Improved quantitative detection of VBNC Vibrio parahaemolyticus using immunomagnetic separation and PMAxx-qPCR

Journal Pre-proof Improved quantitative detection of VBNC Vibrio parahaemolyticus using immunomagnetic separation and PMAxx-qPCR Lichao Zhao, Xinrui Lv, Xiao Cao, Jingfeng Zhang, Xiaokui Gu, Haiyan Zeng, Li Wang PII:

S0956-7135(19)30551-1

DOI:

https://doi.org/10.1016/j.foodcont.2019.106962

Reference:

JFCO 106962

To appear in:

Food Control

Received Date: 2 July 2019 Revised Date:

12 September 2019

Accepted Date: 19 October 2019

Please cite this article as: Zhao L., Lv X., Cao X., Zhang J., Gu X., Zeng H. & Wang L., Improved quantitative detection of VBNC Vibrio parahaemolyticus using immunomagnetic separation and PMAxxqPCR, Food Control (2019), doi: https://doi.org/10.1016/j.foodcont.2019.106962. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. 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. © 2019 Published by Elsevier Ltd.

Improved quantitative detection of VBNC Vibrio parahaemolyticus using immunomagnetic separation and PMAxx-qPCR

Lichao Zhaoa, #, Xinrui Lva, #, Xiao Caoa, Jingfeng Zhanga, Xiaokui Gub, Haiyan Zenga, Li Wanga, * a

Guangdong Provincial Key Laboratory of Nutraceuticals and Functional Foods,

College of Food Science, South China Agricultural University, Guangzhou 510642, China b

Key Laboratory of Biomaterials of Guangdong Higher Education Institutes,

Department of Biomedical Engineering, Jinan University, Guangzhou 510632, China *Corresponding author: Li Wang Mailing address: College of Food Science, South China Agricultural University, 510642, Guangzhou, China. E-mail: [email protected] #

These authors contributed equally to this work and should be considered joint first

authors.

1

1

Abstract

2

Immunomagnetic separation (IMS) is an effective method for specific

3

enrichment and purification of target food-borne pathogens from complex food

4

samples. To detect viable but non-culturable (VBNC) Vibrio parahaemolyticus (V.

5

parahaemolyticus) with greater accuracy and sensitivity, we used an improved

6

propidium monoazide (PMAxx) dye to eliminate dead cell interference in an

7

IMS-PMAxx-real-time (quantitative) polymerase chain reaction (IMS-PMAxx-qPCR)

8

assay. We prepared immunomagnetic beads (IMBs) using streptavidin-conjugated

9

magnetic nanoparticles and biotinylated polyclonal antibodies, and optimized the

10

reaction conditions to establish an IMS method for VBNC V. parahaemolyticus. We

11

determined the optimal antibody amount (30 µg), IMBs volume (150 µL), incubation

12

time (45 min), immunomagnetic separation time (4 min), and separation temperature

13

(25℃). The IMS-PMAxx-qPCR method could detect VBNC V. parahaemolyticus in

14

raw shrimp samples at levels as low as 1.85 CFU/g without any pre-enrichment. The

15

IMS-PMAxx-qPCR assay is highly sensitive, selective, simple, and rapid (< 4 h), and

16

outperformed the conventional PCR based assays. Thus, this method can potentially

17

improve rapid detection of VBNC V. parahaemolyticus in raw shrimp.

18

Key words: Immunomagnetic separation (IMS); Vibrio parahaemolyticus; Viable but

19

non-culturable (VBNC); Real-time (quantitative) polymerase chain reaction PCR

20

(qPCR); Improved propidium monoazide (PMAxx)

21 22 23 24 25 26

2

27

1. Introduction

28

Vibrio parahaemolyticus (V. parahaemolyticus) is a gram-negative, halophilic

29

pathogen found in aquatic environments worldwide (Han et al., 2015; Zhong et al.,

30

2017). It is one of the most common causes of bacterial gastroenteritis associated with

31

the consumption of raw or undercooked seafood. Shrimp is one of the most popular

32

types of seafood in China, and thus is one of the main food vectors for V.

33

parahaemolyticus transmission. V. parahaemolyticus contamination contributes to a

34

high prevalence of foodborne illnesses, outbreaks, and associated mortality (Wang et

35

al., 2015; Xu et al., 2014).

36

In order to prevent the growth and propagation of V. parahaemolyticus and

37

ensure food safety, standardized methods have been adopted for food processing,

38

transportation, and storage, such as refrigeration and high salinity treatment. To cope

39

with these environmental stressors, V. parahaemolyticus can enter into a viable but

40

non-culturable (VBNC) state (Yoon, Moon, Choi, Ryu, & Lee, 2019). In a VBNC

41

state, cells are characterized by a decreased growth rate and metabolism. They may

42

still retain some metabolic activity, pathogenicity, and toxicity (Ayrapetyan & Oliver,

43

2016). However, they cannot be detected by conventional plate count techniques

44

because the cells are not able to develop visible colonies on routine laboratory media.

45

Additionally, the VBNC cells may survive until environmental conditions become

46

favorable for growth and cell division (Li, Mendis, Trigui, Oliver, & Faucher, 2014).

47

Previous research has demonstrated that VBNC V. parahaemolyticus still maintains

48

virulence (Wong, Shen, Chang, Lee, & Oliver, 2004) and can pose a potential threat to

49

environmental and human health. Thus, it is critical to establish a sensitive, accurate,

50

and rapid detection method for monitoring V. parahaemolyticus in a VBNC state in

51

order to ensure food safety.

52

Various molecular biology methods, such as real-time (quantitative) polymerase

53

chain reaction (qPCR) and loop-mediated isothermal DNA amplification (qLAMP),

54

have been used to quantify VBNC state bacteria (Dinu & Bach, 2013; Josefsen et al.,

55

2010; Kibbee & Ormeci, 2017; Morishige, Fujimori, & Amano, 2015; Wang, Zhong, 3

56

& Li, 2012; Zhong, Tian, Wang, & Wang, 2016). However, many of these techniques

57

require an enrichment step due to low levels of VBNC bacteria in a highly complex

58

sample matrix. Traditional enrichment methods, such as membrane filtration and

59

centrifugation (Stevens & Jaykus, 2004), have been reported in the literature.

60

However, these methods have significant limitations because they are based on size

61

and weight, rather than specificity. Compared with traditional enrichment methods,

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immunomagnetic separation (IMS) is more sensitive and has greater specificity.

63

Extremely low signals can be detected from complex biological samples with high

64

background noise (Du et al., 2018). This selectivity is achieved using

65

streptavidin-conjugated magnetic nanoparticles bound to a biotinylated polyclonal

66

antibody. These specific immunomagnetic beads (IMBs) enable selective separation

67

and concentration of trace amounts of target bacteria from a range of sample matrices

68

and background bacteria (Shan et al., 2014; Xiong et al., 2014). However, the IMS

69

technique must be optimized for application to different samples. To date, this

70

technique has not been used to detect VBNC V. parahaemolyticus in raw shrimp

71

samples, and thus factors influencing its specificity and sensitivity in this context are

72

unknown.

73

The aim of this study was to optimize the enrichment conditions of IMS in order

74

to quantify VBNC V. parahaemolyticus in raw shrimp samples. We developed the

75

IMS-PMAxx-qPCR method. PMAxxTM is a modified version of PMA, a nucleic acid

76

dye, that is able to more accurately and quantitatively distinguish living bacteria from

77

dead bacteria (Cao et al., 2019; Randazzo et al., 2018). We compared the sensitivity of

78

IMS-PMAxx-qPCR, PMA-qPCR, qPCR, and traditional culture assay in quantifying

79

VBNC V. parahaemolyticus in actual samples. We demonstrated that our developed

80

assay can be used for rapid and sensitive VBNC state food-borne pathogen screening.

81

Additionally, it can be readily applied in the food industry, government food safety

82

departments, or other relevant organizations.

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2. Materials and methods

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2.1. Bacterial strains and culture conditions 4

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All V. parahaemolyticu strains cryopreserved at -80℃ in glycerin were used to

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inoculate 3% NaCl alkaline peptone water (3% NaCl APW, Huankai Microbial (HM),

87

China) and cultured at 37℃ on a rotary shaker (180 rpm) for 24 h. To determine the

88

bacterial concentration, the cultures were serial-diluted with phosphate buffered saline

89

(PBS, HM, China), used to inoculate 3% NaCl tryptone soy agar (3% NaCl TSA, HM,

90

China), and grown at 37℃ for 24 h. To obtain heat-killed V. parahaemolyticus (ATCC

91

17802) cells, the bacterial suspensions were heated to 85℃ for 10 min. To obtain the

92

VBNC V. parahaemolyticus, 1 mL of the bacterial suspension, reaching the

93

mid-logarithmic growth phase, was harvested and induced, with the method

94

established in our laboratory (Cao et al. 2019). Non-target pathogenic bacteria were

95

grown in Luriae Bertani (LB, HM, China) medium and cultured overnight at 37℃ for

96

24 h.

97

2.2 Preparation of IMBs and IMS Procedure

98

2.2.1 Preparation of IMBs

99

Streptavidin-conjugated magnetic nanoparticles (500 nm, 20 mg/mL, Shanghai

100

So-Fe Biomedical Co., Ltd, Shanghai, China) were condensed in a centrifuge tube

101

with a magnetic separator for 5 min. The storage buffer (PBS, 10% glycerol, Proclin

102

300) was then removed. Uncoated streptavidin-conjugated magnetic nanoparticles

103

were washed three times with 0.01 M PBS containing 0.05% Tween 20 (PBST,

104

pH7.4). The magnetic nanoparticles were then concentrated onto the side of the tube

105

using a magnet and the supernatant was carefully aspirated. To form IMBs,

106

biotinylated polyclonal antibodies (0.15 mg/mL, Wuhan GeneCreate Biological

107

Engineering Co., Ltd, Wuhan, China) were added to coat the magnetic nanoparticles

108

and then incubated on a HS-3 vertical mixer (Ningbo Scientz Biotechnology company,

109

Ningbo, China) at 15 r/min at room temperature for 45 min. After washing three times

110

with PBST, the IMBs were resuspended in PBST with 0.1% BSA and 0.05% NaN3,

111

and stored at 4℃.

112

2.2.2 IMS Procedure 5

113

IMBs were added to 1 mL of PBS containing 105 CFU/mL of V.

114

parahaemolyticus. The mixture was incubated on a rotator at room temperature to

115

form bead-bacteria complexes and then separated using a magnet for 5 min. The

116

complexes were resuspended with 1 mL of PBST and the supernatant was carefully

117

aspirated before PMAxx processing. Then, 500 µL of the solution was used to extract

118

DNA for qPCR amplification. Bacterial solutions not processed by IMS were used as

119

negative controls.

120

2.3 Capture efficiency (CE) calculation

121

CE, defined as the percentage of total bacteria retained on the IMBs, was

122

determined by dividing the number of V. parahaemolyticus isolated by the total

123

number of V. parahaemolyticus present in a sample. CE was calculated using the

124

equation:

125 126

CE (%) = (1 - B/A) × 100%

(a),

where A is the total number of bacteria in the sample (CFU/mL) and B is the

127

number of unbound bacteria in the supernatant (CFU/mL).

128

2.4 Optimization of IMS conditions

129

A range of conditions were studied to determine the optimum capture capacity of

130

the IMBs for V. parahaemolyticus: five different amounts of biotinylated polyclonal

131

antibody (7.5, 15, 22.5, 30, and 60 µg), six IMBs doses (10, 20, 50, 100, 150, and 200

132

µL), five incubation times (15, 30, 45, 60, and 90 min), six immunomagnetic

133

separation times (0.5, 1, 2, 3, 4, and 5 min) and a series of temperatures (4, 25, and

134

37℃). CE was calculated according to the above method.

135

2.5 IMS assay capture specificity and ultrastructure characterization

136

We prepared one V. parahaemolyticus strain (ATCC 17802), along with 7

137

non-target bacterial strains: V. harveyi (SCAUFHSM 011), V. vulnificus (ATCC

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27562), V. alginolyticus (ATCC 33787), Listeria monocytogenes (ATCC 19115),

139

Salmonella typhimurium (ATCC 14028), Staphylococcus aureus (ATCC 25923) and 6

140

Escherichia coli O157:H7 (ATCC 35150). Each bacterial culture was diluted to

141

approximately 105 CFU/mL in PBS and then captured using the standard IMS

142

protocol (above). Results were used to determine assay specificity.

143

We examined pre-capture and post-capture (viable normal and VBNC state

144

bacteria) IMBs using scanning electron microscopy (SEM, Phlilips-FEI, Netherlands)

145

to characterize the VBNC V. parahaemolyticus and bead-bacteria complexes.

146

2.6 Standard curve

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A standard curve was generated by using serial-diluted standards of V.

148

parahaemolyticus during stationary growth phase. The standard curve demonstrated a

149

linear relationship between threshold cycle (Ct) values and Log10 CFU/mL. From

150

each standard, 1 mL was separately treated with PMAxx under optimized conditions,

151

and then the DNA was extracted and used as template for establishing a

152

PMAxx-qPCR standard curve. DNA samples without PMAxx treatment were used to

153

establish a qPCR standard curve.

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2.7 PMAxx treatment

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PMAxx (20 mM, Biotium, Inc.) was dissolved in high purity water to generate a

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2 mM stock solution, which was then stored at -20℃ in the dark. For PMAxx

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treatment, VBNC V. parahaemolyticus suspensions were adjusted to a final

158

concentration of 1 × 105 CFU/mL with PBS. The suspensions were split into two

159

aliquots which were used to prepare viable samples and heat killed samples (85℃, 10

160

min). PMAxx stock solution (8 µL) was added to a 1 mL aliquot of the prepared

161

bacterial suspension and incubated in the dark for 10 min to allow the PMAxx to enter

162

the dead cells. At the end of the 10 min incubation, the cells were placed on ice and

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exposed to HG-EMA nucleic acid light marker (Huguo Science Instrument Co., Ltd.,

164

Shanghai, China) for 10 min. Free PMAxx was removed by centrifuging at 8000 rpm

165

for 5 min, and washed three times with PBS before DNA was extracted for qPCR.

166

Sample solution not treated with PMAxx was used as a negative control.

7

167

2.8 DNA extraction and qPCR

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DNA was isolated from pure cultures and raw shrimp samples using a bacterial

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genomic DNA extraction kit according to the manufacturer’s protocol. Isolated DNA

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was stored at -20℃ until use. The primers used in this study are shown in table 1. V.

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parahaemolyticus primers and probe were designed using Express 3.0.1 software, and

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the sequence specificity of the primers was evaluated using the GenBank Primer

173

BLAST tool. The total PCR volume was 25 µL, which included 12.5 µL AceQ®qPCR

174

Probe Master Mix, 1 µL of each primer (10 µM), 0.5 µL of probe (10 µM), 5 µL of

175

DNA template, and 5 µL of ultra pure water. The PCR amplification conditions were

176

95℃ for 10 min, followed by 45 cycles of 95℃ for 15 s and 60℃ for 1 min. The

177

primers and probe were synthesized by Sangon Biotech (Shanghai) Co., Ltd. All

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qPCR runs were performed using the 7500 Fast Real-Time PCR System.

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2.9 Detection of VBNC V. parahaemolyticus in raw shrimp by IMS-PMAxx-qPCR

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2.9.1 Sample collection

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Raw shrimp samples were purchased from a local supermarket in Guangzhou,

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China, and were determined to be negative for V. parahaemolyticus using standard

183

methods (GB 4789.7-2013, China) and PCR. The raw shrimp samples were stored at

184

4℃ until further processing. The samples were tested using the above two methods

185

and then sterilized if V. parahaemolyticus was detected in order to ensure that

186

subsequent experiments were carried out without V. parahaemolyticus contamination.

187

The raw shrimp samples (25 g) were then homogenized in PBS at a 1:10 ratio for 5

188

min.

189

2.9.2 Determination of IMS-PMAxx-qPCR specificity

190

To determine the specificity, the DNA templates of 25 bacterial strains, 22 V.

191

parahaemolyticus strains and 3 non-V. parahaemolyticus strains, were tested by

192

IMS-PMAxx-qPCR assays. These strains were cultured in 3% NaCI APW at 37℃ for

193

12 h and then diluted in PBS to obtain an approximately 106 CFU/mL bacterial 8

194

suspension. V. parahaemolyticus (ATCC 17802), V. alginolyticus (ATCC 33787), V.

195

vulnificus (ATCC 27562), and V. harveyi (SCAUFHSM 011) were mixed in equal

196

numbers (Table 2) and diluted in PBS to obtain an approximately 106 CFU/mL

197

bacterial suspension. 1-mL bacterial suspension was prepared by inoculating sample

198

homogenate (9 mL). Then, 0.5 mL of mixture was used for IMS-PMAxx test

199

procedures described above. Genomic DNA was extracted and analyzed using qPCR.

200

Bacteria-free samples were used as negative controls.

201

2.9.3 Determination of IMS-PMAxx-qPCR and PMAxx-qPCR sensitivity

202

V. parahaemolyticus was induced into VBNC state and then serial-diluted to final

203

inoculation concentrations of 1.85×106-1.85×10-1 CFU/g using stroke-physiological

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saline solution. After dilution, 0.5 mL of each bacterial solution was mixed with IMBs

205

and captured according to the method described above, followed by PMAxx

206

processing (16 µM final concentration) and qPCR analysis. Bacterial solutions not

207

processed by IMS were used in PMAxx-qPCR sensitivity detection.

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2.9.4 Quantitative detection of VBNC V. parahaemolyticus

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Mixed bacterial solutions of different VBNC V. parahaemolyticus used in this

210

study are listed in Table 3. 1mL of each mixed bacterial suspension was added to 9

211

mL of raw shrimp samples. Mixed bacterial solutions were then treated as described

212

above in section 2.9.2.

213

2.10 Statistical analyses

214

The Ct values, automatically generated through the ABI 7500, expressed as mean

215

± standard deviations. Graphs were plotted by Origin 8.5. Differences value below

216

0.05 was considered statistically significant by performing using SPSS statistical 23.0.

217

All experiments were treated in triplicate to ensure reproducibility of results.

218

3. Results

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3.1 IMS method optimization

9

220

3.1.1 Biotinylated polyclonal antibody optimization

221

The binding ratio of antibodies to magnetic nanoparticles is the most important

222

factor that affects CE of V. parahaemolyticus in the IMS method. IMBs were prepared

223

using different amounts of biotinylated polyclonal antibody, ranging from 7.5 to 60 µg

224

per 0.5 mg streptavidin-conjugated magnetic nanoparticles. Figure 1a shows the

225

relationship between biotinylated polyclonal antibody amount and CE. As the amount

226

of biotinylated polyclonal antibody increased from 7.5 µg to 60 µg, e CE gradually

227

increased, reaching a maximum of 91.8% at 30 µg. Adding more than 30 µg of

228

antibody failed to improve CE, which was stabilized at around 91%. The IMBs

229

prepared using 0.06 mg antibody per 1 mg magnetic nanoparticles was optimum. A

230

3:50 antibodies-to-magnetic nanoparticle mass ratio was determined to be the most

231

economic and efficient, and was thus used for subsequent experiments.

232

3.1.2 IMBs dose optimization

233

We assayed the effects of different IMBs doses (at a 3:50 antibodies-to-magnetic

234

nanoparticles mass ratio) on CE by adding 10, 20, 50, 100, 150, and 200 µL of IMBs

235

to 1 mL of bacteria suspension (1 × 105 CFU/mL in PBS). As shown in Fig. 1b, CE

236

gradually increased from 76.02% to 88.98% as the IMBs dose increased from 10 to

237

150 µL, indicating that sufficient doses of IMBs coupled with the bacteria. However,

238

when the amount of IMBs further increased to 200 µL, CE slightly declined. This may

239

be because excessive IMBs may block the antigen binding sites on the bacterial

240

surface. In addition, some bacteria may be damaged during the separation process,

241

resulting in a low CE of V. parahaemolyticus. Therefore, the addition of 150 µL of

242

IMBs to 1mL of 1 × 105 CFU/mL bacterial suspension was sufficient to ensure a high

243

CE (more than 88%).

244

3.1.3 Incubation time optimization

245

We determined the optimal incubation time for greatest CE in preliminary studies

246

in which bead-bacteria complexes were incubated from 15 min to 90 min. As shown

247

in Fig. 1c, CE increased to 93.75% with a 45 min incubation time, but CE did not 10

248

significantly increase when the incubation time was further extended to 90 min. These

249

results indicate that an incubation time of 45 min is the shortest amount of time to

250

achieve the highest capture efficiency.

251

3.1.4 Optimization of immunomagnetic separation time

252

To determine the effect of immunomagnetic separation time on CE, a range of

253

separation times were applied to one concentration of bacterial suspension prior to

254

PMAxx treatment. The results presented in Fig. 1d show that CE increased from

255

79.54% to 89.67% as the immunomagnetic separation time increased from 1 to 5 min.

256

However, there was no significant difference in CE after 4 min versus 5 min of

257

separation time, thus 4 min was considered to be optimum and used for further assays.

258

3.1.5 Immunoreaction temperature optimization

259

To investigate the influence of temperature on CE, we carried out the experiment

260

under a range of different temperature conditions. We compared CE at refrigeration

261

temperature (4℃, CE = 72.35%), culture temperature (37℃, CE = 89.74%) and room

262

temperature (25℃, CE = 91.62%) (Fig. 1e). Room temperature (25℃) had the greatest

263

CE and was thus used for subsequent experiments.

264

3.2 IMBs specificity

265

To assess the specificity of the IMBs, two V. parahaemolyticus strains (a live

266

strain and a strain in VBNC state) and 7 non-target bacteria strains were tested. As

267

shown in Fig. 2, approximately 88% of V. parahaemolyticus in normal and VBNC

268

states were captured by IMBs, while CEs of non-target bacteria strains were low.

269

However, other V. strains commonly seen in seafood, such as V. harveyi, V. vulnificus,

270

and V. alginolyticus, had a CE of more than 20%, probably due to the presence of

271

similar antigenic surface proteins. However, the probe and primers were sufficiently

272

specific such that the application in shrimp samples was not affected (results shown in

273

Table 2). These results demonstrated that the IMS method had high specificity for V.

274

parahaemolyticus. 11

275 276

3.3 Ultrastructure SEM was used to examine the morphology and size of the IMBs with and

277

without

bound

V.

parahaemolyticus.

The

spherical

shaped

IMBs

had

278

well-proportioned dimensions and good dispersion in the sample solution. IMBs

279

could be separated and concentrated readily from the solution by applying an external

280

magnetic force and then redispersed easily after removing the magnet (Fig. 3a, 3b).

281

The IMBs with smaller diameter (mean diameter of 500 nm) had a higher

282

surface/volume ratio and higher migration efficiency in solution, which can increase

283

opportunities for bacterial contact and higher CE.

284

Figures 3c and 3d show the normal state (rod-like) and VBNC state (coccoid) V.

285

parahaemolyticus attached to the surface of the IMBs to form bead-bacteria

286

complexes. The changed shape of V. parahaemolyticus in the VBNC state is

287

consistent with previous reports (Chen, Jane, Chen, & Wong, 2009). Moreover, each

288

bacterium can complex with several magnetic beads, forming aggregates of magnetic

289

beads and bacteria.

290

3.4 Standard curve

291

To determine the reaction efficiencies of the qPCR and PMAxx-qPCR assays,

292

fresh overnight cultures of V. parahaemolyticus bacterial suspensions were

293

serial-diluted to obtain different concentrations of DNA template used to generate a

294

standard curve for qPCR and PMAxx-qPCR. A good linear relationship was seen

295

between Ct and bacteria concentrations, with R2 values of 0.998 and 0.997 in the

296

range of 1.7 - 7.7 Log10 CFU/mL (Fig. 4). The standard curve could be used to

297

quantify viable V. parahaemolyticus cells.

298

3.5 Evaluation of VBNC V. parahaemolyticus detection using IMS-PMAxx-qPCR

299

3.5.1 Detection specificity

300

In order to reduce the influence of other V. species present in the sample, a probe

301

with high specificity was used for subsequent detection. As shown in table 2, the 12

302

prepared IMBs captured some other V. species, however, the high primer and probe

303

specificity, combined with the IMS-PMAxx-qPCR technique, allowed for specific

304

detection of V. parahaemolyticus in normal and VBNC states. Other non-target

305

bacterial strains were not detected.

306

3.5.2 Detection sensitivity

307

To evaluate the sensitivity of the two detection methods in analyzing

308

contaminated samples, raw shrimp samples spiked with a known number of target

309

bacterial cells were prepared. For PMAxx-PCR analysis, the target bacteria from the

310

artificially contaminated raw shrimp samples were separated and concentrated using

311

the IMS method as described above. Our results showed that the limit of detection

312

(LOD) of PMAxx-qPCR was 18.5 CFU/g VBNC V. parahaemolyticus without

313

enrichment (Fig. 5). When combined with IMS, VBNC V. parahaemolyticus as low as

314

1.85 CFU/g was detected in raw shrimps samples (Fig. 6). Thus, IMS efficiently

315

enriched target bacteria, which improved detection sensitivity. This method detected

316

trace VBNC state pathogenic bacteria in an actual sample in only 4 h.

317

3.5.3 Quantitative detection of different concentrations of VBNC V. parahaemolyticus

318

contamination in raw shrimp samples

319

VBNC V. parahaemolyticus and heat-killed cell suspensions were mixed to

320

obtain six different VBNC V. parahaemolyticus ratios (0%, 0.1%, 1%, 10%, 50%, and

321

100%), which were then tested by plate count, qPCR, PMAxx-qPCR, and IMS-

322

PMAxx-qPCR. As seen in Figure 7, no proportion of VBNC V. parahaemolyticus in

323

raw shrimp could be detected using traditional plate counting methods. This is due to

324

the fact that both VBNC state and heat-killed bacteria cannot grow on a plate.

325

Detection by qPCR was barely affected by the proportion of VBNC state bacteria and

326

was relatively stable (approximately 104 CFU/g). Hence, qPCR was unable to

327

distinguish between dead bacteria and VBNC state bacteria, leading to false positives.

328

Using the PMAxx-qPCR and IMS-PMAxx-qPCR methods, detection increased

329

as the proportion of viable bacteria increased. However, PMAxx-qPCR could not 13

330

quantify the absolutely proportion of living bacteria when the target was under 0.1%.

331

Therefore, IMS-PMAxx-qPCR had higher detection sensitivity than PMAxx-qPCR.

332

In general, when VBNC state and dead bacteria coexist, PMAxx-qPCR and

333

IMS-PMAxx-qPCR can both be used to effectively distinguish them and the dead

334

bacteria contribution can be subtracted to accurately determine the quantity of VBNC

335

state bacteria. In addition, the IMS technique can specifically enrich target bacteria

336

and simplify the sample pretreatment process.

337

4. Discussion

338

Recently, PMA-qPCR has been a popular technique for identifying and

339

quantifying viable V. parahaemolyticus because of its high sensitivity and specificity

340

(Huang, Zheng, Shi, & Chen, 2018; Slimani et al., 2012; Zhu, Li, Jia, & Song, 2012).

341

However, due to the complexity of the sample matrix, this method is limited in its

342

application for actual sample detection of low levels of pathogenic bacteria

343

contamination. Although it has been reported that IMS combined with detection

344

techniques, such as qLAMP (Zeng et al., 2014), PMA-qPCR (Wang et al., 2014), and

345

flow cytometry (Keserue, Baumgartner, Felleisen, & Egli, 2012), can be applied to

346

quantify VBNC state bacteria, IMS has not previously been combined with

347

PMAxx-qPCR to quantify VBNC V. parahaemolyticus in raw shrimp samples. In this

348

study, an IMS-PMAxx-qPCR assay was successfully applied to detect and quantify

349

VBNC V. parahaemolyticus in artificially contaminated raw shrimp.

350

Streptavidin-conjugated magnetic nanoparticles with a diameter of 500 nm were

351

coupled with immune polyclonal antibodies to form IMBs that can specifically attach

352

to a target bacterial strain. To enrich for target pathogenic bacteria, a magnetic field

353

can be applied to concentrate the bead-bacteria complexes, and non-target bacteria

354

and other interfering materials can be removed by washing. In this study, we

355

optimized several conditions in order to increase IMBs CE when applying IMS to

356

VBNC bacteria. We determined the optimal amount of biotinylated polyclonal

357

antibody (30 µg), does of IMBs (150 µL), incubation time (45 min), immunomagnetic 14

358

separation time (4 min), and immunoreaction temperature (25℃) for IMS of VBNC V.

359

parahaemolyticus. Applying IMS simplified the pre-enrichment step, allowing for

360

faster processing and a lower LOD (1.85 CFU/g) in artificially contaminated raw

361

shrimp. Compared with PMAxx-qPCR, IMS-PMAxx-qPCR could quantify the

362

absolute proportion of living bacteria when the target was under 0.1%. The LOD was

363

lower after IMS compared to samples without IMS treatment. A similar phenomenon

364

was reported by Wang et al. (2014) using IMS-SD-PMA-qPCR to detect Escherichia

365

coli O157:H7 in artificially contaminated milk.

366

Primer and probe design is key to achieving specificity in this assay. Our study

367

demonstrated that the IMS method also captured some other V. species (with a CE

368

more than 20%), but the primers and probe used for subsequent detection were

369

designed to specifically target the V. parahaemolyticus gene tlh, which enabled

370

species specificity. We also applied a new photoactivatable dye, PMAxx, which binds

371

less to viable cell DNA, further improving the accuracy of the results.

372

Previous reports using PMA-qPCR methods to detect pathogenic bacteria were

373

generally concerned with optimizing detection and rarely focused on optimizing

374

preprocessing. Elizaquivel, Sanchez, Selma, and Aznar (2012) and Xiao, Tian, Yu,

375

and Wu (2013) developed a PMA-qPCR method for Escherichia coli O157:H7 with a

376

LOD of 20 CFU/mL in fresh-cut vegetable wash water and 102 cell/mL in pure culture.

377

Compared to PMAxx-qPCR, the addition of an IMS preprocessing step makes

378

IMS-PMAxx-qPCR a superior analytical tool for detection of trace microbial

379

contamination in complex samples such as VBNC state foodborne pathogens in raw

380

shrimp.

381

In summary, we established a new IMS method for VBNC V. parahaemolyticus and

382

combined it with PMAxx-qPCR methods to detect VBNC V. parahaemolyticus in raw

383

shrimp samples. We confirmed that the use of IMS in combination with the

384

PMAxx-qPCR assay was more accurate than the PMAxx-qPCR assay alone for

385

detecting VBNC V. parahaemolyticus in raw shrimp. This method offers a means for

386

rapid and sensitive detection of VBNC bacteria in complex food samples without any 15

387

pre-enrichment.

388

Acknowledgments

389

This work was supported by grants from Science and Technology Planning

390

Project of Guangdong Province (2017B020207004), the National Natural Science

391

Foundation of China (31771940).

392

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Methods,

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1

Table 1

2

Primers and probe used in this study Primer sequence（5’ - 3’）

Size(bp)

tlh-F- TTCGTGCGAAAGTGCTTGAG

20

tlh-R- CACGAAACCGTGCTCTTCTG

20

tlh-Probe- FAM-AACGAGTTCATCAAGGCACAAGCG-BHQ

24

Product Length（bp）

143

3

Table 2

4

Evaluation of the specificity of IMS-PMAxx-qPCR assay IMS-PMAxx-qPCR Bacteria species

Strain Results

Ct value

Vibrio parahaemolyticus

ATCC 17802

+

18.50±0.46

VBNC Vibrio parahaemolyticus

ATCC 17802

+

19.71±1.01

Vibrio parahaemolyticus

CGMCC 1.1614

+

20.60±0.21

Vibrio parahaemolyticus

SCAUFHSM 001

+

19.70±1.45

Vibrio parahaemolyticus

SCAUFHSM 002

+

18.64±0.28

Vibrio parahaemolyticus

SCAUFHSM 003

+

20.10±0.11

Vibrio parahaemolyticus

SCAUFHSM 004

+

21.26±0.93

Vibrio parahaemolyticus

SCAUFHSM 005

+

21.20±0.58

Vibrio parahaemolyticus

SCAUFHSM 006

+

19.18±0.63

Vibrio parahaemolyticus

SCAUFHSM 007

+

20.63±1.02

Vibrio parahaemolyticus

SCAUFHSM 008

+

18.91±0.06

Vibrio parahaemolyticus

SCAUFHSM 009

+

20.92±0.42

Vibrio parahaemolyticus

SCAUFHSM 010

+

19.61±0.27

Vibrio parahaemolyticus

SCAUFHSM 1003

+

21.50±0.17

Vibrio parahaemolyticus

SCAUFHSM 1023

+

20.78±018

Vibrio parahaemolyticus

SCAUFHSM 1004

+

19.92±0.30

Vibrio parahaemolyticus

SCAUFHSM 1024

+

18.58±0.20

Vibrio parahaemolyticus

SCAUFHSM 1012

+

20.02±0.26

Vibrio parahaemolyticus

SCAUFHSM 1013

+

19.81±0.14

Vibrio parahaemolyticus

SCAUFHSM 1014

+

20.93±1.01

Vibrio parahaemolyticus

SCAUFHSM 1025

+

18.93±0.09

Vibrio parahaemolyticus

SCAUFHSM 1018

+

20.96±0.14

Vibrio parahaemolyticus

SCAUFHSM 1020

+

21.33±0.86

Vibrio alginolyticus Vibrio harvey Vibrio vulnificus

ATCC 33787

-

37.67±0.56

SCAUFHSM 011

-

38.79±0.21

ATCC 27562

-

39.45±0.69

+

23.38±0.21

-

39.06±1.24

+

20.77±0.20

+

19.47±0.52

+

24.53±1.10

Vibrio parahaemolyticus + Vibrio vulnificus + Vibrio harvey + Vibrio alginolyticus Vibrio alginolyticus + Vibrio harvey + Vibrio vulnificus Vibrio parahaemolyticus + VBNC Vibrio parahaemolyticus + Vibrio harvey Vibrio parahaemolyticus +VBNC Vibrio parahaemolyticus VBNC Vibrio parahaemolyticus + Vibrio alginolyticus + Vibrio harvey + Vibrio vulnificus 5

ATCC, American Type Culture Collection, USA; CGMCC, China General Microbiological

6

Culture Collection Center. SCAUFHSM, food Safety and System Microbiology Laboratory of

7

South China Agricultural University. “＋” indicates positive reaction; “－” indicates negative

8

reaction.

9 10

Table 3 Different proportions of VBNC and dead Vibrio parahaemolyticus cells Sample（CFU/g）

Percentage

of

Vibrio

parahaemolyticus

11

VBNC cells

Heat-killed cells

104

0

100

104

104

50

103

104

10

102

104

1

101

104

0.1

0

104

0

VBNC state（%）

in

1 2

Fig. 1 Optimization of IMS via (a) the amounts of biotinylated polyclonal antibody, (b) the doses

3

of IMBs (3:50 antibodies-to-magnetic beads mass ratio), (c) incubation time, (d) immunomagnetic

4

separation time, (e) immunoreaction temperature. Error bars in diagrams represent standard

5

deviations from three independent replicates.

6 7

8 9

Fig. 2 The specificity of the IMBs

10 11

Fig. 3 Scanning electron microscopy images. (a) Streptavidin-conjugated magnetic nanoparticles;

12

(b) Immunomagnetic beads (IMBs); (c) Enrichment of normal state Vibrio parahaemolyticus by

13

immunomagnetic beads; (d) Enrichment of VBNC Vibrio parahaemolyticus by immunomagnetic

14

beads.

15 16

Fig. 4 Establishing the (a) qPCR and (b) PMAxx-qPCR standard curve according to the

17

relationship between the Ct value and the number viable Vibrio parahaemolyticus. Plotted values

18

indicated the average value and standard deviations obtained from the independent experiments

19

with triplicates.

20 21

Fig. 5 The quantitative effect of artificial contamination sample by PMAxx-qPCR assay.

22

Positive Control;

23

cells

24

1.85×103CFU/g;

25

means fluorescence.

are:

negative control. The concentration of VBNC Vibrio parahaemolytics 1.85×106CFU/g; 1.85×102CFU/g;

1.85×105CFU/g; 1.85×101CFU/g;

1.85×104CFU/g; 1.85×100CFU/g. ∆Rn

26 27 28

Fig. 6 The quantitative effect of artificial contamination sample by IMS-PMAxx-qPCR assay. Positive Control;

29

parahaemolytics cells are:

30

1.85×104CFU/g;

31

1.85×100CFU/g;

negative control. The concentration of VBNC Vibrio 1.85×105CFU/g VBNC Vibrio parahaemolyticus;

1.85×103CFU/g;

1.85×102CFU/g;

1.85×10-1CFU/g. ∆Rn means fluorescence.

1.85×101CFU/g;

32 33

Fig. 7 Evaluation of diagnostic capability of IMS-PMAxx-qPCR assay in artificially contaminated

34

shrimp samples

35

1

Highlights:

2

1. Optimized IMS method to enrich for VBNC Vibrio parahaemolyticus in actual

3

samples.

4

2. LOD using IMS-PMAxx-qPCR in actual samples was determined to be 1.85

5

CFU/g.

6

3. IMS-PMAxx-qPCR is suitable for the detection of trace VBNC microbial

7

contamination.

1