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Detection of <I>Alicyclobacillus</I> Species in Fruit Juice Using a Random Genomic DNA Microarray Chip

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Detection of <I>Alicyclobacillus</I> Species in Fruit Juice Using a Random Genomic DNA Microarray Chip 933 Journal of Food Protection, Vol. 74, No. 6, 2011, Pages 933–938 doi:10.4315/0362-028X.JFP-10-418 Copyright G, International Association for Food Protection Detection of Alicyclobacillus Species in Fruit Juice Using a Random Genomic DNA Microarray Chip JUN HYEONG JANG, SUN-JOONG KIM, BO HYUN YOON, JEE-HOON RYU, MAN BOCK GU, AND HYO-IHL CHANG* College of Life Sciences and Biotechnology, Korea University, 5-1 Anam-dong, Seongbuk-gu, Seoul 136-701, Republic of Korea MS 10-418: Received 27 September 2010/Accepted 9 February 2011 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/74/6/933/1685326/0362-028x_jfp-10-418.pdf by guest on 28 September 2021 ABSTRACT This study describes a method using a DNA microarray chip to rapidly and simultaneously detect Alicyclobacillus species in orange juice based on the hybridization of genomic DNA with random probes. Three food spoilage bacteria were used in this study: Alicyclobacillus acidocaldarius, Alicyclobacillus acidoterrestris, and Alicyclobacillus cycloheptanicus. The three Alicyclobacillus species were adjusted to 2 | 103 CFU/ml and inoculated into pasteurized 100% pure orange juice. Cy5-dCTP labeling was used for reference signals, and Cy3-dCTP was labeled for target genomic DNA. The molar ratio of 1:1 of Cy3-dCTP and Cy5-dCTP was used. DNA microarray chips were fabricated using randomly fragmented DNA of Alicyclobacillus spp. and were hybridized with genomic DNA extracted from Bacillus spp. Genomic DNA extracted from Alicyclobacillus spp. showed a significantly higher hybridization rate compared with DNA of Bacillus spp., thereby distinguishing Alicyclobacillus spp. from Bacillus spp. The results showed that the microarray DNA chip containing randomly fragmented genomic DNA was specific and clearly identified specific food spoilage bacteria. This microarray system is a good tool for rapid and specific detection of thermophilic spoilage bacteria, mainly Alicyclobacillus spp., and is useful and applicable to the fruit juice industry. Alicyclobacillus spp. are acidophilic, thermophilic detection methods require extensive amounts of genetic sporeformers commonly found in acidic fruit juices. They information for spoilage microbes and cannot detect many cause a sour taste and a bad smell and are known to be the spoilage bacteria simultaneously (5, 7, 8, 13). Therefore, main cause of lowered quality in fruit juice (3). Since food there is a critical need for an advanced diagnostic system contains a variety of nutrients, microbial growth can lead to that can rapidly and simultaneously detect various types of adverse changes in taste, smell, and color that negatively food spoilage bacteria. impact food quality. Since information on the genome sequence for specific Spoilage bacteria that form heat-resistant spores can microorganisms that cause food decomposition and poor germinate and grow in industrially sterilized fruit juice, food quality is not always available, genomic DNA from posing a serious threat to the beverage industry. pure cultures and random genomic DNA libraries can be The representative spoilage bacteria Alicyclobacillus built. The DNA chip is made as a product of that library. spp. was first identified in 1971 (6) and was later found to With random priming, hybridization detection of a specific be the cause of decreasing quality in various foods including microorganism can be confirmed (9). As such, DNA fruit juice, ice tea, and canned tomatoes. Classical detection microarray technology can be useful for detecting and methods for spoilage bacteria in various foods are based on analyzing specific microorganisms in foods. the results from enrichment, microscopic observations, and In this study, we developed a DNA chip to detect and biochemical assays. These procedures have several limita- identify Alicyclobacillus spp. A DNA chip containing tions, such as long culture time, dependency on enrichment randomly fragmented genomic DNA of Alicyclobacillus and selective culture, and difficulties in simultaneous spp. was fabricated and assessed for its ability to distinguish detection of spoilage bacteria. Alicyclobacillus spp. from other strains and other bacteria To overcome these problems, molecular-based methods such as Bacillus spp. such as real-time PCR and immunoassay have been developed. These methods offer specificity and high MATERIALS AND METHODS affinity, depending on target genes of specific spoilage Bacterial strains and isolation of genomic DNA. Alicyclo- bacteria. Although these molecular-based methods or bacillus acidocaldarius (ATCC 43030, 27009), Alicyclobacillus immunoassays can shorten the detection time, they also acidoterrestris (ATCC 49025, 49027), and Alicyclobacillus have several drawbacks. For example, real-time PCR cycloheptanicus (ATCC 49029, 49028) were obtained from the American Type Culture Collection (ATCC; Manassas, VA) and grown in ATCC medium 573, 1655, and 1656, respectively, * Author for correspondence. Tel: 82-2-3290-3421; according to the manufacturer’s instructions. Bacillus subtilis E-mail: [email protected]. ATCC 6633 and Bacillus cereus F3812 strains were also used. 934 JANG ET AL. J. Food Prot., Vol. 74, No. 6 FIGURE 1. Schematic of nonsequenced random genomic DNA probes for three different Alicyclobacillus species. All probes were spotted in duplicate. Downloaded from http://meridian.allenpress.com/jfp/article-pdf/74/6/933/1685326/0362-028x_jfp-10-418.pdf by guest on 28 September 2021 Pasteurized 100% pure orange juice was purchased from a genes were prepared by RT-PCR from AGS cancer cell RNA with local grocer. One liter of orange juice was filtered through a 0.45- primers 59-cattctggcccaccaactttgg-39 and 59-tggagatgcaggctc- mm-pore-size membrane filter under vacuum, after which 5 ml each cactttg-39 (Fig. 1). of A. acidocaldarius (ATCC 43030), A. acidoterrestris (ATCC 49025), and A. cycloheptanicus (ATCC 49029) (7, 10, 11) culture Genomic DNA labeling and hybridization. Genomic DNA broth (2 | 103 CFU/ml) were inoculated into 45-ml aliquots of the (50 ng) from all strains, including the reference strains, was labeled pasteurized orange juice and incubated at 47uC for 5 days. The with Cy5-dCTP (Amersham Bioscience, Uppsala, Sweden) for the cells were then recovered by centrifugation at 5,400 | g for reference signals and with Cy3-dCTP by random priming for 10 min. genomic DNA (High Prime DNA labeling kit, Roche, Mannheim, Genomic DNA from Alicyclobacillus spp. was extracted from Germany) (9). Mixtures of genomic DNA (50 ng) from the three 50 ml of culture (Flexible Kit, NucleoGen, Seoul, Korea). DNA reference strains (1:1) used for microarray fabrication were labeled concentration was determined using NanoDrop ND-1000 (Nano- with Cy3-dCTP (Amersham Bioscience) and used as reference Drop Technologies, Inc., Wilmington, DE) by calculating the ratio DNA for signal ratio calculations (Cy3-test/Cy5-reference). of absorbance (260 nm/280 nm) and agarose gel electrophoresis. Bacteriophage MJ1 (10 ng) was included in each labeling reaction The DNA was stored at 220uC until further use. as a positive control. The microarrays were prehybridized in buffer (1.7| SSC [1| SSC is 0.15 M NaCl plus 0.015 M sodium DNA microarray chip fabrication. Genomic DNA from citrate], 0.1% sodium dodecyl sulfate [SDS], 10 mg/ml bovine three different food spoilage bacteria (A. acidocaldarius ATCC serum) for 30 min at 65uC and then hybridized with approximately 43030, A. acidoterrestris ATCC 49025, and A. cycloheptanicus 50 ng of genomic DNA with Cy3-labeled (test)/Cy5-labeled ATCC 49029) was isolated and digested with several pairs of (reference) DNA mixture (1:1) in hybridization buffer (6| SSC, restriction enzymes, i.e., EcoRI/BamHI, HindIII/XhoI, HindIII/ 0.2% SDS, 5| Denhardt’s solution, 0.1 mg/ml denatured salmon SacII, and EcoRI/XhoI. The digested fragments were separated by sperm DNA) at 65uC overnight. Then the microarrays were washed agarose gel electrophoresis, after which bands ranging from 100 bp once with primary wash buffer 1 (2| SSC, 0.1% SDS) at room to 1.5 kb were excised from the gel. QIAquick gel extraction kits temperature to remove the cover glass, followed by 10 min with (QIAGEN, Valencia, CA) were used to elute and purify DNA from buffer 2 (2| SSC, 0.2% SDS prewarmed to 65uC), and finally 1% agarose gels. The genomic DNA fragments were inserted into a twice with wash buffer 3 (0.05| SSC) for 5 min; after this, the pPCR-Script Amp vector (Stratagene, La Jolla, CA) and then chips were dried by centrifugation at 2,200 | g at 4uC for 3 min. transformed into DH5a E. coli (RBC Bioscience, Taipei, Taiwan). All experiments were replicated at least three times. Clones were with amplified with a T3 to T7 promoter primer set. Amplified genomic DNA fragments were purified with a PCR Scanning and image analysis. After hybridization, all purification kit (QIAGEN) (4, 9). After purification, these genomic microarrays were scanned with a GenePix 4000B laser scanner DNA probes were sent to GenoCheck Co. (Gyeonggi, Korea), (Axon Instruments, Foster City, CA). The intensity values of where they were denatured and printed on amine-coated glass fluorescent signals from each spot were measured with GenePix slides in duplicate. In the DNA microarray chip assay, bacterio- Pro 6.0 software (Axon) and normalized by a correction factor phage integrase (MJ 1) gene (14) and peroxisome proliferator- calculated from an internal standard (c ~ Cy5-IS/Cy3-IS). activated receptor c gene (1) were used as positive and negative The intensity values of local background material were controls, respectively. The positive control gene was amplified subtracted from the normalized nonintensive values. To determine from WFC1 DNA with primers 59-cgggatccatgaaacgtgcagcattg-39 positive binding of target genomic DNA, the threshold values were and 59-cgggaattcaccgaatgcatgttcgta-39, and the negative control set up based on average intensity of the negative control and blank J. Food Prot., Vol. 74, No. 6 DETECTION OF ALICYCLOBACILLUS SPP. USING DNA CHIP 935 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/74/6/933/1685326/0362-028x_jfp-10-418.pdf by guest on 28 September 2021 FIGURE 2.
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