BioMed Research International

BioMed Research International / 2013 / Article
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Molecular Approaches for the Classification of Microbial Pathogens of Public Health Significance

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Research Article | Open Access

Volume 2013 |Article ID 295050 |

Masahiro Hayashi, Tatsuya Natori, Sayoko Kubota-Hayashi, Machiko Miyata, Kiyofumi Ohkusu, Keiko Kawamoto, Hisao Kurazono, Souichi Makino, Takayuki Ezaki, "A New Protocol to Detect Multiple Foodborne Pathogens with PCR Dipstick DNA Chromatography after a Six-Hour Enrichment Culture in a Broad-Range Food Pathogen Enrichment Broth", BioMed Research International, vol. 2013, Article ID 295050, 10 pages, 2013.

A New Protocol to Detect Multiple Foodborne Pathogens with PCR Dipstick DNA Chromatography after a Six-Hour Enrichment Culture in a Broad-Range Food Pathogen Enrichment Broth

Academic Editor: Hiroshi Asakura
Received27 Sep 2013
Revised05 Nov 2013
Accepted05 Nov 2013
Published02 Dec 2013


A quick foodborne pathogen screening method after six-hour enrichment culture with a broad-range food pathogen enrichment broth is described. Pathogenic factors of Salmonella enterica, Shigella spp., enteroinvasive Escherichia coli, and enterohemorrhagic E. coli are amplified with a cocktail primer and rapid polymerase chain reaction (PCR), which finishes amplification in 30 min. The PCR amplicon was differentiated with a dipstick DNA chromatography assay in 5–10 min. Starting from a four- to six-hour enrichment culture, this assay was finished within 45 min. Detection sensitivity of this protocol was less than 2.5 CFU/25 g for S. enterica and 3.3 CFU/25 g for enterohemorrhagic E. coli in spiked ground meat experiments.

1. Introduction

Infectious gastroenteritis is a leading cause of morbidity and mortality worldwide, particularly in developing countries [1]. Risk factors for infectious gastroenteritis include exposure to various contaminated food products [2]. Several methods to detect pathogens directly in food samples have been reported [3, 4]; however, most food analysis requires a 25 g food sample. Ideally, pathogen detection in food should be at the single-cell level [5].

Several methods based on polymerase chain reaction (PCR) have been developed to detect a single-cell pathogen from enrichment culture [68]. Cocktail PCR, carried out in a single PCR tube for simultaneous detection of more than one bacterial target, has been investigated as a more cost-effective and time-saving method [9, 10]. However, it is difficult for small food laboratories to use ethidium bromide-based agarose gel. On the other hand, real-time PCR assays employing various types of fluorescence systems allow multiple detection during PCR [1113]. This is an excellent method, but it requires an expensive real-time thermal cycler and reagents. Thus, small laboratories cannot afford this real-time method.

Another aspect of food analysis is the analysis time. Fresh food products must arrive to the market quickly, but current culture-based protocols require several days to confirm that the products are pathogen-free. Confirmation that fresh food is safe before shipping is, therefore, desired, but difficult in practice.

To solve these problems, we developed a quick cocktail PCR and dipstick DNA chromatography to differentiate PCR amplicons for Salmonella enterica, Shigella spp., enteroinvasive Escherichia coli (EIEC), and enterohemorrhagic E. coli (EHEC) from a single enrichment culture broth. In our previous report, we described a food pathogen enrichment (FPE) broth that supports the growth of Campylobacter without adding lysed blood and carbon dioxide [14]. The method detected a few Campylobacter cells in 25 g of chicken within 24 hours and was better than the conventional Bolton-based enrichment culture.

In this report, we describe a new protocol to detect S. enterica, Shigella spp., EIEC, and EHEC from the FPE broth. DNA preparation from the FPE broth was simplified and the cocktail PCR was designed to finish within 30 min. The PCR amplicon was visually differentiated using dipstick DNA chromatography within 5–10 min.

2. Materials and Methods

2.1. Bacterial Strains

The bacterial strains of S. enterica, Shigella spp., EIEC, and EHEC and other strains are listed in Table 1. All strains were supplied from the Gifu Type Culture Collection of the Microbial Genetic Resource Stock Center, Gifu University Graduate School of Medicine (Gifu, Japan), supported by the Ministry of Education, Culture, Sports, Science, and Technology of Japan. All strains were cultured on heart infusion agars (BD, Tokyo, Japan) at 37°C under an aerobic atmosphere overnight. A fresh culture was used for each experiment.

BacteriaSerotypeToxinStrainsNumber of strain

(Enterohemorrhagic E. coli)
O26: H-Shiga toxin 1GTC14538, GTC14548, GTC14605, GTC146064
O26: H11Shiga toxin 1GTC14516, GTC14540, GTC14549, GTC14557, GTC145585
Shiga toxin 1 and 2GTC14515, GTC14539, GTC145673
O111: H-Shiga toxin 1GTC14517, GTC14528, GTC145413
Shiga toxin 1 and 2GTC14508, GTC145822
O115: H10Shiga toxin 1GTC145181
O119: H2Shiga toxin 1GTC145291
O121: H19Shiga toxin 2GTC14530, GTC14577, GTC14601, GTC146024
O128: H-Shiga toxin 1 and 2GTC146031
O157: H7Shiga toxin 2GTC14513, GTC14514,GTC14524, GTC14525, GTC14537 GTC14546, GTC14547, GTC14550, GTC14553, GTC1456010
Shiga toxin 1 and 2GTC14510, GTC14511, GTC14512, GTC14521, GTC14535 GTC14536, GTC14544, GTC14545, GTC14551, GTC14552, 10
O157: H-Shiga toxin 1 and 2GTC14507, GTC14520, GTC14530, GTC14543, GTC14555 GTC14556, GTC14566, GTC14571, GTC14587, GTC1458810
(Enteroinvasive E. coli)
O28: H-GTC14240, GTC14243, GTC14251, GTC14259, GTC142605
O124: H-GTC14241, GTC14242, GTC14245, GTC142624
O136: H-GTC13248, GTC142542
O144: H-GTC14249, GTC14252, GTC142563
O164:H-GTC14244, GTC14246, GTC14247, 3
Salmonella enterica
 subsp.enterica serovar TyphimuriumGTC02557, GTC02561, GTC02562, GTC02563, GTC02564 10
GTC02571, GTC02572, GTC-2573, GTC02574, GTC02575
 subsp.enterica serovar EnteritidisGTC03838, GTC00131, GTC02377, GTC02382, GTC02387 7
GTC02389, GTC02390
 subsp.enterica serovar DublinGTC13214, GTC13215, GTC13216, GTC13217,GTC13218 10
GTC13219, GTC13220, GTC13221. GTC02558, GTC02560
 subsp.enterica serovar Typhi GTC3P001, GTC3P074,GTC3P076, GTC3P081, GTC3P085 10
GTC3P087, GTC3P091, GTC3P095, GTC3P100, GTC3P106
 subsp.enterica serovar ParaTyphi AGTC3P002, GTC3P082, GTC3P0833
Salmonella bongori GTC03793T1
Shigella boydii GTC00779T, GTC01912, GTC01913, GTC01914, GTC01915 8
GTC01915, GTC01916, GTC01917,
Shigella dysenteriae GTC01057T, GTC00786, GTC01929, GTC01930, GTC14808 17
GTC14809, GTC14810, GTC14811, GTC14812, GTC14813,
GTC14814, GTC14815, GTC14816, GTC14817, GTC14818,
GTC14819, GTC14820,
Shigella flexneri GTC 0780T, GTC01918, GTC01920, GTC02007, GTC02008 13
GTC02009, GTC02010, GTC02011, GTC02012, GTC02013,
GTC02015, GTC02016, GTC02014,
Shigella sonnei GTC00781T, GTC01909, GTC01910, GTC01911, GTC01931 7
GTC01932, GTC01933,
Escherichia coli GTC00503 T1
Escherichia albertii GTC 16441T1
Escherichia fergusonii GTC 01720T1
Escherichia vulneris GTC 10613T1
Escherichia hermannii GTC 10612T1
Escherichia blattae GTC 01342T1
Citrobacter freundii GTC 14890T1
Citrobacter diversus GTC 00114T1
Citrobacter rodentium GTC 14911T1
Citrobacter youngae GTC 14914T1
Klebsiella pneumoniae GTC 14868T1
Enterobacter cloacae GTC 00109T1
Enterobacter aerogenes GTC 14962T1
Cronobacter sakazakii GTC 14952T1
Serratia marcescens GTC 146721
Yersinia enterocolitica GTC 00127T1
Pseudomonas aeruginosa GTC 00002T1
Vibrio parahaemolyiticus GTC 020551
Staphylococcus aureus GTC 00286T1

GTC is the Gifu Type Culture Collection supported by the National Bioresource Project (NBRP: of the Ministry of Education, Culture, Sports, Science,and Technology.
“T” after the strain number means type strain.

2.2. Determination of Optimal Enrichment Culture with FPE Broth

FPE broth is designed to support the growth of Campylobacter species without blood and carbon dioxide [14]. This broth was used in the present study to simplify the total food analysis protocol because the FPE broth supported most food borne pathogens in our preliminary experiment. The growth of the foodborne pathogen in the FPE broth was compared with that in conventional selective enrichment broth (Figure 1). In the spiked ground meat experiment, diluted fresh bacterial solution and 25 g of beef were mixed in 225 mL of FPE broth and incubated at 37°C.

2.3. DNA Extraction

DNA was extracted from 1 mL of culture broth using a physical disruption method (MORA-EXTRACT, AMR, Gifu, Japan) according to the manufacturer’s instructions, with a final DNA elution volume of 200–400 μL.

DNA extraction from FPE broth was performed using a simplified protocol. One milliliter of 2 to 18 hours enrichment culture was collected in a 2 mL Eppendorf tube and centrifuged at 12,000 g. The supernatant was completely removed and 1 mL of T10E1 buffer was added and centrifuged under the same conditions. After the complete removal of the supernatant, 200 μL of T10E1 buffer was added to the tube and mixed. The solution was transferred to a tube containing beads and physically disrupted for 1 min with a Disrupter Genie (Scientific Industry Inc., Bohemia, NY, USA). The tube was boiled at 100°C for 3 min. Five microliters of the solution were used for the cocktail PCR assay.

2.4. Cocktail PCR

The cocktail PCR conditions used in the present study are described below. The primers used are described in Table 2. PCR amplification was performed in 10 μL of reaction mixture containing 5 μL of 2× premix Ex Taq (Takara Bio, Shiga, Japan), 2.5 μL of primer mixture, 0.5 μL of distilled water, and 2 μL of DNA template. PCR was carried out using the QuickBath thermal cycler (ThermoGen Ltd., Nagano, Japan) under the following conditions: 95°C for 3 min, 40 cycles of 95°C for 10 s, and 65°C for 10 s. The PCR cycles finished within 30 min.

PathogenTargeted genePrimer nameSequence (5′-3′)

stx2 Forward stx2 Biotin-GATACAGAGAGAATTTCGTC
Shigella spp. and EIECipaH Forward ipaHF Biotin-CTCGCAGAGAAACTTCAGCTCT
Salmonella spp.invA Forward invA Biotin-TGACAGAATCCTCAGTTTTTCA

2.5. Dipstick DNA Chromatography

The 5 terminus of the cocktail PCR amplicon was labeled with biotin, and the 3 terminus was labeled with four different tags. Streptavidin-coated blue latex, kindly provided from Fujikura (Saitama, Japan), bound the biotinylated 5 terminal amplicon and the tagged 3 terminus was bound on the antitag lines printed on the DNA strip (Figure 2).

After PCR, 30 μL of streptavidin-coated blue latex solution was added to the PCR tube, and then the DNA strip was inserted into the PCR tube. The hybridized PCR amplicon was visualized in 5 to 10 min as a blue line, which represented the bound streptavidin-coated blue latex and biotin-labeled 5 terminus of the PCR amplicon.

2.6. A Protocol to Detect Multiple Foodborne Pathogens after 6 Hours Enrichment Culture of Ground Meat

For detection of foodborne pathogens with our protocol, 25 g of beef and 225 mL of FPE broth were homogenized in Stomacher bags (Eiken Chemical Co., Tokyo, Japan). The entire homogenate was transferred to a culture bottle and incubated with shaking at 37°C. After incubation, 1 mL of the supernatant was collected, and DNA was extracted using the physical disruption method described above. Subsequently, 5 μL of the extracted DNA was analyzed by cocktail PCR primers (Table 2) using premix Ex Taq (Takara Bio, Shiga, Japan) and the QuickBath thermal cycler. After 30 min, the PCR amplicon was analyzed by dipstick DNA chromatography. Thirty microliters of streptavidin-coated blue latex solution were added to each tube. Subsequently, the DNA strip was inserted into each tube. After 5–10 min at room temperature, the amplicon bound on the appropriate line on the dipstick surface (Figure 2) was visualized by the blue latex of the biotin-labeled 5 terminus of the PCR amplicon.

2.7. Sensitivity and Specificity of the Cocktail PCR Dipstick DNA Chromatography (CPDC) Assay

To measure the sensitivity of the CPDC assay, purified chromosomal DNA of E. coli O157 GTC14510 and S. enterica serovar Enteritidis GTC03838 were prepared at six different concentrations (2 ng, 200 pg, 20 pg, 2 pg, 200 fg, and 20 fg) and assayed (Table 3). Another sensitivity assay starting from quantitatively diluted culture supernatants was also performed (Table 4). The specificity of the CPDC assay was determined using the 176 strains listed in Table 5.

DNA concentrationEHEC serovar O111, GTC14517 (stx1)EHEC serovar O157, GTC14513 (stx2)Shigella dysenteriae serovar 2, GTC01929 (ipaH)Salmonella enterica serovar Enteritidis GTC03838 (invA)

2 ng/assay++++
200 pg/assay++++
20 pg/assay++++
2 pg/assay++++
200 fg/assay++++
20 fg/assay

Serially diluted purified DNA of each strain was used to count detection sensitivity.

Bacterial concentration (CFU/mL) EHEC O157 GTC14510 Bacterial concentration (CFU/mL)Salmonella enterica serovar
Enteritidis GTC03838
Immunochromat.CPDC assayImmunochromat.CPDC assay

++ ++
++ ++
++ ++
++ ++
++ ++
+ +
+ +

BacteriaSerotype (Virulence factor) Reacted Dipstick lineCPDC assay (positive/strains)

EHECO26: H- (stx1)Line 14/4
O26: H11(stx1)Line 15/5
O26: H11(stx1/2)Line 13/3
O111: H- (stx1)Line 13/3
O111: H- (stx1/2)Line 12/2
O115: H10(stx1)Line 11/1
O119: H2(stx1)Line 11/1
O121: H19(stx2)Line 14/4
O128: H- (stx1/2)Line 11/1
O157: H7(stx2)Line 110/10
O157: H7(stx1/2)Line 110/10
O157: H-(stx1/2)Line 110/10
EIECO28: H- (IpaH)Line 25/5
O124: H- (IpaH)Line 24/4
O136: H- (IpaH)Line 210/10
O144: H- (IpaH)Line 23/3
O164:H- (IpaH)Line 23/3
Shigella boydii (IpaH)Line 28/8
Shigella dysenteriae (IpaH)Line 216/16
Shigella flexneri (IpaH)Line 213/13
Shigella sonnei (IpaH)Line 27/7
Salmonella enterica
 subsp.enterica serovar Typhimurium (InvA)Line 310/10
 subsp.enterica serovar Typhi (InvA)Line 310/10
 subsp.enterica serovar Enteritidis (InvA)Line 37/7
 subsp.enterica serovar Dublin (InvA)Line 310/10
 subsp.enterica serovar paratyphi A (InvA)Line 33/3
Salmonella bongoriGTC 03793 T(InvA)Line 31/1
Escherichia coli GTC00503 T N*0/1
Escherichia albertiiGTC 16441 T N0/1
Escherichia fergusoniiGTC 01720 T N0/1
Escherichia vulneris GTC 10613 T N0/1
Escherichia hermanniiGTC 10612 T N0/1
Escherichia blattaeGTC 01342 T N0/1
Citrobacter freundiiGTC 14890 T N0/1
Citrobacter diversusGTC 00114 T N0/1
Citrobacter rodentium GTC 14911 T N0/1
Citrobacter youngaeGTC 14914 N0/1
Klebsiella pneumonia GTC 14868 T N0/1
Enterobacter cloacaeGTC 00109 T N0/1
Enterobacter aerogenesGTC 14962 T N0/1
Cronobacter sakazakiiGTC 14952 T N0/1
Serratia marcescensGTC 14672 N0/1
Yersinia enterocoliticaGTC 00127 T N0/1
Vibrio parahaemolyticus GTC02055 N0/1
Pseudomonas aeruginosaGTC 00002 T N0/1
Staphylococcus aureusGTC 00286 T N0/1

N*: no positive line.
Reacted line no. 1 is stx1 and 2 for EHEC, line 2 is ipaH for Shigella spp. and EIEC, and line 3 is for invA for Salmonella.

2.8. Evaluation of CPDC Assay with Spiked Ground Meat

The CPDC assay after enrichment culture in FPE broth was compared with a commercial immunochromatography system (Wako Pure Chem. Industries, Ltd., Osaka, Japan). Ground beef collected from local supermarkets was immediately transported to our laboratory in an insulated cooler box at 4°C. However, the isolation frequency of the target Shigella spp., Salmonella enterica, and E. coli O157 : H7 was less than 0.1% by culture-based conventional methods in our preliminary experiments. We decided, therefore, to evaluate the CPDC assay with spiked ground meat experiments. Ground meat was collected from a supermarket and confirmed target to be pathogen free by conventional methods. One milliliter of a mixed culture containing Shigella dysenteriae, S. enterica subspecies enterica serovar Enteritidis, and E. coli O157:H7 at three different concentrations were mixed with 25 g of ground meat, and 225 mL of FPE broth was then added. The total volume was incubated at 37°C. Immediately, and 4 hours, 6 hours, 8 hours, and 18 hours, 1 mL of the enrichment was used for extraction and then used in the CPDC assay. Another aliquot was used for the commercial immunochromatography kit for S. enterica serovar Enteritidis and E. coli O157.

3. Results and Discussion

Conventional culture has been the “gold standard” method for the detection of enteric bacterial pathogens. The advantages of this method include identification, facilitation of outbreak management, and generation of an antimicrobial susceptibility profile [15]. However, this conventional method has many disadvantages. Many different enrichment broths and solid media are used to screen for all possible foodborne pathogens, and time-consuming protocols are prepared to generate a result. FPE broth is designed to support Campylobacter without adding lysed blood and carbon dioxide. Campylobacter, however, is a slow-growing organism and needs 24 hours to reach 104 CFU/mL. Therefore, addition of selective antibiotics to the FPE broth was essential to suppress contaminating other bacteria for 24 hours enrichment culture. FPE broth could also support the growth of Listeria without adding blood, but the growth of Listeria is also slow, needing 24 hours to reach 104 CFU/mL (unpublished data).

The growth of pathogens in conventional enrichment culture and FPE broths were measured (Figure 1). Approximately 1–10 bacteria were spiked in 225 mL of enrichment broth. S. enterica and E. coli reached 104 CFU/mL after 6-hours incubation in FPE broth, buffered peptone water broth, and mEC broth, as shown in Figure 1. V. parahaemolyticus reached 104 CFU/mL after 4-hours incubation in both FPE and alkaline peptone broths. Based on these data, we selected 6 hours incubation for the CPDC assay.

In the present study, cocktail PCR was capable of simultaneously determining the presence of Salmonella spp., Shigella spp., EIEC, and EHEC by targeting invA, ipaH, stx1, and stx2 genes (Figure 2).

To evaluate the detection limit of the CPDC assay for each pathogen, 2 ng to 20 fg of DNA per reaction was prepared. The sensitivity and specificity of this assay are shown in Tables 35. The detection limit was 200 fg for each pathogen per CPDC assay (Table 3). The presence of the products with expected sizes was also confirmed by agarose gel electrophoresis, and nonspecific products were not observed (data not shown). The specificity of this CPDC assay was evaluated using 157 target strains (45 strains of Shigella spp., 54 strains of EHEC, 17 strains of EIEC, and 41 strains of Salmonella spp.) and 19 nontarget strains shown in Table 5. The detection limit of Salmonella and Escherichia from FPE culture supernatant (Table 4) was 103 CFU/mL, while the commercial immunochromatography kit required 105 CFU/mL. No false positive lines appeared on the dipstick DNA chromatography for any of the nontarget strains.

Immunochromatography is a simple technology to detect antigen in culture supernatant. However, immunochromatography targeting EHEC serotypes is not useful for testing food, because many kinds of E. coli serotypes produce shiga toxins. Thus, it is practically difficult to cover all of the EHEC serotypes by immunochromatography. Our method targeted both shiga toxin 1 and shiga toxin 2 toxins and detected non-O157 shiga toxin-producing serotypes (O26, O111, and O121). Three serotype (O45, O103, and O145) strains were not used because they are not available from our collection.

Shiga toxin 1 and shiga toxin 2 PCR products were designed to be bound on line 1 of the dipstick (Figure 2). The two genes were not equally amplified. The signal of the shiga toxin 2 amplicon was always bigger than the signal of the shiga toxin 1 amplicon.

The invasion plasmid antigen H (ipaH) gene is often used to diagnosis dysentery [16], because ipaH is carried by all four Shigella species as well as EIEC. In our cocktail primer, therefore, we selected the ipaH-specific primer to detect both Shigella and EIEC. The ipaH amplicon was designed to react on the second line of the dipstick chromatography strip, as shown in Figure 2. The CPDC assay was found to be effective at detecting Shigella species and EIEC from 4 to 6 hours FPE broth.

The CPDC assay required 4 to 6 hours FPE broth for the detection of S. enterica serovar Enteritidis. The detection limit of chromatography for Salmonella was, however, 105 CFU/mL in culture supernatant. To reach this cell number in FPE broth, incubation for more than 6 hours was necessary (Table 6). There is another disadvantage to using immunochromatography. The commercially available immunochromatography kits for Salmonella serovars are limited. The products only detect serovar Enteritidis. Thus, it is difficult to screen many different Salmonella serotypes simultaneously, such as serovar Typhimurium, serovar Choleraesuis, serovar Dublin, serovar Typhi, and others.

Inoculated level
(CFU/25 g)
CPDC assayImmunochromatography
Enrichment time (h)Enrichment time (h)

Enterohemorrhagic E. coli O157 GTC14510

Salmonella enterica serovar Enteritidis GTC03838

The CPDC assay for Salmonella was evaluated by spiked ground meat experiments with three different inoculation levels from 2.5 to 10 CFU/25 g (Table 6). The CPDC assay detected target pathogens on the third line of a dipstick DNA strip from 4 to 6 hours culture with FPE broth. On the other hand, the commercial immunochromatography kit only detected antigens from 18 hours enrichment culture because the method requires 105 CFU/mL of organism (Tables 4 and 6).

Multiplex PCR to detect many Salmonella serovars has been reported [17, 18]; however, we selected the invA gene for our assay because all Salmonella serovars carry this gene [19].

Internal amplification control (IAC) is designed to bind to line 4 of the dipstick chromatography strip to check for the presence of PCR inhibitor and false negatives [20]. A general guideline proposed for PCR testing of foodborne pathogens also requires the presence of IAC in the reaction mixture [21].

Systematic review of clinical implications, public health considerations, and the cost effectiveness of rapid diagnostic tests for detection and identification of bacterial pathogens in feces and food suggests that adoption of rapid test methods, especially for PCR, in combination with a routine culture is unlikely to be cost-effective [7, 22]. However, as the cost of rapid technologies decreases, total replacement with rapid technologies may be feasible.

The clinical impact of the decreased turnaround time means that bacterial diarrhea is more promptly ruled out using the CPDC assay compared to using conventional culture in small laboratories. This reduces the expenditure of infection control resources and, in particular, in cases of sporadic diarrhea, helps to reduce the requirement for scarce isolation rooms. In addition, the earlier availability of results is helpful in community-based management of outbreaks.

For detection of Salmonella spp., Shigella spp., EIEC, and EHEC, the overall time to confirm a positive result by conventional culture plus immunochromatography is at least two working days. Generating a negative report requires 48 hours. In contrast, a report can be generated for the CPDC assay within one working day. One advantage of an early laboratory report is early judgment of contamination, which can prevent food poisoning and additional outbreaks. The method also contributes to the quick shipment of fresh food products to the markets.

4. Conclusion

Cocktail PCR targeting multiple foodborne pathogens and simple dipstick DNA chromatography to differentiate the PCR product was designed. The method was applied to detect pathogens in ground meat after 6 hours enrichment broth, which supports the growth of broad range foodborne pathogens. This single tube PCR and enrichment method help to simplify food analysis protocol. As a result, the method offers rapid report to food suppliers and helps the quick shipment of safety-confirmed food products to markets.


This work was partially funded by the Regional Innovation Strategy Support Program of the Ministry of Education, Culture, Sports, Science, and Technology of Japan, and Grant for Health and Labour Sciences Research, Research on Global Health Issues supported by the United States-Japan Cooperative Medical Science Program.


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Copyright © 2013 Masahiro Hayashi et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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