

Page 24 of 34

(41) Kell AJ, Somaskandan K, Stewart G, Bergeron MG, Simard B. Superparamagnetic

548

Nanoparticle-Polystyrene Bead Conjugates as Pathogen Capture Mimics: A Parametric

549

Study of Factors Affecting Capture Efficiency and Specificity. Langmuir 2008;24:3493-

550

502.

551

(42) Liu G, Lin Y. Nanomaterial labels in electrochemical immunosensor and

552

immunoassays. Talanta 2007;74:308–17.

553

(43) Ravindranath SP, Wang Y, Irudayaraj J. SERS driven cross-platform based

554

multiplex pathogen detection. Sens Actuators B Chem 2011;152:183–90.

555

(44) Taylor AD, Ladd J, Yu Q, Chena S, Homola J, Jiang S. Quantitative and

556

simultaneous detection of four foodborne bacterial pathogens with a multi-channel SPR

557

sensor. Bios Bioelec 2006;22:752–58.

558

(45) Viswanathana S, Rani C, Ho JA. Electrochemical immunosensor for multiplexed

559

detection of food-borne pathogens using nanocrystal bioconjugates and MWCNT

560

screen-printed electrode. Talanta 2012;94:315– 19.

561

(46) Duan N, Wu S, Dai S, Miao T, Chen J, Wang Z. Simultaneous detection of

562

pathogenic bacteria using an aptamer based biosensor and dual fluorescence resonance

563

energy transfer from quantum dots to carbon nanoparticles. Microchim Acta 2014; DOI

564

10.1007/s00604-014-1406-3. Accepted article.

565

(47) Hsing I, Xu Y, Zhao W. Micro- and Nano- Magnetic Particles for Applications in

566

Biosensing. Electroanalysis 2007;19:755 – 68.

567

(48) Zhao Y, Ye M, Chao Q, Jia N, Ge Y, Shen H. Simultaneous Detection of

568

Multifood-Borne Pathogenic Bacteria Based on Functionalized Quantum Dots Coupled

569

with Immunomagnetic Separation in Food Samples. J Agric Food Chem 2009;57:517–

570

24.

Ac ce

pt

ed

M

an

us

cr

ip t

547

25

Page 25 of 34

(49) Yang L, Li Y. Simultaneous detection of Escherichia coli O157:H7 and

572

Salmonella typhimurium using quantum dots as fluorescence labels. Analyst

573

2006;131:394–401.

574

(50) Wu S, Duan N, Shi Z, Fang C, Wang Z. Simultaneous Aptasensor for Multiplex

575

Pathogenic Bacteria Detection Based on Multicolor Upconversion Nanoparticles

576

Labels. Anal Chem 2014;86:3100−07.

577

(51) Rodríguez-Lázaro D, Lombard B, Smith H, Rzezutka A, D´Agostino M, Helmulth

578

R, et al. Trends in analytical methodology in food safety and quality: monitoring

579

microorganisms and genetically modified organisms. Trends Food Sci Technol 2007;18

580

(6):306–19.

581

(52) Kawasaki S, Horikoshi N, Okada Y, Takeshita K, Sameshima T, Kawamoto S.

582

Multiplex

583

monocytogenes, and Escherichia coli O157:H7 in Meat Samples. J Food Prot

584

2005;68(3):551-6.

585

(53) Garrido A, Chapela M, Román B, Fajardo P, Vieites JM, Cabado AG. In-house

586

validation of a multiplex real-time PCR method for simultaneous detection of

587

Salmonella spp., Escherichia coli O157 and Listeria monocytogenes. Int J Food

588

Microbiol 2013;164:92–98.

589

(54) Köppel R, Kuslyte AR, Tolido I, Schmid J, Marti G. Nonaplex real-time PCR

590

detection of Listeria monocytogenes, Campylobacter, Salmonella and enteropathogene

591

E. coli after universal enrichment in food samples. Eur Food Res Technol

592

2013;237:315-22.

Simultaneous

Detection

of Salmonella spp., Listeria

ed

M

for

Ac ce

pt

PCR

an

us

cr

ip t

571

26

Page 26 of 34

(55) Li Y, Li Y, Zheng B, Qu L, Li C. Determination of foodborne pathogenic bacteria

594

by multiplex PCR-microchip capillary electrophoresis with genetic algorithm-support

595

vector regression optimisation. Anal Chim Acta 2009; 643:100–07.

596

(56) Hong B, Jiang L, Hu Y, Fang D, Guo H. Application of oligonucleotide array

597

technology for the rapid detection of pathogenic bacteria of foodborne infections. J

598

Microbiol Methods 2004;58:403 –11.

599

(57) Cho I, Irudayaraj J. In-situ immuno-gold nanoparticle network ELISA biosensors

600

for pathogen detection. Int J Food Microbiol 2013;164:70–05.

601

(58) Z. Fu, X. Zhou, D. Xing, Rapid colorimetric gene-sensing of food pathogenic

602

bacteria using biomodification-free gold nanoparticle. Sens Actuators B Chem

603

2013;182: 633–41.

604

(59) Wang H, La Y, Wang A, Slavik M. Rapid, Sensitive, and Simultaneous Detection

605

of Three Foodborne Pathogens Using Magnetic Nanobead–Based Immunoseparation

606

and Quantum Dot–Based Multiplex Immunoassay. J Food Prot 2011;74(12):2039-47.

607

(60) Cho I, Mauer L, Irudayaraj J. In-situ fluorescent immunomagnetic multiplex

608

detection of foodborne pathogens in very low numbers. Biosens Bioelectron

609

2014;57:143–48.

610

(61) Zhang D, Huarng MC, Alocilja EC. A multiplex nanoparticle-based bio-barcoded

611

DNA sensor for the simultaneous detection of multiple pathogens. Bios Bioelec

612

2010;26:1736–42.

Ac ce

pt

ed

M

an

us

cr

ip t

593

613

27

Page 27 of 34

Table 1 Preenrichment

Total assay time

LOD

Ref.

Agarose gel

artificially contaminated pork samples

overnight

~ 1h 40 min

9-670 CFU mL-1

[23]

Agarose gel

artificially contaminated meat samples

24 h

~ 1h 35 min

10–17 CFU g1 sample

[24]

>15 h

~ 1h 45 min

30h

1h 10 min

qPCR for Salmonella spp., E. coli, L. monocytogenes

Fluorescence

qPCR for Salmonella spp., E. coli, L. monocytogenes

Fluorescence

Nonaplex qPCR for L. monocytogenes, Campylobacter, Salmonella, enteropathogenic E. coli

Fluorescence

IMS-qPCR assay for Salmonella spp., Shigella spp., S. aureus

Fluorescence

IMS -mPCR assay for S. Typhimurium, E. coli, L. monocytogenes

Agarose gel

agarose gel electrophoresi s and GoldView™ agarose gel stained with GoldView™

Oscillatory-flow multiplex PCR PCR for S. enterica, E. coli, L. monocytogenes

Microchip capillary electrophoresis for V. parahemolyticus, Salmonella, E. coli, Shigella DNA microarray for Salmonella spp., Shigella spp., L. monocytogenes, E. coli

Capillary electrophoresis

Chemilumines -cence

DNA microarray for E. coli, S. enterica, L. monocytogenes, C. jejuni

Fluorescence

DNA microarray for 12 different bacterial strains using QDs

Fluorescence

[25]

1 CFU in 25 g sample

[52]

30h

55 min

5 CFU in 25 g sample

[53]

20 h

60 min

Multiplexed detection of foodborne pathogens based on magnetic particles.

This paper addresses the novel approaches for the multiplex detection of food poisoning bacteria, paying closer attention to three of the most common ...
722KB Sizes 3 Downloads 13 Views