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Rapid and quantitative detection of Vibrio parahemolyticus by the mixed-dye-based loop-mediated isothermal amplification assay on a self-priming compartmentalization microfluidic chip Bo Pang, Xiong Ding, Guoping Wang, Chao Zhao, Yanan Xu, Kaiyue Fu, Jingjing Sun, Xiuling Song, Wenshuai Wu, Yushen Liu, Qi Song, Jiumei Hu, Juan Li, and Ying Mu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b03655 • Publication Date (Web): 03 Dec 2017 Downloaded from http://pubs.acs.org on December 4, 2017

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Journal of Agricultural and Food Chemistry

Rapid and quantitative detection of Vibrio parahemolyticus by the mixed-dye-based loop-mediated isothermal amplification assay on a self-priming compartmentalization microfluidic chip

Bo Pang1,#, Xiong Ding2,#, Guoping Wang2, Chao Zhao1, Yanan Xu2, Kaiyue Fu1, Jingjing Sun2, Xiuling Song1, Wenshuai Wu2, Yushen Liu1, Qi Song2, Jiumei Hu2, Juan Li1, *, Ying Mu2,*.

#

These authors contributed equally to this work.

1

Department of Hygienic Inspection, School of Public Health, Jilin

University, Changchun 130021, Jilin, P.R. China 2

Research Center for Analytical Instrumentation, Institute of

Cyber-Systems and Control, State Key Laboratory of Industrial Control Technology, Zhejiang University, Hangzhou, 310058, P. R. China.

*

Corresponding Author:

Juan Li, Phone: +86 431 85619437, E-mail: [email protected]. Ying Mu, Phone: +86 571 88208383, E-mail: [email protected].

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Abstract:

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Vibrio parahemolyticus (VP) mostly isolated from aquatic products is

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one of the major causes of bacterial food poisoning events worldwide,

4

which could be reduced using a promising on-site detection method.

5

Herein, a rapid and quantitative method for VP detection was developed

6

by applying mixed-dye-loaded loop-mediated isothermal amplification

7

(LAMP) assay on self-priming compartmentalization (SPC) microfluidic

8

chip, termed on-chip mixed-dye-based LAMP (CMD-LAMP). Compared

9

to conventional approaches, CMD-LAMP was advantageous on the limit

10

of detection which reached down to 1×103 CFU/ mL in food

11

contaminated

12

Additionally, due to the use of mixed-dye and SPC chip, the quantitative

13

result could be easily acquired avoiding the requirement of sophisticated

14

instruments and tedious operation. Also, CMD-LAMP was rapid and

15

cost-effective. Conclusively, CMD-LAMP has a great potential in

16

realizing the on-site quantitative analysis of VP for food safety.

samples

without

the

pre-enrichment

of

bacteria.

17 18 19 20

Keywords: Microfluidic chip, Vibrio parahemolyticus, loop-mediated isothermal amplification, on-site detection

21 22


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1. Introduction

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Vibrio parahaemolyticus (VP) is a major foodborne pathogenic

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bacterium responsible for worldwide outbreak of seafood and

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ready-to-eat food poisoning events which causes acute gastroenteritis,

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dysentery, and diarrhea.1-5 Commonly, the infected person by VP has the

28

clinical symptoms like abdominal pain, fever and vomiting, but those

29

affected severely may become unconsciousness and even die.4 Since first

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reported in the 1953, 6 VP has been considered as the foremost foodborne

31

pathogen especially in China. During the past two decades, the number of

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VP-caused foodborne bacterial outbreak in China was on the rise clearly,

33

which was from 31.1% between 1991 and 20017 to over 70% between

34

1998 and 2013.8, 9 According to the data from the China National Center

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for Food Safety Risk Assessment (CFSA) and reported literature, the VP

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infection number per year was calculated as 4.95 million person-time, 9

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which has been a major threat to human health. Therefore, to efficiently

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prevent and control the infectious diseases, developing a rapid and

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quantitative method for VP detection has aroused the great concern.

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At present, culture-based assay, polymerase chain reaction (PCR) and

41

loop-mediated isothermal amplification (LAMP) are the common VP

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detection methods. In China, the culture identification has been defined as

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the national standard method (GB 4789.7-2013), namely “gold standard”,

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while PCR and LAMP as the professional standard methods (SN/T 3 ACS Paragon Plus Environment


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4063-2016; SN/T 2745.5-2011). Culture-based method possesses the

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advantage of giving the most accurate result, but it is not comparable to

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PCR in terms of analytical sensitivity and detection time. However, both

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of culture-based method and PCR require professional experimenters and

49

sophisticated instruments that are not readily available in the remote

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region. Also, culture process of plate counting and the electrophoresis

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after conventional PCR reaction are very tedious and time-consuming.10,

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11

Alternatively, as the burgeoning nucleic acid amplification technique

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without thermal cycle, LAMP partially addresses the problems with its

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rapidness, high specificity and sensitivity. Unfortunately, LAMP

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compromises to be developed as a quantitative detection method.12 In

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addition, due to its use of single dye (e.g. calcein or SYBR Green I),

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LAMP fails to clearly indicate the color change in weakly positive

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reaction, leading to false negative errors. Moreover, the Chinese National

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Food Safety Standard (CNFSS): Limit of Pathogen in Foods (GB

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29921-2013) has defined that the highest safety limit value (M) of VP is

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1000 CFU/ mL. Consequently, to reach the detectable level, all of the

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three methods above and even some cutting-edge technologies have to

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enrich the target bacteria beforehand.1,

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inevitably raises the time and cost of detection. Thus, overcoming above

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obstacles demands for a new method to rapidly and quantitatively detect

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VP.

13-17

However, pre-enrichment


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As a metal ion indicator in LAMP, hydroxyl naphthol blue (HNB) was

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usually used alone to achieve visual product detection based on the color

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change of reaction solution under the natural light. However, since the

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color change especially in weakly positive reaction was hard to be

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distinguished by naked eyes, its detection sensitivity was lower than that

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of fluorescence-based product detection.18 In 2015, Ding and the

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co-workers19 first reported that when excited by a 455 nm blue light,

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HNB could emit a red fluorescence with an intensity peak at 610 nm in

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the isothermal reaction solution. Owing to the decreased Mg2+ after

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amplification, the red fluorescence of HNB weakened, which was just

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opposite with the enhanced green fluorescence of SYBR Green I.19

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Inspired by this, a mixed dye containing HNB and SYBR Green I was

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developed for LAMP.20 Compared to single dye, the mixed dye could

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improve the detection sensitivity and avoided the empirical preset of

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cutoff intensity values, making the result more accurate. However, this

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mixed-dye-based LAMP was still not appropriate for quantitative

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detection.

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To further improve the sensitivity and realize quantitative detection, a

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self-priming compartmentalization (SPC) microfluidic chip was applied

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in LAMP.21 SPC chip evacuated in a vacuum could form a negative

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pressure environment and partitioned the LAMP reaction solution to

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thousands of reaction units. After incubation, the units containing the 5 ACS Paragon Plus Environment


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target sequence(s) appeared positive, while the units without enough

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target appeared negative. The number of positive units could be counted,

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which possessed a linear relationship with the concentrations of samples.

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Therefore, SPC chip could be used to quantify. Theoretically, if the

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number of micro-reaction chambers was large enough or the volume of

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them was small enough, one positive plot represented one target molecule,

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which meant that SPC chip even had the potential in realizing single

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molecule detection.22, 23 To date, SPC chip-based LAMP has been applied

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for β-actin DNA detection.21 However, to our knowledge, the SPC-based

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detection associated with the public health has not been reported yet, such

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as the detection of VP.

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In this study, a novel rapid and quantitative detection of VP was first

101

developed by using mixed-dye-loaded LAMP assay on a SPC

102

microfluidic chip, namely the on-chip mixed-dye-based LAMP

103

(CMD-LAMP). The CMD-LAMP has a great potential in realizing the

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rapid and on-site quantitative analysis of VP for food safety in China and

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around the world.

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2 Materials and Methods

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2.1. Bacterial strains

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A total of ten bacterial strains were used, including one standard Vibrio

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parahemolyticus strain (ATCC 17802) and five isolation strains of VP as

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target and other four common non-target foodborne pathogenic bacteria, 6 ACS Paragon Plus Environment

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Salmonella typhimurium (ATCC 14028), Shigella flexneri (ATCC 12022),

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Listeria monocytogenes (ATCC 43251), Staphylococcus aureus (ATCC

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23213). All the strains were stored in the Department of Hygienic

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Inspection, School of Public Health, Jilin University (Changchun, China)

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at -80 ºC.

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2.2. Bacteria culture and DNA templates preparation

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According to the CNFSS: Food Microbiology Testing (GB 4789), VP

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was cultured in alkaline peptone water (APW) with 3.0 % NaCl at 37 ºC

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with shaking at 200 rpm for 8 hours. For enumeration, the culture was

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serially diluted with PBS and incubated on thiosulfate citrate bile salts

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sucrose (TCBS) agar at 37 ºC for 22–24 hours. The other four bacteria

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were grown and counted using Luria-Bertani (LB) medium and agar.

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Based on the counting results, the VP was serially diluted with deionized

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water and the non-target bacteria were diluted to 1×107 CFU/ mL and

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1×105 CFU/ mL.

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The DNA templates were extracted by using Mag-MK Bacterial

127

Genomic DNA extraction kit (# B518725, Sangon, China) and all

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procedures were strictly adhered to the instruction. The purity and

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concentration of DNA were judged by the ratio of A260 and A280 and the

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ratio of A260 and A230, which were measured by compatible BioTek

131

detection system (Synergy H1M, BioTek, USA).

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2.3 Fabrication of microfluidic chip 7 ACS Paragon Plus Environment


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Based on the previous report, 21 SPC microfluidic chips were fabricated

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to develop CMD-LAMP method for VP detection. The SPC chips were

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made of multilayer silicone elastomer polydimethylsiloxane (PDMS)

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bonded on glass coverslips and were fabricated with soft lithography

137

technique. Detail was described in the supplementary material.

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2.4 Off-chip mixed-dye-based LAMP reaction

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The off-chip mixed-dye-based LAMP (MD-LAMP) reaction was

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carried out in sterilized 200 µL PCR tubes. Because of species specificity

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and existing widely,24 the tlh gene of VP was chosen as the template to

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develop the reaction. The sequence information of six matched primers

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(FIP, BIP, F3, B3, LF, LB), shown in Table S-1, was acquired from

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previous literature12 and all the primers were synthesized and purified by

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the Sangon Biotech Ltd.. A total of 10 µL optimized assay each tube was

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prepared and it consisted of the following components: 0.8 M betaine

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(Sangon, China), 1.4 mM each of Deoxynucleoride Solution (New

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England BioLabs, USA), 1 µL 10 × IsoAmp Buffer (New England

149

BioLabs, USA), 6 mM MgSO4 Solution (New England BioLabs, USA),

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0.2 µL 50×SYBR Green I (Life Technologies, USA), 1.6 µM each of FIP

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and BIP, 0.2 µM each of F3 and B3, 0.8 µM each of LF and LB, 3.2 U Bst

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2.0 WarmStart DNA polymerase (New England BioLabs, USA) and 2 µL

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extracted DNA template. To find out suitable dose of HNB, various

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concentrations of HNB (Lemongreen, China) (100, 150, 200, 250, 300, 8 ACS Paragon Plus Environment

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350 and 400 µM) were added into the described mixture. The reaction assay was incubated at 63 ºC for 60 min and heated at 80

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º

C for 10 min to inactivate the enzyme in the 7900HT Fast Real-Time

158

PCR System (ABI, USA). The product of MD-LAMP reaction was run

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on the 2% agarose gel electrophoresis marked by SYBR Green I. For

160

evaluating this novel home-made assay, conventional LAMP and PCR

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were conducted simultaneously to compare with it. Protocol of these two

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methods was described in the supplementary material.

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2.5 On-chip mixed-dye-based LAMP reaction.

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The CMD-LAMP assay (10 µL) was carried out in the make-up SPC

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chip. First, the chip was evacuated using the vacuum pump at 5 kPa for

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60-80 minutes to remove the air in the chip. Then, the chip was taken to

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the normal atmosphere and the transparent adhesive tape covering inlet

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was punctured. 10 µL reaction solution was pipetted and inserted into the

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inlet. Because of the draught head, the solution was dispensed into the

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chambers in five minutes approximately. The CMD-LAMP assay was the

171

same as the MD-LAMP assay. Similarly, various concentrations of HNB

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(50, 150, 250, 350 µM) were also tested. Two replicates of 5×104 CFU/

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mL of VP template were conducted to find out the proper HNB

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concentration and three replicates were tested to determine the sensitivity

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and specificity. And next, the silicone oil containing PDMS A and B

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(silicone oil: A: B= 12: 2: 1) was inserted following the reaction solution. 9 ACS Paragon Plus Environment


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When all of the chambers were filled by reaction solution and separated

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by the silicone oil, the sealing mixture with a ratio of PDMS A: B= 2:1

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was added into the inlet and outlet. Finally, the surface of the SPC chip

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was covered by transparent adhesive tape again and the chip could be

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incubated for CMD-LAMP reaction. The chip was incubated at 63 ºC for

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60 min and heated at 80 ºC for 10 min to inactivate the enzyme in the

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PCR machine (LongGene, China) with a flat-bed heating block.

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2.6 Food contaminated samples treatment

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Fresh shrimp was purchased from a local market in Changchun, China

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and quickly brought to the laboratory in an ice box. The sample was

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treated following the literature.25-27 First, the shrimp was grinded and

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spread on a Petri dish without cap, followed by exposure to UV

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disinfection light for 2 hours in order to avoid the interference from the

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naturally accumulated VP in the shrimp. Then, 10g of sanitized shrimp

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paste was added into 100 mL of APW with 3.0 % NaCl and the mixture

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was homogenized. To determine the linear correlation between bacterial

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concentration and the response value of CMD-LAMP method in this

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shrimp homogenate, spiked samples were prepared by adding standard

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strain of VP to the matrix with different final concentrations (2.5×102

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CFU/ mL, 1.0×103 CFU/ mL, 4.0×103 CFU/ mL, 1.6×104 CFU/ mL,

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6.4×104 CFU/ mL, 2.56×105 CFU/ mL and 1.024×106 CFU/ mL) as

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determined by plate counting. The shrimp homogenate without bacteria 10 ACS Paragon Plus Environment

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was chosen as the control. Afterwards, the protocol of DNA extraction

200

and CMD-LAMP was the same as described above.

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Similarly, shrimp homogenate with 1×103 CFU/ mL isolation strain of

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VP was prepared. For better simulating the practical situation, the mixture

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was placed at the room temperature for 6 hours and it was plate counted

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before DNA extraction to compare with the result of CMD-LAMP. The

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subsequent operation was the same as described above.

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2.7 Data collection and statistical analyses

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The emission spectra analysis and the images acquisition of

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MD-LAMP reaction, CMD-LAMP reaction and electrophoresis were

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performed by Maestro In-vivo Imaging system (CRi, USA). The

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fluorescence was excited by a blue light at 455 nm with an exposure time

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of 5000 ms. The images under the natural light were obtained by a smart

212

phone (Apple, USA).

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The excited fluorescent intensity and the number of positive signal

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plots on the chip were expressed as the mean± standard deviation (x ̅±

215

SD). Probit analysis was used to estimate 95% probability limit of

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detection (LOD) with SPSS Statistics Software Version 22.0 (IBM,

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USA).

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3 Results

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3.1 Self-priming compartmentalization (SPC) microfluidic chip

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The SPC chip used for VP detection contained 1056 uniform 11 ACS Paragon Plus Environment


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rectangular micro-chambers which were interlacedly located on 16

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branch channels converged at the outlet (Fig. 1A). The length and width

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of the micro-chambers were both 200 µm, and their height was 150 µm.

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The width of primary channels connecting with the inlet and branch

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channels connecting with the micro-chambers were 100 µm and 50 µm.

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All of the channels were 50 µm high. The bottom of the chip was sealed

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by a piece of 0.17 mm thick coverslip and the total size of it was 29 mm

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× 15 mm × 4.5 mm, which was similar with a coin size (Fig. 1B).

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The self-priming compartmentalization was realized by the air pressure

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difference between atmosphere and the degassed chip. Because of the air

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permeability of PDMS, the negative pressure could sustain a long enough

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time for reaction solution and silicon oil flowing into the chip. First, the

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reaction solution was sucked into the micro-chambers from the inlet

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driven by the air pressure. Then, the following silicon oil was self-primed

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into the chip replacing the redundant reaction solution in the channels.

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Finally, all the channels were filled with silicon oil, which made each

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micro-chamber filled with reaction solution as a separate unit (Fig. 1C

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and Fig. 1D).

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3.2 MD-LAMP Assays

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As reported previously, 20 the mixed-dye-based dual fluorescence was

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greatly influenced by HNB rather than SYBR Green I. Thus, the

242

concentration of HNB was only investigated to develop MD-LAMP 12 ACS Paragon Plus Environment

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assays. As suggested in Fig. S-1, 350 µM of HNB was the optimized

244

concentration to constitute the mixed dye. When excited by a 455 nm

245

blue light, positives emitted green fluorescence and non-target controls

246

(NTCs) were orange red, which could be easily judged by naked eyes.

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In the emission spectra from 500 to 650 nm, the fluorescence intensity

248

at 540 nm (FI540) of positive decreased and the fluorescence intensity at

249

610 nm (FI610) of NTC increased (Fig. S-1). Thus, the result was defined

250

as positive when the FI540 was greater than the FI610. To investigate the

251

sensitivity of MD-LAMP, a series of VP templates with ten-fold dilution

252

concentrations were detected. As shown in Fig. 2A, the detection

253

sensitivity reached down to 1×104 CFU/ mL. In addition, Fig. 2B showed

254

that MD-LAMP had high detection specificity, since the green

255

fluorescence only occurred in the reactions with target bacteria, which

256

was further confirmed by 2% agarose gel electrophoresis (Fig. S-2).

257

Due to the ambiguous result with 1×103 CFU/ mL VP (Fig. 2A), the

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LOD of MD-LAMP was further investigated. Meanwhile, the

259

conventional LAMP and PCR was tested. Their LODs in 95% probability

260

were calculated through probit analysis. As displayed in Table 1, the LOD

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of MD-LAMP was approximately 14528.001 CFU/ mL, which was better

262

than those of conventional LAMP and PCR. However, this LOD still

263

didn’t meet the VP maximum limits of 1000 CFU/ mL defined by the

264

CNFSS. To address this issue, a microfluidic chip was fabricated to 13 ACS Paragon Plus Environment


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develop CMD-LAMP.

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3.3 Mixed Dye for CMD-LAMP

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Because the microcosmic on-chip reaction was different from the

268

macroscopical off-chip reaction, the concentration of HNB should be

269

re-optimized to achieve the optimal mixed dye for CMD-LAMP. Two

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parallel samples with 5×104 CFU/ mL Vibrio parahemolyticus was used

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to the optimization experiment (Fig. 3). As depicted in Fig. 3B, 150 µM

272

HNB could obviously increase the color change between the positive plot

273

and the negative plot. When the concentration of HNB was 50 µM, due to

274

the strong green background fluorescence which mainly produced from

275

SYBR Green I combined with the non-amplification DNA template of

276

sample, it was hard to indicate the result. As for the relatively high

277

concentration like 250 µM and 350 µM, HNB could cover the

278

fluorescence intensity at 540 nm and made the green fluorescence weak.

279

What’s more, the strong red fluorescence of HNB influenced the result

280

judgment, which might make parts of positive plots be misclassified as

281

the false negative. Regarding the above, 150 µM of HNB was determined

282

to perform CMD-LAMP reaction.

283

3.4 Sensitivity and specificity of CMD-LAMP reaction

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With the optimized assay, three concentrations of pure culture VP were

285

detected by SPC microfluidic chip to roughly determine the sensitivity.

286

As shown in Fig.4, 1.7± 0.6, 33.3± 8.3 and 277.0± 35.5 positive plots 14 ACS Paragon Plus Environment

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were easily counted with the concentration of 1×103 CFU/ mL, 1×104

288

CFU/ mL and 1×105 CFU/ mL, respectively. There was a good

289

proportional relation (R2= 0.9995). In the positive control for 1×108 CFU/

290

mL VP detection, almost all the chambers were positive attributed to the

291

limitation of 1056 micro-chambers (Fig. 4D). In the NTCs, there was no

292

positive signal observed (Fig. 4E). Thus, the results demonstrated that the

293

CMD-LAMP reaction was feasible to complete the VP quantitative

294

determination and the LOD was approximated 1×103 CFU/ mL.

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Furthermore, the specificity of CDM-LAMP was tested with other four

296

common non-target food-borne pathogens. As shown in Fig.5, only the

297

detection of VP displayed a positive result, proving that CDM-LAMP was

298

capable of realizing on-chip VP detection with high specificity.

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3.5 Real application of CMD-LAMP in food contaminated samples

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To validate the applicability of the CMD-LAMP method, templates

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extracted from food contaminated samples with different concentrations

302

of VP were tested to fit the regression line equation. The acquired

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equation was similar to it of pure culture VP detection (y= 0.0023x+ 0.54,

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R2= 0.9993 vs y= 0.0028x+ 1.41, R2= 0.9995, Fig. S-3 vs Fig. 4).

305

Meanwhile, as shown in Fig. S-3C, 2.7± 1.2 positive plots could be

306

counted with the concentration of 1×103 CFU/ mL, which verified that

307

the LOD of CMD-LAMP could reach down to 1×103 CFU/ mL in the

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complex matrix. 15 ACS Paragon Plus Environment


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What’s more, simulating the practical situation, five isolation strains of

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VP in the shrimp homogenate were measured by CMD-LAMP. As shown

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in Tab. 2 and Fig. S-4, comparing with the result of “gold standard”, plate

312

count method, the recovery rate was in an excellent range from 93.38% to

313

103.61% and the relative standard deviation (RSD) of the measured value

314

was less than 9.32%, demonstrating that this approach was stable and

315

promising in actual samples detection.

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4 Discussion

317

VP-caused food safety is always of over-whelming public concern.

318

Therefore, developing new methods to detect VP rapidly and

319

quantitatively is focused on by the scientists especially in the developing

320

countries, such as China. Because of the advantages of isothermal nucleic

321

acid amplification, LAMP for VP detection was first reported in 200812

322

and subsequently it was widely developed to target other genes of VP.28-32

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However, these traditional LAMP approaches compromised on

324

quantitative analysis and detection of VP with low concentration, which

325

had to be quantified and pre-enriched through tedious bacteria culture.16,

326

33-37

To address these obstacles, the combination of microfluidics and

327

LAMP is an alternative way, but currently reported methods are still not

328

appropriate for the prospective on-site VP detection.38, 39

329

Herein, in this study, a novel rapid VP quantitative detection method

330

based on MD-LAMP assay using SPC microfluidic chip is reported for 16 ACS Paragon Plus Environment

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the first time. Compared with traditional approaches and other LAMP

332

methods for VP detection, CMD-LAMP shows its competitive superiority

333

(Tab. S-2, Tab. S-3). First, the use of the mixed dye and SPC microfluidic

334

chip

335

pre-enrichment process, the LOD reaches down to 1×103 CFU/ mL,

336

which is the highest safety limit value (M) of VP in Chinese seafood and

337

ready-to-eat food. Second, for the unique fluorescence property of mixed

338

dye, the result of the reaction can be easily identified no matter off-chip

339

or on-chip detection. Under the blue excitation light at 455 nm, the

340

distinguishable color change of positive and negative sample reduces the

341

misjudgment of the low-concentration sample. Third, the result of

342

CMD-LAMP detection can be quantitatively analyzed directly. Within the

343

detection range, the exact bacteria concentration of the sample can be

344

calculated accurately by counting the positive plots on the chip. Last but

345

not least, this approach is cost-effective and requires only less than two

346

hours to complete all the protocol from DNA extraction to result readout.

greatly

improves

the

sensitivity.

Without

any

bacteria

347

Additionally, in the outbreak of food safety event, the time to identify

348

and quantify pathogens greatly determines the degree of harm diffusion

349

and influences the treatment of infectious diseases. As shown in Fig. 6, a

350

new emergency coping strategy for VP infections were proposed based on

351

CMD-LAMP. Taking this rapid and quantitative VP detection approach,

352

the government can quickly response to pandemic threat in two hours. 17 ACS Paragon Plus Environment


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Thus, CMD-LAMP has huge potential in food safety screening.

354

Acknowledgments

355

We are grateful for the help of Mr. Kun Xu, Ms. Juan Wang and Ms.

356

Qing Zhen from School of Public Health, Jilin University, China as well

357

as Mr. Xiujun Yang and Ms. Wei Zhao from Jilin Province Center for

358

Disease Control and Prevention, China during the process of the

359

experiment and thank all the participants for their support.

360

Funding Sources

361

This work was supported by the National Natural Science Foundation

362

of China (No: 81473018, 81502849), Jilin Province Science and

363

Technology Development Plan Item (No: 20170204003SF), and the Open

364

Research Project of the State Key Laboratory of Industrial Control

365

Technology, Zhejiang University, China (No: ICT1600203, ICT170293).

366

Supporting Information

367

Brief statement in nonsentence format listing the contents of the

368

material supplied as Supporting Information.

369

Conflict of interest

370

The authors declare no competing financial interest.


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17. Oh, S. J.; Park, B. H.; Jung, J. H. Centrifugal loop-mediated isothermal amplification microdevice for rapid, multiplex and colorimetric foodborne pathogen detection. Biosens. Bioelectron. 2016, 75, 293-300. 18 Ding, X.; Nie, K.; Shi, L. Improved detection limit in rapid detection of human enterovirus 71 and coxsackievirus A16 by a novel reverse transcription-isothermal multiple-self-matching-initiated amplification assay. J. Clin. Microbiol. 2014, 52, 1862-1870. 19. Chen, J.; Ji, X.; He, Z. Smart Composite Reagent Composed of Double-Stranded DNA-Templated Copper Nanoparticle and SYBR Green I for Hydrogen Peroxide Related Biosensing. Anal. Chem. 2017, 89, 3988-3995. 20. Ding, X.; Wu, W.; Zhu, Q. Mixed-Dye-Based Label-Free and Sensitive Dual Fluorescence

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25. Park B.; Choi SJ. Sensitive immunoassay-based detection of Vibrio parahaemolyticus using capture and labeling particles in a stationary liquid phase lab-on-a-chip. Biosens. Bioelectron. 2017, 90, 269-275. 26. Wu S.; Wang Y.; Duan N. Colorimetric Aptasensor Based on Enzyme for the Detection of Vibrio parahemolyticus. J. Agric. Food Chem. 2015, 63, 7849-54. 27. Sha Y.; Zhang X.; Li W. A label-free multi-functionalized graphene oxide based electrochemiluminscence immunosensor for ultrasensitive and rapid detection of Vibrio parahaemolyticus in seawater and seafood. Talanta 2016, 147,220-5. 28. Yamazaki, W.; Kumeda, Y.; Misawa, N.; Nakaguchi, Y.; Nishibuchi, M., Development of a loop-mediated isothermal amplification assay for sensitive and rapid detection of the tdh and trh genes of Vibrio parahaemolyticus and related Vibrio species. Applied and environmental microbiology 2010, 76, 820-8. 29. Nemoto, J.; Sugawara, C.; Akahane, K.; Hashimoto, K.; Kojima, T.; Ikedo, M.; Konuma, H.; Hara-Kudo, Y., Rapid and specific detection of the thermostable direct hemolysin

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Figure captions: Fig. 1. Schematic illustration showing the structure and mechanism of the SPC microfluidic chip for digital VP detection: (A) Schematic diagram of the SPC microfluidic chip; (B) Photograph of the real product; (C) Photograph of the chip before sample introduction under the microscope. The micro-chambers and branch channels could be observed; (D) Photograph of the chip after sample introduction under the microscope. Because of the filled silicon oil, the branch channels could hardly to recognized, but the separate micro-chambers filled with reaction solution could be identified precisely. Fig. 2. Sensitivity and specificity of MD-LAMP for VP detection. (A) Sensitivity of the MD-LAMP method: 1, 108 CFU/ mL; 2, 107 CFU/ mL; 3, 106 CFU/ mL; 4, 105 CFU/ mL; 5, 104 CFU/ mL; 6, 103 CFU/ mL; NTC, nuclease-free water; (B) Specificity of the MD-LAMP method: 1, Vibrio parahemolyticus; 2, Salmonella typhimurium; 3, Shigella flexneri; 4, Listeria monocytogenes; 5, Staphylococcus aureus; NTC, nuclease-free water. The images were captured under the blue light (λ= 455 nm) excitation. Error bars represented the standard deviation of FI540 (yellow bar) and FI610 (blue bar) at three duplicate samples. The concentration of each bacterium was 1×107 CFU/ mL.



Fig. 3. Investigation on the optimal concentration of HNB for CMD-LAMP reaction: (A) 50 µM HNB; (B) 150 µM HNB; (C) 250 µM HNB; (D) 350 µM HNB. Two replicates of VP template (5×104 CFU/mL) were tested. Fig. 4. The CMD-LAMP reactions with different concentration of VP: (A) 1×103 CFU/mL; (B) 1×104 CFU/mL; (C) 1×105 CFU/mL; (D) Positive control: 1×108 CFU/mL; (E) Negative control: nuclease-free water; (F) The linear relationship between number of positive plots and concentrations. Error bars represented the standard deviation value of each test with three replicates. Fig. 5. The CMD-LAMP reactions with different bacteria: (A) Vibrio parahemolyticus; (B) Salmonella typhimurium; (C) Shigella flexneri; (D) Listeria monocytogenes; (E) Staphylococcus aureus; (F) Negative control: nuclease-free water. Three replicates were tested and the concentration of each bacterium was 1×105 CFU/ mL. Fig. 6. Schematic illustration of a new emergency coping strategy based on CMD-LAMP.


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Tables：：

Tab. 1. Comparison of the MD-LAMP method and the conventional methods. Number of tests with Positive Target bacteria

signal/ Total test number

/NTC (CFU/ mL)

Mixed-dye

Conventional

Conventional

LAMP

LAMP

PCR

1000000

9/9

9/9

9/9

100000

9/9

9/9

9/9

10000

8/9

6/9

6/9

5000

4/9

1/9

4/9

1000

2/9

0/9

3/9

500

1/9

1/9

0/9

100

1/9

1/9

0/9

50

0/9

0/9

0/9

10

0/9

0/9

0/9

5

0/9

0/9

0/9

1

0/9

0/9

0/9

nuclease-free water

0/9

0/9

0/9

LOD (CFU /mL)

14528.001

15053.531

15536.200



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Tab. 2. Detection of the VP in food contaminated samples. Plate count method

Number of positive

CMD-LAMP method

Recovery

RSD

(× 1000 CFU/ mL)

plots in CMD-LAMP

(× 1000 CFU/ mL)

(%)

(%)

Isolation-1

47.67± 4.73

104.00± 9.64

44.98± 4.19

94.37

9.32

Isolation-2

23.00± 2.65

53.00± 3.61

22.81± 1.57

99.16

6.88

Isolation-3

102.33± 5.51

220.33± 8.50

95.56± 3.70

93.38

3.87

Isolation-4

57.33± 7.09

127.00± 3.61

54.98± 1.57

95.90

2.86

Isolation-5

20.33± 5.03

49.00± 4.00

21.07± 1.74

103.61

8.26

Samples


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Figures:

Fig. 1.



Fig. 2.


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Fig. 3.



Fig. 4.


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Fig. 5.



Fig. 6.


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