International Journal of Food Microbiology 185 (2014) 27–32

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International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro

High-throughput detection of food-borne pathogenic bacteria using oligonucleotide microarray with quantum dots as fluorescent labels Aihua Huang a,b,1, Zhigang Qiu a,1, Min Jin a, Zhiqiang Shen a, Zhaoli Chen a, Xinwei Wang a, Jun-Wen Li a,⁎ a b

Tianjin Key Laboratory of Risk Assessment and Control Technology for Environment and Food Safety, Institute of Health and Environmental Medicine, Tianjin, 300050, P. R. China Logistics College of Chinese People’s Armed Police Forces, Tianjin, 300162, P. R. China

a r t i c l e

i n f o

Article history: Received 19 November 2013 Received in revised form 16 April 2014 Accepted 14 May 2014 Available online 21 May 2014 Keywords: Microarray Detection Quantum dots Hybridization Probes Bacteria

a b s t r a c t Bacterial pathogens are mostly responsible for food-borne diseases, and there is still substantial room for improvement in the effective detection of these organisms. In the present study, we explored a new method to detect target pathogens easily and rapidly with high sensitivity and specificity. This method uses an oligonucleotide microarray combined with quantum dots as fluorescent labels. Oligonucleotide probes targeting the 16SrRNA gene were synthesized to create an oligonucleotide microarray. The PCR products labeled with biotin were subsequently hybridized using an oligonucleotide microarray. Following incubation with CdSe/ZnS quantum dots coated with streptavidin, fluorescent signals were detected with a PerkinElmer Gx Microarray Scanner. The results clearly showed specific hybridization profiles corresponding to the bacterial species assessed. Two hundred and sixteen strains of food-borne bacterial pathogens, including standard strains and isolated strains from food samples, were used to test the specificity, stability, and sensitivity of the microarray system. We found that the oligonucleotide microarray combined with quantum dots used as fluorescent labels can successfully discriminate the bacterial organisms at the genera or species level, with high specificity and stability as well as a sensitivity of 10 colony forming units (CFU)/mL of pure culture. We further tested 105 mock-contaminated food samples and achieved consistent results as those obtained from traditional biochemical methods. Together, these results indicate that the quantum dot-based oligonucleotide microarray has the potential to be a powerful tool in the detection and identification of pathogenic bacteria in foods. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Rapid detection of food-borne pathogenic bacteria is of great importance for public health. To minimize the prevalence of food-borne diseases and reduce microbial contaminations in food supplies, effective monitoring of the occurrence and distribution of bacterial pathogens in foods is essential. Currently, conventional methods commonly used in the field are based on cultivation of target pathogens or indicator microorganisms on specific media. However, these methods require several days for completion and are time-consuming. In addition, the culturebased methods sometimes lack specificity in selecting or identifying unknown pathogens in foods (Abubakar et al., 2007; Suo et al., 2010). More recently, several DNA-based methods have been developed to detect pathogenic bacteria. The polymerase chain reaction (PCR) is a very convenient and important technique for the detection of DNA of a specific microorganism (Horakova et al., 2008; Liu et al., 2008; Llop et al., 1999; Mao et al., 2007; Sawada et al., 1992; Takaishi et al., 2003; Versalovic et al., 1995). A number of methods based on probes, ⁎ Corresponding author. Tel.: +86 22 84655345; fax: +86 22 84655017. E-mail address: [email protected] (J.-W. Li). 1 The first two authors contributed equally to this work.

http://dx.doi.org/10.1016/j.ijfoodmicro.2014.05.012 0168-1605/© 2014 Elsevier B.V. All rights reserved.

restriction fragment length polymorphisms, and multiplex PCR have been reported in previous studies to detect pathogenic bacteria (de Las Rivas et al., 2005; Kim et al., 2007; Naravaneni and Jamil, 2005; Priest et al., 1994). Real-time PCR systems for quantitative analyses of pathogenic bacteria have also been developed (Fricker et al., 2007; Fukushima et al., 2007; Lopez and Pardo, 2010). Although these DNA-based methods have been used to detect pathogenic bacteria, they share a common limitation when faced with the complicated distribution of various strains, species, and genera of pathogenic bacteria. Moreover, these approaches cannot simultaneously detect multiple pathogenic bacteria in parallel using a single experimental cycle. However, this high degree of parallelism can be achieved using microarray technology. Microarray technology is based on the hybridization of oligonucleotide probes on a slide to another nucleotide population. In recent years, microarray technology has enabled high-throughput detection of multiple pathogens in a large number of samples and has been widely applied to DNA detection and genotyping because of its miniature arrangement, high performance, and ease of automation (Call et al., 2003; Cremonesi et al., 2009; Drost et al., 2009; Hacia et al., 2000; Huang et al., 2006; Jin et al., 2006; Kim et al., 2003; Park et al., 2004; Pasquini et al., 2008; Peplies et al., 2003; Tiberini et al., 2010). The labeled molecules used

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in DNA microarrays to detect binding events are usually fluorescent organic dyes. Although they provide a sensitive, safe, and low cost detection system, they suffer from several limitations. First, organic dyes are sensitive to photo bleaching and are often not bright enough for the quantification of a specific signal over background. Second, the fluorescence spectrums of organic dyes are not symmetric and each fluorophore is characterized by its specific optimal wavelength of excitation, which limits their multiplexing capabilities (Resch-Genger et al., 2008). Therefore, there is an urgent requirement to develop DNA microarrays that do not rely on organic dyes. Current advances in nanotechnology have provided a novel and promising class of semiconductor nanocrystal quantum dots (QDs). QDs possess a number of advantages over traditional dyes, such as a high quantum yield, long photostability, and high extinction coefficient (Chan et al., 2002; Tan et al., 2002). Several groups have reported the application of QDs in nucleic acid diagnostic methods. For example, QDs have been used to detect complementary target nucleic acid sequences in fluorescent in situ hybridization (FISH) assays (Pathak et al., 2001). Single nucleotide polymorphism (SNP) discrimination and two-color, two-target detection systems have also been demonstrated with QD labels in a microarray format (Gerion et al., 2003). QDs have also been used with FISH to label the HER2gene locus in breast cancer cells (Xiao and Barker, 2004). QDs have been used successfully for the detection of Cryptosporidium parvum (Lee et al., 2004), Escherichia coli O157 (Hahn et al., 2005) and Mycobacterium spp. (Gazouli et al., 2010). Moreover, it has been shown that two food-borne strains Escherichia coli O157:H7 and Salmonella typhimurium labeled with QDs were identified simultaneously (Zhao et al., 2009). These experiments showed that QDs have superior sensitivity and photostability compared to conventional dyes. However, few studies have examined the detection of pathogenic bacteria from foods using an oligonucleotide microarray with QDs as fluorescent labels. In the present study, we established a sensitive and specific oligonucleotide microarray using QDs as fluorescent labels to detect 11 common species of food-borne pathogenic bacteria that are the most common in food or most dangerous to human health (Gomes et al., 2013; Haagsma et al., 2013; Kozak et al., 2013). We also evaluated the assay by testing for specific bacterial strains within real samples.

broth (Becton Dickinson, Kansas, USA) or 25 ml of milk in 225 ml of universal pre-enrichment broth (Becton Dickinson, Difco). The preparations were then incubated at 37 °C overnight and DNA was extracted. 2.3. Preparation of mock-contaminated food samples To test the effectiveness of this novel procedure in a real-world application, we introduced bacteria into the samples to represent mock-contaminated food samples. In these experiments, only those food samples that were confirmed to be negative for pathogens by both culture and PCR methods were used. A total of 105 food samples from different local manufacturers were used in this experiment, including 35 pork, 42 chicken, 23 fish, and 5 milk samples. Food samples were mock contaminated by inoculating each sample with 102–106 CFU of a strain prior to homogenization and enrichment. We found that only 2 h of incubation was required to enrich bacteria to detectable levels, without the need to isolate the organism. 2.4. Extraction of bacterial DNA from pure cultures and foods For pure cultures, DNAs were extracted using the boiling method as previously described (Afghani and Stutman, 1996). DNAs from various bacteria species in food samples were extracted using the QIAamp DNA Mini Kit (Qiagen, GmbH, Germany) according to the manufacturer’s instructions and were used as templates for amplification. 2.5. Sequencesof the primers and probes The universal primers were based on the conserved region of the 16S rRNA gene. The forward primer sequence was 5′-aactggaggaaggtggggat-3′, and the reverse primer sequence was 5′-aggaggtgatccaaccgca-3′. Primers were synthesized by Invitrogen (USA) and the forward primer was labeled with biotin at the 5′ end. The oligonucleotide probes were targeted to the variability of the 16S rRNA gene regions (Wang et al., 2007). Probes (Table 3) were synthesized by Invitrogen (USA) and modified with NH2 to increase binding to the glass slide as well as hybridization intensity.

2. Materials and methods 2.6. PCR amplification of the target gene 2.1. Strains The standard bacteria strains, including food-borne and nosocomial pathogens, used in this study were obtained from the Chinese Medical Culture Collection Center (CMCC, Beijing China) and are listed in Table 1. The isolated strains used in this study were isolated from foods and are listed in Table 2. The negative control strains tested included Aspergillus fumigatus (CMCC A1), Fonsecaea pedrosoi (CMCC D6a), Sporothrix schenckii (CMCC D1), Mucor racemosus (CMCC 33440), Candida albicans (CMCC C1a), Microsporum canis (CMCC M3b), Citrobacter spp. (CMCC 48025), Clostridium perfringens (CMCC 64615), Aeromonas hydrophila (CMCC 12017), and Bacillus cereus (CMCC 63303). Bacteria cultures were serially diluted to the appropriate inoculum levels and confirmed by plating in standard plate count agar (PCA) in triplicate. Artificially inoculated samples were tested using conventional culture methods with serological confirmation and the VITEK test system (BioMerieux SA, France). All fungi were inoculated onto potato dextrose agar slants at 30 °C for 72–120 h and were stored at 4 °C until further use. 2.2. Preparation and cultivation of bacteria in food samples Food samples (pork, chicken, fish, and milk) were purchased from local supermarkets. For bacterial detection from raw foods, cultures were pre-enriched by homogenizing 25 g of meat in 225 ml of nutrient

PCR was performed in 50 μl containing 5 μl 10 × buffer (Takara), 200 μM dNTP mixture (Takara), 0.1 U/μl Takara Taq (5 U/μl), 1 μM forward primer, 0.1 μM reverse primer, and 2 μl supernatant containing bacterial DNA. Sterile distilled water was added to a final volume of 50 μl. The PCR mixtures were denatured at 95 °C for 5 min, followed by 30 cycles of 95 °C for 30 s, 50 °C for 30 s, and 72 °C for 30 s. The PCR product was verified using 1% agarose gel electrophoresis and visualized with ethidium bromide. 2.7. Generation of oligonucleotide microarrays In the present study, 14 oligonucleotide probes were used (Table 3) to design a microarray model for the detection of bacteria pathogens (Fig. 1A). Four categories of designed oligonucleotide probes were identified based on their efficacies. Category 1 included oligonucleotide probes 3–12, which is a cluster of genus- or species-specific probes used to identify bacteria at the genus or species level. Category 2, composed of oligonucleotide probe 1, is a G+ probe shared by all G+ bacteria and used to detect all types of G+ bacteria. Category 3, which included oligonucleotide probe 2, is a G− probe shared by all G− bacteria and was used to detect all types of G− bacteria. Category 4 included oligonucleotide probes 13 and 14, which were positive and negative control probes that were used to reflect the effectiveness of the hybridization system and to serve as reference coordinates for scanning.

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2.10. Scanning of the microarray for fluorescent signals and scoring of hybridization results

Table 1 Standard strains used in the present studya. Species

Strain no.

Salmonella spp.

50001, 50004, 50013, 50019, 50041, 50042, 50043, 50047, 50051, 50073, 50082, 50086, 50093, 50096, 50098, 50099, 50104, 50105, 50106, 50109, 50112, 50115, 50120, 50124, 50145, 50201, 50210, 50220, 50304, 50306, 50307, 50309, 50310, 50313, 50315, 50320, 50322, 50326, 50327, 50333, 50355, 50337, 50358,50338, 50354, 50355, 50360, 50402, 50708, 50709, 50710, 50719, 50730, 50732, 50733, 50735, 50739, 50774, 50783, 50825, 50835, 50853, 50854, 50864 51081, 51100, 51207, 51227, 51233, 51252, 51253, 51255, 51258, 51259, 51262, 51307, 51315, 51334, 51335, 51336, 51424, 51464, 51570, 51571, 51572, 51573, 51575,51582, 51583, 51584, 51585, 51610 44102, 44109, 44110, 44113, 44126, 44127, 44149, 44155, 44156, 44186, 44216, 44336, 44338, 44344, 44505, 44561, 44752, 44813, 44824 49027, 49101, 49102, 49103 26001, 26003, 26101, 26111, 26113 52202, 52203, 52206, 52207, 52211, 52218 54003 20502, 20507 33252 32210 32221, 32223 11610, 11611, 11609, 17002, 11608

Shigella spp.

Escherichia coli spp.

Proteus spp. Staphylococcus aureus Yersinia enterocolitica Listeria monocytogenes Vibrio parahaemolyticus Campylobacter jejuni β-hemolytic Streptococcus Enterococcus faecalis Vibrio fluvialis Total number a

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Number 64

Slides were inserted into a PerkinElmer ScanarrayGx (PerkinElmer, USA) to scan the area of the slide containing the microarray. The scanned images were uploaded as tiff files into the Proscanarray express software (PerkinElmer, USA) and examine for fluorescence intensity.

3. Results 3.1. Assessment of the primers

28

19

4 5 6 1 2 1 1 2 5 138

All standard strains listed in this table were obtained from CMCC.

Oligonucleotides were bound to the slides as follows: 5 μl of each oligonucleotide (50 μM) was spotted onto a glass slide using an arrayer (PixSys 5500 Workstation, Cartesian Technologies, Irvine, CA). When all of the oligonucleotides were applied, the glass slides were incubated at room temperature for 24 h to permit thorough drying of the DNA onto the surface of the slides. After drying, the slides were washed in 0.2% SDS for 5 min, distilled water for 5 min, sodium borohybride solution (1.3 g Na2BH4 dissolved in 375 ml phosphate buffered saline followed by the addition of 125 ml pure ethanol) for 5 min, 0.2% SDS for 2 min, and finally distilled water for 2 min.

2.8. Hybridization The amplicons labeled using biotin were hybridized to the oligonucleotide microarrays with the following protocol. Amplicons (1 μl) were added to a tube containing 4 μl hybridization solution (UniHyb, Tele Chem International, USA), heated to 95 °C for 10 min, and then immediately placed on ice. The mixture in the tube was then transferred to the microarray and incubated for 1 hat 50 °C in a hybridization cassette (TeleCHem USA). Following hybridization, unbound amplicons were washed at 25 °C, using buffer A [1× saline-sodium citrate (SSC) plus 0.2% SDS] for 1 min, wash buffer B (0.1 × SSC + 0.2% SDS) for 1 min, and finally wash buffer C (0.1 × SSC) for 1 min.

2.9. QD labeling The above microarray was incubated with 20nM CdSe/ZnSQDs coated with streptavidin (emission 525 nm, Jia Yuan, Wuhan, China) for 1 h at room temperature. A final wash step of 10 min in buffer (1× SSC + 0.2% SDS) followed by 10 min in 0.2% SDS was required in order to remove unbound labels.

The universal primers were assessed under the same conditions using the target genes from 138 standard strains (Table 1) and 78 isolated strains (Table 2). All bacterial pathogens tested generated PCR products with bands of 200–500 bp.

3.2. Hybridization from enrichment broths of standard bacteria From the hybridization signals of the four categories of oligonucleotide probes, the target pathogenic bacteria within a given specimen were easily identified (Fig. 1B). Strains belonging to Vibrio parahaemolyticus, Vibrio fluvialis, Yersinia enterocolitica, Proteus sp., Staphylococcus aureus, Enterococcus faecalis, Campylobacter jejuni, β-hemolytic Streptococcus, and Listeria monocytogenes were clearly discriminated using this approach, while strains belonging to Escherichia coli, Shigella spp., and Salmonella spp. were differentiated as one class of pathogenic bacteria.

3.3. Sensitivity of the oligonucleotide microarray using QDs as fluorescent labels Vibrio parahaemolyticus and Staphylococcus aureus were used to evaluate array sensitivity. Freshly cultured bacteria (106 CFU/ml) were serially diluted 10-fold until there was 1 cell per ml (as determined by the CFU count) in a 0.1 M PBS buffer (pH 7.3). DNA was then extracted and amplified using PCR. The PCR products were hybridized with the oligonucleotide microarray. These processes were repeated in triplicate for each diluted concentration. The results showed that sensitivity of the oligonucleotide microarray was 10 CFU/ml for the pure culture (Fig. S1).

3.4. Reproducibility of the oligonucleotide microarray using QDs as fluorescent labels To evaluate the reproducibility of the assay, Listeria monocytogenes, Vibrio parahaemolyticus, and Staphylococcus aureus were detected using the oligonucleotide microarray. The experiment was repeated five times under the same conditions for each bacterium. Importantly, the coefficients of variation of the signal-to-noise ratio were all less than 10% (Table S1).

3.5. Detection of isolated pathogenic bacteria from food samples Seventy-eight strains of pathogenic bacteria isolated from food samples listed in Table 2 were processed and hybridized as described above and then identified according to their specific hybridization maps. All 78 strains were distinguished using their hybridization maps. The comprehensive identification results generated from classical methods were regarded as the final standards. All of the hybridization assay results were 100% (78/78) consistent with those of the conventional methods.

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A. Huang et al. / International Journal of Food Microbiology 185 (2014) 27–32 Table 2 Isolated strains from food samples used in this study. Species

Number

Salmonella spp. Shigella spp. Escherichia coli spp. Proteus spp. Staphylococcus aureus Yersinia enterocolitica Listeria monocytogenes Vibrio parahaemolyticus Campylobacter jejuni β-hemolytic Streptococcus Enterococcus faecalis Vibrio fluvialis Total number

8 8 10 5 11 9 8 6 1 1 5 6 78

3.6. Detection of mock-contaminated food samples A total of 105 mock-contaminated food samples were tested using this method. The results obtained using the array methods were completely consistent with the results obtained with traditional culture and biochemical identification methods (Table S2). These results indicated that the QDs-based oligonucleotide microarrays can successfully detect pathogens in contaminated food samples. 4. Discussion DNA microarrays using fluorescent organic dyes have recently been more widely used for the detection of food-borne pathogens (Caspers et al., 2011; Kim et al., 2003; Rasooly and Herold, 2008; Suo et al., 2010). However, there are significant limitations to using fluorescent organic dyes, such as rapid photo bleaching, a narrow excitation spectrum, and low signal intensities (Resch-Genger et al., 2008). In this study, we provided an oligonucleotide microarray with quantum dots used as fluorescent labels to detect food-borne pathogenic bacteria. Using QDs as fluorescent labels of chip technology could overcome the disadvantages of traditional DNA microarrays that use fluorescent organic dyes. Our study demonstrated that a series of species-specific hybridization profiles can be obtained using this oligonucleotide microarray and CdSe-ZnS core-shell quantum dots as fluorescent labels. A wide variety of food-borne pathogenic bacteria can be clearly discriminated using this method. A total of 138 standard pathogen strains were processed and identified to test the specificity of this method. Highly specific hybridization signals were obtained for the strains, and no signals appeared for the negative control strains in any of the reactions. Furthermore, the method was validated using 78 strains of bacteria isolated from food samples, and species-specific hybridization signals were also obtained with this method. In order to assess its potential application in contaminated foods, we tested this technique on 105 Table 3 Oligonucleotide probes used in this study. Target

Sequence (5′-3′)

All Gram-positive bacteria All Gram-negative bacteria E. coli, Shigella spp., and Salmonella spp. Proteus spp. Campylobacter jejuni Listeria monocytogenes Enterococcus faecalis Yersinia enterocolitica Vibrio parahaemolyticus Vibrio fluvialis β-hemolytic Streptococcus Staphylococcus aureus Positive control Negative control

gacataaggggcatgatgatgatttgacgt gtcgtaagggccatgatgacttgacgt acgacgcactttatgaggtccgcttg ttcaccgtagcattctgatctacgatta cgaactgggacatattttatagatttgc actgagaatagttttatgggattag cctcgcggtctagcggctcgttgtactt tacgacagactttatgtggtccgcttgc ggattcgctcactttcgcttgttggctg tcactttcgcaagttggccgccctctgc agattggctttaagagattagcttgccg gctcctaaaaggttactccaccggct atccccaccttcctccagtt agcgattccttgctcctgagcaacaac

mock-contaminated food samples, and obtained data that were very consistent with those generated from traditional culture and biochemical identification methods. Together, these results indicate that this diagnostic platform possesses the ability to detect and differentiate pathogens in food samples. Although the coefficients of variation of our experiment were higher than 5%, they could also be accepted as many studies have reported (Kim et al., 2014; Leporati et al., 2014; Chung et al., 2014; Maruyama et al., 2013). The reason for the higher coefficients of variation in our study may be due to random error generated in the processes of QD labeling and washing, which were performed manually. If these operations are completed by a machine with a standardized procedure, then the coefficient of variation may be significantly reduced. One of the key parameters in pathogen detection assays is sensitivity, since in most food samples pathogens are often present at very low levels. Therefore, the ability to detect bacteria that are present at low concentrations has remained a key parameter used to evaluate detection methods. Although some microarrays coupled with fluorescent organic dyes have been reported for the detection of food-borne pathogens, their detection thresholds have been far from satisfactory. For example, our laboratory (Wang et al., 2007) reported on the development and application of a 16S rRNA gene-based microarray using Cy3 as a label for the detection of food-borne pathogens. The sensitivity of that assay was 102 CFU/ml of pure culture. The 16S–23S rRNA gene internal transcribed spacer was used as a marker for the development of a microarray that could detect 10 different pathogenic bacteria associated with powder infant formula contamination. The microarray sensitivity was found to be 104 CFU/mL of pure culture (Wang et al., 2009). Panicker et al. developed a gene-specific DNA microarray coupled with multiplex PCR for the comprehensive detection of pathogenic Vibrios. Amplified PCR products were hybridized to arrays at 50 °C and detected using tyramide signal amplification with the Alexa Fluor 546 fluorescent dye. The detection sensitivity for pure cultures was 102–103 CFU/ml (Panicker et al., 2004). Dazhi et al. developed a Cy3-based DNA microarray combined with tyramide signal amplification to simultaneously detect six species of pathogenic bacteria. The detection sensitivity was 103 CFU/ml for pure cultures (Jin et al., 2008). Recently, QDs have been used for microarray studies to improve sensitivity. Shingyoji et al. (2005) demonstrated that protein detection with QD conjugates can be achieved with a sensitivity of 1 pmol. Moreover, Liang et al. (2005) developed a microarray for microRNA expression analysis using QDs as labels and found that the detection sensitivity was only 0.4 fmol of microRNA. In order to improve the sensitivity of the DNA microarray, we labeled it with QDs, which contributed to a sensitivity of 10 CFU/mL. Importantly, this approach was more sensitive than the studies described above. QDs have similar quantum yields (approximately 70%), but much larger extinction coefficients compared to organic dyes, and their brightness has been shown to be 20–100 times higher. This increased brightness allows for a decrease in the quantity of fluorescent labels needed in order to limit nonspecific binding, which has been shown to occur when highly concentrated QD conjugates are used on microarrays (Karlin-Neumann et al., 2007). As QDs are bigger than organic dyes, the complexes of QDs with modified nucleotides seem to be too bulky for correct uptake by polymerases, and thus, their insertion rate is both low and not uniform. For this reason, we adapted the biotin–streptavidin system to bind streptavidin-coated QDs with biotinylated nucleotides. Although the use of the streptavidin–biotin system involves an additional step for probe-labeling, the complexes formed with labeled QDs were easily detected in the oligonucleotide microarrays, and the fluorescence intensity of the detected signals was strong. One shortcoming of our persistent experiment was the use of a 16S single probe for the identification of E. coli, Shigella spp. and Salmonella spp. These three strains of bacteria share nearly identical sequences within the amplified segments of target16S rRNA genes, which resulted

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Fig. 1. The design and results of microarray. (A) Layout of the oligonucleotide probes. 1, positive control; 2, negative control; 3, Gram-positive; 4, Gram-negative; 5, Vibrio parahaemolyticus; 6, Vibrio fluvialis; 7, Campylobacter jejuni; 8, Proteus spp.; 9, Yersinia enterocolitica; 10, Listeria monocytogenes; 11, Escherichia coli, Shigella spp., and Salmonella spp.; 12, Enterococcus faecalis; 13, Staphylococcus aureus; and 14, β-hemolytic Streptococcus. (B) Typical hybridization profiles on the oligonucleotide microarray. 1, Vibrio parahaemolyticuse (CMCC20502); 2, Vibrio fluvialis (CMCC11610); 3, Campylobacter jejuni (CMCC33252); 4, Proteus spp. (CMCC49027); 5, Yersinia enterocolitica (CMCC52203); 6, Listeria monocytogenes (CMCC54003); 7, Enterococcus faecalis (CMCC32223); 8, Staphylococcus aureus (CMCC26101); 9, β-hemolytic Streptococcus (CMCC32210); 10, Escherichia coli (CMCC44102); 11, Shigella spp. (CMCC51081); 12, Salmonella spp. (CMCC50001).

from interspecies ambiguity in phylogenic topology derived from evolutionary analysis. The hybridization maps derived from the 16SrRNA probes of Shigella spp., E. coli, and Salmonella spp. are too similar to differentiate. A DNA microarray to discriminate Salmonella and Shigella was developed in our previous study (Wang et al., 2007) using invA and virA genes to design specific oligonucleotide probes that could distinguish Salmonella and Shigella from other enteric bacteria. In future work we should change the fluorescent organic dyes of this microarray into QDs using the method described in this study. 5. Conclusions The development of more efficient labels for oligonucleotide microarray applications based on QDs was investigated in the present study. We found that the QD-based microarray system can successfully discriminate food-borne bacterial pathogens at the genus or species level. The sensitivity was 10 CFU/ml of pure culture. This QD-based oligonucleotide microarray is a rapid, simple, and sensitive detection method that is potentially an ideal alternative to fluorescent organic dye-based detection in oligonucleotides microarrays. Acknowledgements This research was supported by the National High-Tech Rand D Program of China (863 program) (grant no. 2009AA06Z404) and the Tianjin Key Project of Scientific and Technical Supporting Program (grant no. 11ZCKFSF01100). Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.ijfoodmicro.2014.05.012. References Abubakar, I., Irvine, L., Aldus, C.F., Wyatt, G.M., Fordham, R., Schelenz, S., Shepstone, L., Howe, A., Peck, M., Hunter, P.R., 2007. A systematic review of the clinical, public health and cost-effectiveness of rapid diagnostic tests for the detection and identification of bacterial intestinal pathogens in faeces and food. Health Technol. Assess. 11, 1–216.

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High-throughput detection of food-borne pathogenic bacteria using oligonucleotide microarray with quantum dots as fluorescent labels.

Bacterial pathogens are mostly responsible for food-borne diseases, and there is still substantial room for improvement in the effective detection of ...
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