Rapid and accurate detection of viable Vibrio parahaemolyticus by sodium deoxycholate-propidium monoazide-qPCR in shrimp

Journal Pre-proof Rapid and accurate detection of viable Vibrio parahaemolyticus by sodium deoxycholate-propidium monoazide-qPCR in shrimp Nan Ling, Jinling Shen, Jingjing Guo, Dexin Zeng, Jianluan Ren, Lixin Sun, Yuan Jiang, Feng Xue, Jianjun Dai, Baoguang Li PII:

S0956-7135(19)30472-4

DOI:

https://doi.org/10.1016/j.foodcont.2019.106883

Reference:

JFCO 106883

To appear in:

Food Control

Received Date: 6 April 2019 Revised Date:

4 September 2019

Accepted Date: 6 September 2019

Please cite this article as: Ling N., Shen J., Guo J., Zeng D., Ren J., Sun L., Jiang Y., Xue F., Dai J. & Li B., Rapid and accurate detection of viable Vibrio parahaemolyticus by sodium deoxycholate-propidium monoazide-qPCR in shrimp, Food Control (2019), doi: https://doi.org/10.1016/j.foodcont.2019.106883. 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.

1

Rapid and accurate detection of viable Vibrio

2

parahaemolyticus by sodium deoxycholate-propidium

3

monoazide-qPCR in shrimp

4

Nan Linga#, Jinling Shenb#, Jingjing Guoa#, Dexin Zenga,c#, Jianluan Rena, Lixin

5

Sune, Yuan Jiangb,d, Feng Xuea*, Jianjun Daia , Baoguang Lif

6

a

MOE Joint International Research Laboratory of Animal Health and Food

7

Safety, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing,

8

210095, China

9 10 11 12 13 14 15 16

b

Shanghai Academy of Inspection and Quarantine, Shanghai, 200135, China

c

Animal, Plant and Food Inspection Center of Nanjing Customs, Nanjing,

210095, China d

Animal, Plant and Food Inspection Center of Shanghai Customs, Shanghai,

200135, China e

Jiangsu International Travel Health Care Center, Nanjing, 210019, China

f

Division of Molecular Biology, Center for Food Safety and Applied Nutrition,

U.S. Food and Drug Administration, Laurel, MD 20708, USA

17 18

Running

Title:

19

parahaemolyticus

SD-PMA-qPCR

for

selective

detection

20 21

* Corresponding authors: Feng Xue, [email protected]

22 23

#

These authors equally contributed to this work.

24 25 26

1

of

viable

V.

27

Highlights

28 29

Specific primers and probe targeting a unique fragment in the toxR gene were

30

assessed for detection of V. parahaemolyticus by qPCR.

31

The optimal conditions for treatment of V. parahaemolyticus with SD and PMA

32

were investigated.

33

A SD-PMA-qPCR assay was developed and proved to be specific and

34

sensitive for detection viable cells from mixtures of viable and dead cells.

35

The assay has been applied to detect V. parahaemolyticus in spiked shrimp

36

samples.

2

37

Abstract

38

Vibrio parahaemolyticus is an important human pathogen causing a variety of

39

life-threatening diseases and is widely distributed in marine and estuarine

40

environments. The objective of this study was to develop a sensitive, specific,

41

and accurate method by using sodium deoxycholate (SD)-propidium

42

monoazide (PMA)-qPCR (SD-PMA-qPCR) for selective detection of viable V.

43

parahaemolyticus cells in shrimp. A qPCR assay was developed by targeting a

44

unique fragment in the toxR gene in V. parahaemolyticus. The qPCR assay

45

demonstrated superior specificity (100%) on V. parahaemolyticus strains (n =

46

70) and non-V. parahaemolyticus strains (n = 37) examined in the inclusivity

47

and exclusivity tests; and the limit of detection (LOD) of the assay reached 5 ×

48

101 CFU/ml. To remedy the drawback of PCR, SD-PMA treatment was

49

incorporated with the qPCR assay. The optimized PMA treatment conditions

50

were determined as follows, 40 µM PMA and 3-min light exposure at 40 w. The

51

maximum removal efficiency of non-viable cell DNA was achieved by an

52

optimal amplicon (262 bp) of qPCR for PMA treatment with SD at an optimal

53

concentration

54

SD-PMA-qPCR assay for detection of viable V. parahaemolyticus cells in

55

shrimp. Consequently, the SD-PMA-qPCR assay could accurately detect as

56

low as 5 × 101 CFU/g of V. parahaemolyticus in the presence of a large number

57

of non-viable cells (5 × 107 CFU/g) in spiked shrimp with a 4-h enrichment. In

58

summary, the qPCR assay based on the target gene, toxR, is sensitive and

59

specific; treatment of non-viable cells with SD and PMA improved the removal

60

efficiency of DNA of non-viable cells; and the SD-PMA-qPCR assay developed

61

in this study is a specific and accurate detection method for viable V.

62

parahaemolyticus, providing an effective and rapid means for detection of

63

viable V. parahaemolyticus in food.

(0.02%

wt/vol).

Furthermore,

we

have

applied

the

64 65

Keywords: Vibrio parahaemolyticus, propidium monoazide (PMA), sodium

66

deoxycholate (SD), SD-PMA-qPCR, foodborne pathogens, viable cells, shrimp, 3

67

limit of detection (LOD).

68 69

1. Introduction

70

Vibrio parahaemolyticus is a Gram-negative pathogenic bacterium and a

71

major foodborne pathogen known to cause gastroenteric infections (Su & Liu,

72

2007). This is pathogen is often isolated from seawater, sediment, and a

73

variety of seafood including oyster, clam, scallop, octopus, shrimp, crab,

74

lobster, and crawfish (Letchumanan, Chen, & Lee, 2014; Shen et al., 2009). It

75

is the most prevalent species among over 30 Vibrio species reported and has

76

become a major food safety concern in many Asian countries. In the coastal

77

cities in China, 23.12% outbreaks of foodborne diseases are related to V.

78

parahaemolyticus due to high seafood consumption (Wu et al., 2018). This

79

pathogen has not only become an important food safety issue, but it is also a

80

serious medical and public health problem. To handle outbreaks in a timely

81

and efficient manner in the future, it is necessary to have sensitive, specific

82

and reliable methods for detection of V. parahaemolyticus.

83

To date, the traditional culture method remains the most common

84

detection method, which mainly includes the steps of enrichment, selective

85

culture separation, biochemical identification, etc. Culture based methods

86

have the advantages of strong specificity and acceptable sensitivity in

87

detection of microorganisms. However, these methods also have drawbacks

88

as they are time-consuming and cumbersome (Zeng, Chen, Jiang, Xue, & Li,

89

2016). qPCR (Bustin et al., 2009) is a faster, more sensitive, less

90

labor-intensive assay and has been widely used in detection of various

91

foodborne pathogens (Li & Chen, 2012, 2013; Schnetzinger, Pan, & Nocker,

92

2013; Willenburg & Divol, 2012; Xiao, Zhang, Sun, Pan, & Zhao, 2015; Zi et al.,

93

2018). Several qPCR-based methods have been reported for rapid and

94

sensitive detection of V. parahaemolyticus (Blackstone et al., 2003; Cai, Jiang,

95

Yang, & Huang, 2006; Kim et al., 1993; Lee, Pan, & Chen, 1995; Liu et al.,

4

96

2012; Makino et al., 2003; Tada et al., 1992; Venkateswaran, Dohmoto &

97

Harayama, 1998; Zhang et al., 2015). These assays manage to identify V.

98

parahaemolyticus, however, there is room for improvement in their specificity.

99

These studies showed that the thermostable direct hemolysin (TDH) gene of V.

100

parahaemolyticus has various degrees of homology to the TDH gene of other

101

species in the Vibrio family (Tada et al.,1992). Therefore, it is necessary to

102

select a more specific and stable genetic marker for detection of V.

103

parahaemolyticus.

104

Another challenge for accurate detection of viable V. parahaemolyticus in

105

food is PCR’s inability to differentiate DNA from non-viable cells and viable

106

cells in amplification (Zeng, Chen, Jiang, Xue, & Li, 2016). Propidium

107

monoazide (PMA) has been combined with qPCR to overcome this

108

shortcoming of PCR assay (Li & Chen, 2012, 2013; Scariot, Venturelli, Prudê

109

ncio, & Arisi, 2018; Nocker, Chenung, & Camper, 2006; Schnetzinger, Pan, &

110

Nocker, 2013; Yuan, Guolu, Mengsh, & Azlin, 2018). However, it was reported

111

that for some pathogens, PMA alone cannot completely inhibit the DNA

112

amplification in non-viable cells, probably because the damaged cell debris

113

can prevent the penetration of PMA into the cell membranes (Wang et al.,

114

2015). Sodium deoxycholate (SD), an anionic surfactant, can destroy the

115

membranes of the damaged or non-viable cells to enhance permeability of

116

cells. Therefore, treatment with SD prior to treatment of PMA can facilitate

117

PMA to penetrate non-viable or damaged cells more effectively and thus more

118

completely remove the DNA from the non-viable cells (Wang et al., 2015). In

119

the present study, we developed a qPCR assay and combined it with an

120

improved PMA treatment to accurately detect viable V. parahaemolyticus cells

121

in shrimp.

122

2. Materials and methods

123

2.1 Bacterial strains and culture conditions

124

V. parahaemolyticus ATCC 17802, a reference strain used throughout the 5

125

study, was inoculated in 3% NaCl alkaline peptone water (NAPW) medium and

126

incubated at 37°C with shaking at 180 rpm for 6 h. The bacterial culture was

127

centrifuged at 5000 × g for 5 min and washed twice with phosphate buffered

128

saline (PBS), then the bacteria were resuspended in PBS. The cell density of

129

the suspension was then measured by BioTek Synergy at OD600nm. To count

130

bacterial cells, cultures were serially diluted in NAPW medium in 10-fold

131

increment and plated overnight on nutrient agar plates at 37°C. The results

132

showed that the concentration of a culture at OD600nm = 0.5 was plate counted

133

to be equivalent to 5 × 108 CFU/ml. These used in the inclusivity and

134

exclusivity tests were also grown in NAPW broth at 37°C with shaking at 180

135

rpm for 6 h.

136

2.2 Primers and probes

137

In order to identify a sensitive, specific and reliable genetic marker for

138

detection of V. parahaemolyticus by qPCR, the tdh, tlh, trh, toxR, and VP1332

139

genes were assessed and compared in this study. We found all these genes of

140

V. parahaemolyticus demonstrated various degrees of homology with other

141

species in the Vibrio family; whereas a fragment near the 5’-end of the toxR

142

gene was found unique in V. parahaemolyticus by BLAST analysis. Therefore,

143

the unique fragment in the toxR gene was selected as a genetic marker for the

144

detection of V. parahaemolyticus by qPCR. Studies have shown that PMA has

145

different binding efficiency to DNA of different lengths (Li & Chen, 2012, 2013).

146

Six pairs of primers were designed to study the relationship between the PMA

147

binding efficiency among different PCR amplicons targeting the same area but

148

varying in length. To monitor possible inhibitory factor(s) present in samples,

149

an internal amplification control (IAC) was designed for the assay. The primers

150

and probe for the IAC were designed based on the pUC57 sequence (Table 1).

151

2.3 Preparation of non-viable V. parahaemolyticus

152

V. parahaemolyticus ATCC 17802 was inoculated at OD600nm = 0.5. The

153

cultures were washed three times with PBS by centrifuging at 5,000 × g for 5 6

154

min at room temperature. The diluted cell suspensions were equally divided

155

into two sets of aliquots. One set of the aliquots for non-viable cells was heated

156

at 70, 80, 90, and 100°C for 5 min, respectively; and the other set for viable

157

cells was not heated. Plate count and qPCR assay were performed on both

158

sets. Based on the results of plate count and qPCR, when the heat

159

temperature (80°C) was used to make non-viable cells, a minimal Cq

160

difference between viable and non-viable V. parahaemolyticus was achieved

161

(data shown) in the PMA-qPCR assay.

162

2.4 Optimization of PMA treatment

163

The optimal PMA concentration was obtained by incubating with 106

164

non-viable cells/ml at 80℃ for 5 min. PMA (Biotium, Hayward, CA, USA) was

165

dissolved in water to make stock solution at 10 mM and then diluted with water

166

to 1 mM as work solution. Appropriate volumes of PMA (1 mM) were added to

167

1 ml of non-viable cells to the final concentrations of 0, 10, 20, 30, 40, 50, and

168

80 µM, respectively (Figure 2). The PMA-treated samples were incubated at

169

room temperature in the dark for 5 min, with shaking gently three to four times,

170

3 s each time. The samples were then exposed to light with six intensities (0,

171

20, 40, 60, 80, and 100 w) and incubated with five durations (1, 3, 5, 7, and 9

172

min). The treatment of viable cells was the same as non-viable cells. DNA was

173

extracted using TIANamp bacterial DNA kit (Tiangen Biotech Beijing Co., Ltd.,

174

Beijing, China) according to the manufacturer’s instructions.

175

2.5 Optimization of SD concentration

176

To select suitable SD concentration for cell treatment, the experiment was

177

divided into two groups: group I was assessed for the SD inhibitory effect on

178

viable cells; and group II was assessed for the SD effective concentration for

179

treatment of non-viable cells. In group I, each concentration of SD (0, 0.02,

180

0.04, 0.08, 0.10, and 0.50%) was added separately to 1-ml of viable bacterial

181

suspensions (5 × 106 CFU/ml) and incubated at 37°C for 20 min. The treated

182

bacteria were 10-fold serially diluted and plated onto nutrient agar plates for 7

183

viable cell count. In group II, an optimized SD concentration (0.02%) was

184

added to non-viable cells and incubated at 37°C with shaking at 180 rpm for 20

185

min. The samples were then incubated with 40 µM PMA in the dark at room

186

temperature for 5 min with intermittent shaking. Finally, the cells were exposed

187

to light at 40 w for 5 min. The PMA-treated samples were subjected to DNA

188

extraction using the same DNA kit as mentioned above. The resulting DNA

189

template was used for qPCR analysis.

190

2.6 DNA extraction and qPCR assay

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One ml of bacterial culture (OD600 = 0.5, equivalent to 5.0 × 108 CFU/ml)

192

was treated with PMA and extracted for DNA. The DNA concentrations were

193

determined using a spectrophotometer (NanoDrop Technology, Wilmington,

194

DE, USA). qPCR was performed in a total volume of 20 µl using the 7500

195

Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The PCR

196

reaction mix consisted of 10 µl of Premix Ex Taq Master Mix (Takara, Dalian,

197

China), 800 nM forward and reverse primers, 400 nM probe, 200 nM ROX

198

Reference Dye II (Takara, Dalian, China), 2 µl of template DNA, and 5.8 µl of

199

nuclease-free water to make up 20 µl as the final reaction volume. The final

200

concentrations for the IAC in the PCR mixture were as follow: 100 nM forward

201

and reverse primers, 50 nM probe, and roughly 300 copies of plasmid pUC57

202

DNA as template. The qPCR amplification included initial pre-denaturation at

203

95°C for 30 s, then 95°C for 5 s, and followed by 62°C for 34 s (40 cycles).

204

2.7 Inclusivity and exclusivity tests

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The V. parahaemolyticus strains in the inclusivity test included a reference

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strain (ATCC17802) and diverse V. parahaemolyticus strains (n = 70) with

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different multi-locus sequence types and antimicrobial resistance profiles,

208

which were isolated from various food categories in a two-year survey. The

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exclusivity test contained different species of non-V. parahaemolyticus strains

210

(n = 37), including common foodborne pathogens (Table 2). All strains were

211

from Nanjing Agricultural University. 8

212

2.8 Sensitivity test and limit of detection

213

A mid-exponential phased culture (OD600 = 0.5) of V. parahaemolyticus

214

grown at 37°C (5 - 6 h) was centrifuged. A 10-fold serial dilution of the culture

215

was made. Bacterial DNA was extracted with TIANamp bacterial DNA kit and

216

used as template in the qPCR assay.

217

2.9

218

SD-PMA-qPCR

Detection

of

V.

parahaemolyticus

by

qPCR,

PMA-qPCR,

and

219

This experiment was divided into three groups. The Cq values of viable

220

cells and non-viable cells in each group were compared. In the first group,

221

neither the viable nor non-viable cells were treated by PMA or SD-PMA. In the

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second group, 1 ml of viable and non-viable cells (5 × 106 CFU/ml) were

223

treated with 40 µM PMA in the dark and exposed to light at 40 w for 3 min,

224

respectively. In the third group, 1 ml of viable and non-viable cells (5 × 106

225

CFU/ml) were treated with 0.02% SD, incubated at 37℃ with shaking at 180

226

rpm for 20 min, and then treated with 40 µM PMA in the dark and exposed to

227

light at 40 w for 3 min. DNA extraction and qPCR were performed in the same

228

way as mentioned above for all the three groups.

229

2.10 Detection viable cells from mixtures of viable and non-viable cells by the

230

SD-PMA-qPCR assay

231

The optimized SD-PMA-qPCR assay was used to detect viable V.

232

parahaemolyticus in a mixture of viable and non-viable cells. Viable V.

233

parahaemolyticus cells ranging from 5 × 101-107 CFU/ml were equally divided

234

into two sets of aliquots: one set was treated with SD-PMA; and the other set

235

was not treated as a control. To make the mixtures of viable and non-viable

236

cells, viable cells of different concentrations (5 × 101-107 CFU/ml) were mixed

237

with 5 × 107 CFU/ml non-viable cells respectively, and then the mixtures were

238

equally divided into two set of aliquots: one set was treated with SD-PMA, and

239

the other set was not treated as control. One ml of cell suspension was used 9

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for DNA extraction using TIANamp bacterial DNA kit and analyzed by qPCR as

241

mentioned above.

242

2.11 Application of SD-PMA-qPCR for detection of viable V. parahaemolyticus

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in spiked shrimp

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Raw shrimp was purchased from a local supermarket and confirmed to be

245

free of V. parahaemolyticus using standard culture methods (ISO, 2007). The

246

shrimp samples were spiked with two types of inoculums: viable cells of (5 ×

247

101 CFU/g) and a mixture viable cells (5 × 101 CFU/g) and non-viable cells (5 ×

248

107 CFU/g). The spiked sample (25 g) was mixed with 225 ml of 3% NAPW

249

medium blending at low speed for 2 min and incubated at 37°C for 6 h. Two ml

250

of incubated samples were collected after 0-h, 2-h, 4-h, 6-h, and 8-h incubation.

251

The samples were centrifuged at 600 × g for 5 min to remove tissue and fat.

252

The cells in the supernatant were precipitated, washed twice with PBS, and

253

resuspended in 1 ml of PBS. SD-PMA treatment of samples was done before

254

DNA extraction. SD-PMA-qPCR was performed as described above.

255

2.12 Statistical analysis

256

Statistical analysis was performed using SPSS 17.0 software. Value

257

differences were compared using the least significant difference (LSD) method

258

at p = 0.05 (Zhang et al., 2015).

259

3. Results

260

3.1 Sensitivity and specificity of the qPCR assay

261

Several genetic markers have been used in PCR assays for detection of V.

262

parahaemolyticus, including tdh, trh, 16srDNA, pR72H, gyrB, toxR, and

263

vp1332 (Blackstone et al., 2003; Kim et al., 1993; Lee, Pan, & Chen, 1995;

264

Makino et al., 2003; Tada et al., 1992; Venkateswaran, Dohmoto, & Harayama,

265

1998). However, as targets, those genes, sometimes, failed to specifically

266

identify V. parahaemolyticus (Kim et al., 1993; Shirai et al., 1990; Tada et al.,

267

1992; Venkateswaran & Harayama, 1998). This prompted us to assess the 10

268

suitability of using the toxR gene as a genetic marker for detection of V.

269

parahaemolyticus in qPCR. The expression of T3SS1 is regulated by the toxR

270

and calR genes, and the toxR gene indirectly inhibits the expression of

271

T3SS1-related genes by directly activating the transcriptional expression of the

272

calR gene. The toxR gene is a species-specific gene of Vibrio (George et al.,

273

2017). A roughly 300-bp fragment located in the toxR gene was found to be a

274

unique sequence in V. parahaemolyticus by BLAST analysis. Therefore, the

275

unique fragment of the toxR gene was selected as the genetic marker in this

276

study. The qPCR results showed that the slope of the standard curve was

277

-3.4005. The amplification efficiency (E = 10-1/slope-1) of the corresponding PCR

278

calculated in this method were 95.0%. There was a good linear correlation

279

between Cq values and bacterial concentrations in a range of 5 × 100-107

280

CFU/ml from V. parahaemolyticus with R2 values of 0.999. The resultant limit

281

of detection (LOD) of the q-PCR was 5 × 101 CFU/ml for V. parahaemolyticus

282

(Figure 1)

283

In the exclusivity test, all the non-target strains (n = 37) produced negative

284

results; while in the inclusivity test, all the target strains (n = 70) were tested

285

positive by the qPCR assay. The qPCR assay demonstrated 100% specificity

286

in detection of V. parahaemolyticus.

287

3.2 Optimization of the PMA treatment

288

As shown in Figure 2A, the abscissa was PMA concentration of 10, 20, 30,

289

40, 50, and 80 µM, respectively. The ordinate was the average Cq value

290

difference, which is the Cq value of viable cells with PMA treatment minus the

291

Cq value of non-viable cells of the same concentration (5 × 106 CFU/ml) with

292

PMA treatment. It can be seen clearly that the optimal PMA concentration is 40

293

µM (p < 0.05). For light exposure optimization, the largest Cq value difference

294

was observed when the light exposure intensity was set at 40 w (p < 0.05)

295

(Figure 2B). The effect of amplicon length (70 - 349 bp) in qPCR was shown in

296

Figure 2C, demonstrating a strong relationship with the removal efficiency of

11

297

non-viable cell DNA in PMA treatment. However, no significant difference was

298

found between amplicon lengths of 262 and 349 bp. Figure 2D shows the

299

schematic effect of light exposure duration on the Cq value difference. There

300

was no significant difference between 3 and 7 min of incubation time, so, 3 min

301

was selected as the incubation time.

302

3.3 Optimization of the SD concentration

303

The maximum SD concentration without affecting viable cells was

304

determined by assessing the enhanced PMA penetrability to non-viable cells.

305

SD concentration from 0.02% to 0.50% demonstrated various degrees of

306

efficacy in removal DNA of non-viable cells in qPCR, and concentration ≥ 0.1%

307

demonstrated some effect on the amplification of viable cells (Table 3).

308

Hence, the optimized concentration of SD was selected as 0.02%, as at this

309

concentration, SD notably enhanced PMA’s inhibitory effect on DNA

310

amplification of non-viable cells without affecting viable cells (Table 4).

311

3.4 Comparison of qPCR, PMA-qPCR, and SD-PMA-qPCR assays in

312

detection of V. parahaemolyticus

313

There was no significant difference between the viable cells treated with

314

SD-PMA, PMA, or untreated cells. However, there was a significant difference

315

(p < 0.05) between the non-viable cells treated with SD-PMA, PMA, and the

316

untreated cells as shown in Table 4. The treatment with SD-PMA was the most

317

effective way for the selective detection of viable V. parahaemolyticus.

318

3.5 Differentiation of viable cells from mixtures of viable and non-viable cells in

319

SD-PMA-qPCR

320

The comparison of viable cells treated with and without SD-PMA was

321

made in qPCR and shown in Figure 3. The Cq values of the SD-PMA-treated

322

samples decreased as the number of viable cells increased. The SD-PMA

323

treated samples showed similar Cq values to the Cq values of samples with

324

the same viable cell concentrations that were not treated with SD-PMA (NC). 12

325

The effect of SD-PMA on mixtures of viable cells and non-viable cells was

326

shown in Figure 4. The Cq values from the SD-PMA treated samples

327

increased as the number of viable cells decreased. The smaller the proportion

328

of viable cells was, the bigger Cq value differences between the SD-PMA

329

treated and untreated samples became, whereas the Cq values of the samples

330

without SD-PMA treatment did not show much notable changes. This indicated

331

that SD-PMA inhibited the DNA amplification of non-viable cells and the qPCR

332

result exclusively reflected the amount of DNA of viable cells. Thus, this

333

SD-PMA-qPCR assay can accurately detect viable cells from mixtures of

334

viable and non-viable cells.

335

3.6 Detection of viable V. parahaemolyticus cells in spiked shrimp

336

The shrimp samples spiked with 5 × 101 CFU/g of V. parahaemolyticus

337

viable cells were positive by the SD-PMA-qPCR after a period of enrichment

338

time (Figure 4A). In the case of 0-h enrichment, the Cq values for SD-PMA

339

treated and untreated samples were both greater than 35, which were

340

generally considered negative. The Cq values (36.81) for SD-PMA treated

341

samples were slightly higher than the Cq value (34.32) for untreated sample

342

after a 2-h enrichment; while with a 4-h enrichment, the Cq values for the

343

SD-PMA treated and untreated samples were 25.46 and 25.39, respectively.

344

These results showed that the SD-PMA-qPCR was able to detect 5 × 101

345

CFU/g V. parahaemolyticus in the shrimp samples and, SD-PMA treatment of

346

samples did not affect the amplification of the DNA of viable cells.

347

Furthermore, the SD-PMA-qPCR assay was successfully detected low

348

number of viable cells (5 × 101 CFU/g) spiked in shrimp in the presence of a

349

large number of non-viable cells (5 × 107 CFU/g) (Figure 4B). With a 4-h

350

enrichment, a Cq value of 25.36 was detected in the sample with viable cells (5

351

× 101 CFU/g) mixed with non-viable cells (5 × 107 CFU/g). The Cq values for

352

0-h, 2-h, 4-h, and 6-h enrichment of SD-PMA untreated samples were 29.12,

353

25.57, 21.78, and 18.61, respectively. Obviously, these qPCR results were

13

354

heavily affected by the presence of non-viable cells in the samples. However,

355

the Cq values of the samples treated with SD-PMA yielded Cq values of 37.22,

356

33.23, 25.36, and 21.91, respectively. This result seemed to have depicted a

357

cell growth curve, i.e., as the incubation time went on, the viable cells

358

increased proportionally. It also showed that the DNA of non-viable cells in the

359

samples did not affect the outcome of the detection, suggesting that the

360

SD-PMA treatment effectively inhibited the amplification of the DNA of

361

non-viable cells in the samples. No obvious differences were observed on the

362

Cq values between the viable cells and the mixtures of viable and non-viable

363

cells after a 4-h enrichment, suggesting that the SD-PMA-qPCR assay

364

selectively detected 5 × 101 CFU/g viable V. parahaemolyticus cells in the

365

shrimp samples.

366

4. Discussion

367

Outbreaks of V. parahaemolyticus cause many problems in human health,

368

food safety and animal husbandry development. It is of great significance to be

369

able to rapidly and accurately detect V. parahaemolyticus in food. In the

370

present study, we developed a novel qPCR assay in conjunction with SD-PMA

371

treatment of samples for accurate detection of viable V. parahaemolyticus in

372

raw shrimp. It not only allows for rapid and accurate detection of V.

373

parahaemolyticus, but also for detecting low concentration of viable V.

374

parahaemolyticus from shrimp samples. One of the focuses of this study was

375

to select a specific target gene for development of a sensitive and specific

376

qPCR assay for detection of V. parahaemolyticus. The inclusivity and the

377

exclusivity tests in this study demonstrated that the qPCR assay with the

378

unique fragment in the toxR gene as target is specific and sensitive, showing a

379

LOD of 5 × 101 CFU/ml in the qPCR assay without cross-reactivity with any

380

non-V. parahaemolyticus strains (Figure 1). Also, inclusion of IAC in the qPCR

381

assay can help monitor possible inhibitory factor(s) in amplification to minimize

382

false negative results.

14

383

The other focus of the present study was to selectively detect viable V.

384

parahaemolyticus cells from non-viable cells. We incorporated a sample

385

treatment procedure with PMA in the qPCR assay to overcome a major

386

drawback of the conventional and qPCR assays, i.e., inability to differentiate

387

DNA of viable and non-viable cells in amplification. PMA does not significantly

388

affect the amplification of DNA of viable cells in PCR (Zi et al., 2018). Amplicon

389

length has been found to be critical in removal efficiency of DNA of non-viable

390

cells (Li & Chen, 2012, 2013), which is corroborated by our finding on the

391

relationship between PMA’s removal efficiency of DNA of non-viable cells and

392

the amplicon length in qPCR. Specifically, the PMA’s removal efficiency by and

393

large increased with the amplicon length as shown in Figure 2. However, when

394

the amplicon length exceeded 262 bp, this tendency appeared diminished

395

(Figure 2). Thus, we selected an amplicon of 262-bp in length as the optimal

396

amplicon. In addition, we observed that non-viable or injured cells with

397

complete membranes could impede PMA’s permeation to cells. To solve this

398

problem, previously, SD was used to enhance PMA’s permeation to damaged

399

cell membranes (Nkuipou-Kenfack, Engel, Fakih, & Nocker, 2013). In this

400

study, we assessed the toxic effect of SD treatment on viable cells and found

401

that different concentrations of SD demonstrated variable degree of inhibitory

402

effects on viable cells of V. parahaemolyticus as shown in Table 3.

403

Comparative analysis indicated the optimized SD (0.02%) should be used in

404

conjunction with PMA treatment and that SD-PMA treatment is more

405

advantageous for the selective detection of viable V. parahaemolyticus

406

compared with qPCR or PMA-qPCR assays (Table 4).

407

In this study, we used SD-PMA treatment to distinguish viable cells from

408

non-viable cells in qPCR assay (Figure 3). The Cq value between the SD-PMA

409

treated and untreated viable cells did not show much differences, indicating

410

that SD-PMA did not affect the DNA amplification of viable cells. In contrast,

411

the Cq values of mixtures of viable cells and non-viable cells treated by

412

SD-PMA were much higher than those of the untreated; while the Cq values of 15

413

the SD-PMA treated mixtures of viable cells and non-viable cells were similar

414

to those of viable cells. These results indicated that the SD-PMA assay

415

accurately distinguishes viable cells from non-viable cells in the amplification.

416

Furthermore, we have applied this SD-PMA-qPCR assay in selective

417

detection of viable V. parahaemolyticus cells in spiked shrimp. The detection

418

results indicated that the SD-PMA-qPCR assay can selectively detect 5 × 101

419

CFU/g viable V. parahaemolyticus from shrimp samples after a 4-h enrichment

420

(Figure 4). Despite the presence of a large number of non-viable cells (5 × 107

421

CFU/ml) in the samples, the detection results seemed to have reflected the

422

actual viable cell numbers in the samples, suggesting that the DNA from

423

non-viable cells was completely excluded in the PCR amplification.

424

PMA-qPCR method has been used for detection of viable V.

425

parahaemolyticus (Niu et al., 2018; Cao et al., 2017), and SD has been used to

426

facilitate PMA to penetrate non-viable or damaged cells more effectively (Liang

427

et al., 2019). In this study, we incorporated a SD-step in the PMA treatment

428

procedure and, the PMA’s removal efficiency of DNA of non-viable cells was

429

notably improved without stretching the experimental process of the assay

430

(within 1.5-h). Furthermore, we have determined the optimal amplicon length

431

(262 bp) in qPCR for PMA treatment, which may serve as a guidance for probe

432

design in selective detection of viable cells of V. parahaemolyticus and

433

potentially other Gram-negative bacterial pathogens by PAM-qPCR.

434

In summary, in this study, a qPCR assay was developed by using a unique

435

fragment in the toxR gene as the detection target. The assay proved to be

436

sensitive and specific for detection of V. parahaemolyticus. Incorporation of a

437

SD-step in the PMA treatment procedure notably improved PMA’s removal

438

efficiency of DNA of non-viable cells. As a result, the superb sensitivity and

439

specificity of the SD-PMA-qPCR assay were evidenced by the accurate

440

detection of 5 × 101 CFU/g viable V. parahaemolyticus in spiked shrimp in the

441

presence of 5 × 107 CFU/ml non-viable cells in the samples. Thus, this

442

SD-PMA-qPCR assay may provide a sensitive, specific, and accurate means 16

443

for detection of viable V. parahaemolyticus in food.

444 445

Acknowledgements

446

This study was funded by the National Key Research and Development

447

Program of China (31871893), the National Key Research and Development

448

Program of China (2018YFC1603600, 2017YFF0208600), Jiangsu Agricultural

449

Independent Innovation Project (SCX(18) 2011)，Science and Technology

450

Joint Project of the Yangzte River Delta (No.17395810102),The National

451

“Youth Top-notch Talent” Support Program

452

Nanjing Agricultural University Scientific Research Grants Project (804121),

453

Central Guidance for Local Science and Technology Development (No.

454

YDZX20173100004528), Jiangsu Collaborative Innovation Center of Meat

455

Production and Processing. The authors gratefully thank professors Xue, F

456

and Li, B for their guidance.

(W0270187), Introduction of

457 458

Ethics approval and consent to participate

459

Not applicable.

460

Competing interests

461

The authors declare that they have no competing interests.

462

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583

Figure Legends

584 585

Figure 1. Standard curves of qPCR assay using toxR gene as a target. Each

586

bar represents the average Cq value ± standard deviation of a triplicate.

587 588

Figure 2. PMA pretreatment on non-viable cells: PMA concentration (A), light

589

exposure intensity (B), effect of the PCR amplicon length (C), and light

590

exposure duration (D). Each bar represents the average of Cq value ±

591

standard deviation of a triplicate.

592 593

Figure 3. Differentiation of viable cells in mixtures of viable and non-viable

594

cells by SD-PMA-qPCR. Two sets of the cell dilutions were treated with

595

SD-PMA or left untreated prior to DNA preparation (A). Two sets of the cell

596

dilutions were mixed with 5 × 106 non-viable cells/ml (B). The cell mixtures

597

were treated with SD-PMA or left untreated prior to DNA preparation. Each bar

598

represents the average of Cq value ± standard deviation of a triplicate.

599 600

Figure 4. Selective detection of low numbers of viable V. parahaemolyticus

601

cells spiked in shrimp by SD-PMA-qPCR assay. Homogenates of shrimp

602

samples were inoculated with 5 × 101 CFU/g V. parahaemolyticus cells as

603

control (A); and shrimp samples were simultaneously inoculated with 5 ×107

604

non-viable cells/g and 5 × 101 CFU/g V. parahaemolyticus cells (B). The

605

incubated samples were collected in a time course as indicated. Cells

606

recovered from the shrimp samples were treated with SD-PMA or left

607

untreated prior to DNA preparation. Each bar represents the average of Cq

608

value ± standard deviation of a triplicate.

22

609 Table 1. Amplicons and their primers and probes targeting the ToxR gene in qPCR. Amplicon name

Primer/probe sequence (5'--- ---3')

ToxR-1

Forwad AACGATCGTAGAGCCGTCTT

Amplicon size (bp) 70

Reverse AGGTACTACTGGCGCTTCT Probe FAM-ACGCAATCGTTGAACCAGAAGCGCCA-TAMRA ToxR-2

Forwad AACGATCGTAGAGCCGTCTT

103

Reverse AGGATTCACAGCAGAAGCCA Probe FAM-ACGCAATCGTTGAACCAGAAGCGCCA-TAMRA ToxR-3

Forwad AACGATCGTAGAGCCGTCTT

158

Reverse GCAGTACGCAAATCGGTAGTA Probe FAM-ACGCAATCGTTGAACCAGAAGCGCCA-TAMRA ToxR-4

Forwad AACGATCGTAGAGCCGTCTT

202

Reverse CTCACCAATCTGACGGAACTG Probe FAM-ACGCAATCGTTGAACCAGAAGCGCCA-TAMRA ToxR-5

Forwad AACGATCGTAGAGCCGTCTT

262

AGGCAACCAGTTGTTGATTTG Probe FAM-ACGCAATCGTTGAACCAGAAGCGCCA-TAMRA ToxR-6

Forwad AACGATCGTAGAGCCGTCTT

349

Reverse AGGCAACCAGTTGTTGATTTG Probe FAM-ACGCAATCGTTGAACCAGAAGCGCCA-TAMRA IAC

Forwad CGGTGGAAACTACCAAGCTG Reverse TTTCGCCGTTGGTGTTCTTT Probe HEX-ACGCATTTCACCGCTCCACCGG-TAMRA

610 611

23

93

Table 2. Inclusivity and exclusivity tests for specific detection of V. parahaemolyticus by qPCR.

612

Bacterial species

Name of strains*

V. parahaemolyticus V. parahaemolyticus V. parahaemolyticus V. parahaemolyticus V. cholerae V. mimicus V. fluvialis V. vulnificus V. metschnikovii V. fischeri V. harveyi V. anguillarum V. alginolyticus Bacillus cereus group Bacillus subtilis Yersinia enterocolitica Listeria monocytogenes Salmonella Enteritidis Campylobacter jejuni Proteus vulgaris Proteus mirabilis Enterococcus faecalis Escherichia coli O157:H7 Escherichia coli Pseudomonas aeruginosa Pseudomonas putida Citrobacter freundii Staphylococcus epidermidis Staphylococcus aureus Staphylococcus warneri Hafnia alvei Klebsiella pneumoniae Aerococcus viridans Rhodococcus coprophilus Erysipelothrix rhusiopathiae Enterobacter aerogenes Streptococcus pyogenes Streptococcus dysgalactiae Shewanella algae Shigella dysenteriae Serratia fonticola

ATCC 33847 ATCC 17802 CICC 10522 VP1-VP67 BNCC232030 CICC10474 CICC21612 CICC10383 CICC21660 BNCC188419 BNCC336937 BNCC170086 ATCC33787 ATCC11778 Isolate ATCC23715 ATCC19119 CICC24119 ATCC33291 CMCC49027 CMCC49005 ATCC19433 ATCC43859 ATCC25922 ATCC27853 Isolate ATCC10787 ATCC12228 ATCC25923 Isolate Isolate Isolate Isolate Isolate Isolate Isolate Isolate Isolate Isolate Isolate Isolate

No. of strains tested

Detection result

1 1 1 67 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

*All strains used in this study were from College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, China.

24

+ + + + -

Table 3. Treatment of viable and non-vible V. parahaemolyticus cells with SD and its effect in the PMA-qPCR. SD concentration Cell treatment

Cells were not treated with SD

0.02%

0.04%

0.08%

0.10%

0.50%

Viable cell number (x 10 ) assessed by plant count

5.8±0.7

3.8±0.7

4.3±0.7

2.7±0.5*

2.8±0.1**

6.0±0.6

Cq value on the non-viable (heat-killed) cells

35.78±0.38*

33.63±0.27

34.51±0.19

36.01±0.85*

35.56±0.44*

34.13±0.19

6

* Indicates a significant difference from the negative control (p < 0.05). ** Indicates a very significant difference compared to the negative control (p < 0.01).

25

Table 4. qPCR detection of viable and non-viable V. parahaemolyticus cells treated by PMA or SD-PMA. Cell treament

Viable cells

Non-viable cells

SD-PMA

22.74±0.18a

35.78±0.38b

PMA

22.53±0.25

34.13±0.20

Cells were not treated by PMA or SD

22.50±0.14

22.90±0.38

a Cq value refers to the average ± standard deviation of triplicate. b Bold-faced number refers to a significant difference (p < 0.5).

26

27

B

A

C

D

28

29

30

Table 2. Inclusivity and exclusivity tests for specific detection of V. parahaemolyticus by qPCR. Bacterial species

Name of strains*

V. parahaemolyticus V. parahaemolyticus V. parahaemolyticus V. parahaemolyticus V. cholerae V. mimicus V. fluvialis V. vulnificus V. metschnikovii V. fischeri V. harveyi V. anguillarum V. alginolyticus Bacillus cereus group Bacillus subtilis Yersinia enterocolitica Listeria monocytogenes Salmonella Enteritidis Campylobacter jejuni Proteus vulgaris Proteus mirabilis Enterococcus faecalis Escherichia coli O157:H7 Escherichia coli Pseudomonas aeruginosa Pseudomonas putida Citrobacter freundii Staphylococcus epidermidis Staphylococcus aureus Staphylococcus warneri Hafnia alvei Klebsiella pneumoniae Aerococcus viridans Rhodococcus coprophilus Erysipelothrix rhusiopathiae Enterobacter aerogenes Streptococcus pyogenes Streptococcus dysgalactiae Shewanella algae Shigella dysenteriae Serratia fonticola

ATCC 33847 ATCC 17802 CICC 10522 VP1-VP67 BNCC232030 CICC10474 CICC21612 CICC10383 CICC21660 BNCC188419 BNCC336937 BNCC170086 ATCC33787 ATCC11778 Isolate ATCC23715 ATCC19119 CICC24119 ATCC33291 CMCC49027 CMCC49005 ATCC19433 ATCC43859 ATCC25922 ATCC27853 Isolate ATCC10787 ATCC12228 ATCC25923 Isolate Isolate Isolate Isolate Isolate Isolate Isolate Isolate Isolate Isolate Isolate Isolate

No. of strains tested 1 1 1 67 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

Detection result + + + + -

*All strains used in this study were from College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, China.

Table 1. Amplicons and their primers and probes targeting the ToxR gene in qPCR. Amplicon name

Primer/probe sequence (5'--- ---3')

ToxR-1

Forwad AACGATCGTAGAGCCGTCTT

Amplicon size (bp) 70

Reverse AGGTACTACTGGCGCTTCT Probe FAM-ACGCAATCGTTGAACCAGAAGCGCCA-TAMRA ToxR-2

Forwad AACGATCGTAGAGCCGTCTT

103

Reverse AGGATTCACAGCAGAAGCCA Probe FAM-ACGCAATCGTTGAACCAGAAGCGCCA-TAMRA ToxR-3

Forwad AACGATCGTAGAGCCGTCTT

158

Reverse GCAGTACGCAAATCGGTAGTA Probe FAM-ACGCAATCGTTGAACCAGAAGCGCCA-TAMRA ToxR-4

Forwad AACGATCGTAGAGCCGTCTT

202

Reverse CTCACCAATCTGACGGAACTG Probe FAM-ACGCAATCGTTGAACCAGAAGCGCCA-TAMRA ToxR-5

Forwad AACGATCGTAGAGCCGTCTT

262

AGGCAACCAGTTGTTGATTTG Probe FAM-ACGCAATCGTTGAACCAGAAGCGCCA-TAMRA ToxR-6

Forwad AACGATCGTAGAGCCGTCTT

349

Reverse AGGCAACCAGTTGTTGATTTG Probe FAM-ACGCAATCGTTGAACCAGAAGCGCCA-TAMRA IAC

Forwad CGGTGGAAACTACCAAGCTG Reverse TTTCGCCGTTGGTGTTCTTT Probe HEX-ACGCATTTCACCGCTCCACCGG-TAMRA

93

Table 3. Treatment of viable and non-vible V. parahaemolyticus cells with SD and its effect in the PMA-qPCR. SD concentration Cell treatment

Cells were not treated with SD

0.02%

0.04%

0.08%

0.10%

0.50%

Viable cell number (x 106) assessed by plant count

5.8±0.7

3.8±0.7

4.3±0.7

2.7±0.5*

2.8±0.1**

6.0±0.6

Cq value on the non-viable (heat-killed) cells

35.78±0.38*

33.63±0.27

34.51±0.19

36.01±0.85*

35.56±0.44*

34.13±0.19

* Indicates a significant difference from the negative control (p < 0.05). ** Indicates a very significant difference compared to the negative control (p < 0.01).

Table 4. qPCR detection of viable and non-viable V. parahaemolyticus cells treated by PMA or SD-PMA. Cell treament

Viable cells

Non-viable cells

SD-PMA

22.74±0.18

PMA

22.53±0.25

34.13±0.20

Cells were not treated by PMA or SD

22.50±0.14

22.90±0.38

a

a Cq value refers to the average ± standard deviation of a triplicate. b Bold-faced number refers to a significant difference (p < 0.5).

35.78±0.38

b

Highlight 1. The design of specific gene primers and probes for qPCR of V. parahaemolyticus 2. The optimal conditions for the SD-PMA-qPCR for V. parahaemolyticus were studied. 3. SD-PMA-qPCR can detect viable cells from mixtures of viable and dead cells. 4. In the spiked shrimp samples, SD-PMA-qPCR can selectively detect target bacteria.

Conflict of Interest and Authorship Conformation Form Please check the following as appropriate:

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All authors have participated in (a) conception and design, or analysis and interpretation of the data; (b) drafting the article or revising it critically for important intellectual content; and (c) approval of the final version.

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This manuscript has not been submitted to, nor is under review at, another journal or other publishing venue.

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The authors have no affiliation with any organization with a direct or indirect financial interest in the subject matter discussed in the manuscript

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The following authors have affiliations with organizations with direct or indirect financial interest in the subject matter discussed in the manuscript:

Author’s name Nan Ling Jinling Shen Jingjing Guo Dexin Zeng Jianluan Ren Lixin Sun Yuan Jiang Feng Xue Jianjun Dai Baoguang Li Administration

Affiliation College of Veterinary Medicine, Nanjing Agricultural University Shanghai Academy of Inspection and Quarantine College of Veterinary Medicine, Nanjing Agricultural University College of Veterinary Medicine, Nanjing Agricultural University College of Veterinary Medicine, Nanjing Agricultural University Jiangsu International Travel Health Care Center, Shanghai Academy of Inspection and Quarantine College of Veterinary Medicine, Nanjing Agricultural University College of Veterinary Medicine, Nanjing Agricultural University Center for Food Safety and Applied Nutrition, U.S. Food and Drug