WO2005103294A1

WO2005103294A1 - Genomic microarray for detecting lactic acid bacteria and method for diagnosing lactic acid bacteria using it

Info

Publication number: WO2005103294A1
Application number: PCT/KR2005/000569
Authority: WO
Inventors: Yong-Ha Park; Jin-Woo Bae; Sung-Keun Rhee; Ja Ryeong Park; Byung-Chun Kim
Original assignee: Korea Research Institute Of Bioscience And Biotechnology
Priority date: 2004-04-24
Filing date: 2005-03-02
Publication date: 2005-11-03
Also published as: KR20050103085A

Abstract

Disclosed is a genomic microarray for detecting lactic acid bacteria using microarray techniques. The genomic microarray is capable of detecting the presence of lactic acid bacteria in a sample or identifying the bacteria to at least the species level when genomic DNA extracted directly from the sample is hybridized to the genomic microarray possessing genomic DNA from lactic acid bacteria as probes. Also, disclosed is a method of detecting lactic acid bacteria using the genomic microarray of lactic acid bacteria. The genomic microarray of lactic acid bacteria allows rapid detection and identification of lactic acid bacteria present in a variety of samples, including samples from human and animal gastrointestinal tracts, food samples and environmental samples, the lactic acid bacteria including lactic acid-producing bacteria, for example, Carnobacterium, Lactobaacillus, Lactococcus, Streptoccccus, Enterococcus, Oenococcus, Leuconostoc, Pediococcus, Bifidobacterium and Weissella, and propionic acid or citric acid-fermenting bacteria, for example, Prop.ionibacterium.

Description

GENOMIC MICROARRAY FOR DETECTING LACTIC ACID BACTERIA AND METHOD FOR DIAGNOSING LACTIC ACID BACTERIA USING IT

Technical Field

The present invention relates to a genomic microarray for detecting lactic acid bacteria and a method of detecting lactic acid bacteria using the same.

Background Art

Lactic acid bacteria are microbes producing lactic acid, which inhibits the growth of pathogens and harmful bacteria, by lactic acid fermentation, and are thus useful for producing various food items, including dairy products, kimchi and brewing products. Since lactic acid bacteria inhabit in the intestine of mammals and prevent abnormal fermentation by harmful bacteria, they are the most significant group of probiotic organisms. Many efforts have been made to detect or identify lactic acid bacteria in foods containing lactic acid bacteria, such as cheese, kimchi (spicy fermented cabbage) and wine, silage, or human feces. Over 100 different kinds of cheese are currently produced using lactic acid bacteria, including actococcus lactis subsp. lactis, Lac. lactis subsp. cremoris, Leuconostoc mesenteroides subsp. mesenteroid.es, Leuc. mesenteroides subsp. cremoris, Streptococcus salivarius subsp. thermophilus, and

Lactobacillus delbrueckii subsp. delbrueckii, lactis and bulgaricus. Cheese comes to have unique taste and flavor, which vary depending on the species of lactic acid bacteria and the kinds of raw milk. Fermented dairy products are manufactured by fermenting several kinds of animal milk using lactic acid- fermenting microbes as starters . Legal requirements for fermented dairy products vary according to nations. In Korea, they must contain over 3.0% non-fat milk solids and over 10 million viable lactic acid bacteria cells/ml. On the other hand, important factors in the manufacture of fermented dairy products are starter selection and inhibition of acidity increase during storage. In particular, since plain-type products are manufactured using only milk-derived components and their flavor and physical properties are thus determined by properties of starters used, vast time and labor are consumed screening suitable strains from nature. Recently, with advances in genetic recombination technologies, breeding efforts for starter strains have been made using molecular breeding based on genetic recombination techniques. In order to prevent the reduction of viable cells during storage and the increase of acidity of fermented dairy products, affecting the flavor of the fermented products, monitoring of starter strains injected at early stages is very important. Thus, lactic acid bacteria must be accurately detected in a short time. Another representative food containing lactic acid bacteria is kimchi. When raw materials for making kimchi are blended without particular starters, lactic acid bacteria grow rapidly. In kimchi, Pediococcus species are dominant at early stages of fermentation, and members of the genera Leuconostoc and Lactobacillus become dominant as fermentation progresses. It is necessary to construct a database including information for bacteria species contained in delicious kimchi by assessing such changes of lactic acid bacteria in kimchi, or to monitor changes in lactic acid bacteria through detection of lactic acid bacteria in kimchi for quality control of kimchi. It is also necessary to monitor lactic acid bacteria in the gastrointestinal tract of humans and animals . The small intestine and large intestine function to digest ingested food and absorb nutrients, as well as playing critical roles in immune function. When beneficial bacteria are dominant in the intestine, spoilage of ingested food is prevented, and immune function is activated, leading to the prevention of infectious diseases, suppression of aging and better health. Lactic acid bacteria as probiotics, used for such purpose, are marketed in various forms of health supplement foods in which the bacteria are used alone or in combination with other bacteria. After such lactic acid bacteria are ingested, bacteria populations resident in the intestinal tract of humans and animals should be accurately monitored and detected to evaluate their probiotic effects . Also, the use of lactic acid bacteria is required for quality improvement when silage (winter feed for dairy cows) and bread are made. In this case, changes in lactic acid bacteria should also be observed. Most conventional methods of identifying lactic acid bacteria include cultivating lactic acid bacteria in selective media for several days and observing grown bacteria. However, these cultivation-based methods have many limitations, including the followings. They may fail to identify lactic acid bacteria that are capable of growing in samples but cannot grow in media outside of the sample environment. Major bacterial identification errors may occur according to cultivation conditions. Also, bacterial identification by these methods is performed only to the species level . For more precise bacterial identification,, analysis of physiological and biochemical properties, 16S rDNA sequencing, SDS-PAGE of proteins, and randomly amplified polymorphic DNA fingerprinting have been used. However, these methods are time-consuming and labor-intensive. Recently, some attempts for rapid analysis of diversity of several microbial populations have been made using molecular biological methods . These methods are based on direct analysis of DNA in the environment and do not require cell cultivation. For example, in order to identify lactic acid bacteria in a cheese manufacturing process, Ogier et al. used TTGE (temporal temperature gradient gel electophoresis) that is based on electrophoretic separation of PCR fragments of 16S rRNA genes (Appl. Environ. Microbiol., 2002, Aug; 68(8): 3691-701), and another research group employed DGGE (Denaturing Gradient Gel Electrophoresis) (Hertel et. al., Berl Munch Tierarztl Wochenschr, 2003, Nov- Dec; 116(11-12): 517-23). Also, identification of lactic acid bacteria present in samples was performed based on PCR- RFLP (Restriction Fragment Length ^' Polymorphism) and FISH (fluorescence in situ hybridization) (Jang et. al., J. Microbiol Methods, 2003, Oct; 55(1): 295-302; Randazzo et. al., Int. J. Food Microbiol. , 2004, Jan., 1; 90(1): 9-14; and Blasco et. al., FEMS Microbiol. Lett. 2003, Aug., 8; 225(1): 115-23) . However, these methods have limited applications when one researcher simultaneously analyzes a plurality of samples. Thus, there is an urgent need for the development of a new technique in the post-genomic era. That is, too much time is required for performing DNA amplification, cloning and DNA sequencing for all samples, which range from hundreds to thousands in number. Also, most methods are dependent on PCR amplification of specific DNA from samples, which .may result in a very large deviation that leads to incorrect results (Cottrell et al., 2000; Speksnijder et al., 2001; Wang and Wang 1996; POLZ et al., 1998; Lueders et al., 2003; Ishii and Fukui 2001; Becker et al., 2000). One recently developed method overcoming these problems involves screening microorganisms in the environment using a microarray. DNA microarrays, which combine molecular biological knowledge with mechanical automation and electronic control techniques, contain hundreds to hundreds of thousands of DNA, which are deposited in a very small space. That is, a DNA microarray contains a massive population of DNA sequences that are attached in high density for gene search. As one of various microarrays, an array of multiple DNA molecules that are spotted on a substrate such as glass slides in a predetermined array is a technique that is very useful for simultaneously interpreting gene expression, variation, polymorphism, etc. Representative conventional genetic engineering techniques replicable with such microarrays include Southern blotting, Northern blotting, PCR, mutation search and DNA sequencing. The largest difference between these techniques and microarrays is that microarrays can simultaneously analyze at least hundreds of genes in a short time. Microarrays have another great advantage in that the composition of existing microorganisms can be accurately assessed by analyzing DNA in environmental samples without PCR amplification. Therefore, microarrays make it possible to rapidly, accurately analyze a plurality of samples at one time even with very small amounts of DNA in environmental samples because they allow high- density attachment of very small amounts of the genetic material. Most microarrays developed so far, in which genes of one individual are individually placed on each spot of a chip, are mainly used for analyzing the expression of intracellular genes in specific situations, or microbial functional genomics, for example, to study new metabolic pathways or regulatory networks. Recently, several research groups in the world apply basic knowledge and findings obtained from these studies to environmental microbiological monitoring, especially, microbial population analysis in environmental samples. One strategy for microbial population analysis includes constructing a phylogenetic framework based on nucleotide sequence information of SSϋ (small subunit) rDNA obtained from RDP DB (ribosomal database project database) , and designing oligonucleotide probes specific for each microorganism population based on nucleotide sequence conservation. This strategy has an advantage in that an array prepared by one event of designing and construction is applicable to other types of ecosystems and environments without cultivation of each microbial strain every time. However, this method has a significant drawback of having very low resolution. Taking into consideration that microorganisms are classified as different species in 98% sequence similarity levels, 50-mer oligo arrays have a difficulty in that each probe should express a different species due to a one-base difference. Also, the limitation of such oligomer chips is clearer taking into consideration that many important features of microorganisms, sucn as an ability to degrade pollutants, pathogenicity of strains and production of physiologically active substances or antibiotics, are determined below species levels. In fact, most current publications involving microbial analysis in the environment using microarray techniques only analyzed groups above species by PCR. In addition to microarrays, RSGP (Reverse Sample Genome Probing) , which is a technique for accurately identifying microorganisms in environmental samples, was developed by Voordouw (Appl Environ Microbiol. 1991 November 57 (11) : 3070-3078) . RSGP analysis is a method of analyzing metagenome extracted from the environment via Southern hybridization using a nitrocellulose filter on which the entire genome of each microorganism is attached. This technique has been reported to have a resolution allowing microbial identification in the environment at the subspecies level. However, because genomes should be obtained through cultivation, only slightly over 20 probes were reported to be prepared. Also, RSGP analysis is a representative macroarray method of over 100 cm², which requires a massive amount of environmental DNA every time probes are prepared. Thus, RSGP analysis is difficult to apply to practical analysis of environmental microorganisms because it is time-consuming, labor-intensive and requires expensive analysis instruments and high-cost probe preparation. Thus, in order to achieve the practical use of RSGP analysis, there is a need to develop RSGP via microarrays of lower than 1 cm² in order to greatly reduce the cost incurred by every analysis of DNA extraction from environmental samples and DNA labeling, and in order to establish rapid, accurate and large-scale analysis techniques overcoming the limits of oligomer chips. In microarrays using microbial genomes, resolution is determined by similarity between microbial genomes. At high levels of similarity, since it is difficult to find suitable conditions for obtaining a high resolution and cross-reactivity may occur upon hybridization and washing steps, a process and conditions of microarray preparation should be changed according to target analytes . In particular, unlike microbial analysis in general environments (e.g., soil) in which different types of microorganisms are present and microbial genomes to be analyzed do not have high similarity, in samples expected to contain lactic acid bacteria, types of microorganisms do not vary much, but high similarity may be present between existing microbial genomes. Unlike conventional nucleic acid microarrays using pure cultures, since samples containing lactic acid bacteria, for example, kimchi, cheese and samples from the gastrointestinal tract, contain many substances other than lactic acid bacteria, it is difficult to isolate pure genomes of lactic acid bacteria from such samples containing interfering substances, and in particular, extracted genomes are not easy to label when contaminated with sugars from samples . Therefore, there is a need for a method of rapidly and accurately detecting lactic acid bacteria in a variety of samples while saving time, effort and cost and overcoming high genomic similarity between lactic acid bacteria .

Disclosure of the Invention

It is therefore an object of the present invention to provide a genomic microarray of lactic acid bacteria, which is capable of overcoming low sensitivity occurring when oligomers present in trace amounts in the environment are detected through the use of whole genomes of lactic acid bacteria, has high specificity allowing microbial identification even at the subspecies level, does not need cost-, time- and labor-consuming probe preparation essential for the preparation of oligomer DNA chips and is thus cost-effective, and is capable of directly detecting lactic acid bacteria in environmental samples without DNA amplification due to its high sensitivity and high specificity. It is another object of the present invention to provide a method of preparing the genomic microarray of lactic acid bacteria. It is a further object of the present invention to provide a method of detecting lactic acid bacteria present in a sample using the genomic microarray of lactic acid bacteria.

Brief Description of the Drawings

The above and other objects, features and other advantages of the present invention will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which: FIG. 1 is a photograph of a microarray of the present invention, which has been printed with 616 (154x4) genome probes from 154 bacterial strains listed in Table 1, including lactic acid bacteria and Escherichia coli and Bacillus subtilis as reference strains, post-processed, and stained with a fluorescent dye, PicoGreen; FIG. 2 shows the results of hybridization of only the E. coli genome to a microarray of the present invention, displaying that only hybridization signals for the E. coli genome are produced with no cross-hybridization on the array; FIG. 3 shows the results of hybridization of only the

Enterococcus mundtii genome to a microarray of the present invention, displaying that only hybridization signals for the E. mundtii genome are produced with no cross- hybridization on the array; and FIG. 4 shows the results of hybridization of only the

Weissella confusa genome to a microarray of the present invention, displaying that only hybridization signals for the W. confusa genome are produced with no cross- hybridization on the array.

Best Mode for Carrying Out the Invention

In one aspect, the present invention relates to a genomic microarray for detecting lactic acid bacteria, which is printed with genomic DNA of lactic acid bacteria. The term "lactic acid bacteria", as used herein, refers to microorganisms that produce mainly lactic acid while producing other acids, such as acetic acid and propionic acid, by degrading carbohydrates such as glucose or lactose. Lactic acid bacteria morphologically belong to Gram-positive Bacillus or Micrococcus, and have physiological properties of being anaerobic and not producing catalase. In a preferred aspect, lactic acid bacteria include the genera Carnobacterium, Lactobacillus , Lactococcus _r Streptococcus, Enterococcus, Oenococcus, Leuconostoc Pediococcus , Bifidobacterium, - Weissella, and

Propioniba cteri um . Genomes printed onto the microarray of the present invention are used as probes . The whole genomic DNA of lactic acid bacteria, or a substantial portion thereof, for example, 95% or higher, preferably 97% or higher, and more preferably 99% or higher, may be used. This is because genomic DNA may be degraded by ultrasonic waves or physical force used in a genomic DNA extraction process. Since the microarray of the present invention is printed with genomic DNA as described above, a nucleic acid sequence required when a fragment is used does not need to be specified. The term "microarray", as used herein, refers to a one-dimensional or two-dimensional array that is divided into segments on a solid support to give separated regions having predetermined areas . Typically, a microarray means a biochip in which over thousands or tens of thousands of nucleic acids or proteins are arranged at regular intervals and which is capable of analyzing a target analyte to assess binding patterns thereof. Representative biochips are nucleic acid chips and protein chips . In the present invention, the biochip particularly indicates a nucleic acid chip, and the nucleic acid refers to particularly genomic DNA. In the present invention, a chip is interchangeably used with a microarray, and a nucleic acid microarray is interchangeably used with a genomic microarray. The density of a microarray is determined according to the total number of nucleic acids to be detected, which is located on the surface of a solid support, preferably over 50/cm², more preferably over 100 /cπX, and evern more preferably over 500/crX The position of each probe can be identified when the whole genomic DNA isolated from a variety of lactic acid bacteria or a substantial portion thereof is printed onto predetermined positions of the solid support of the present invention. Thus, by assessing the binding of DNA of the lactic acid bacteria to a specific position of the microarray, the presence of lactic acid bacteria in a sample may be detected, and the bacteria may be identified to at least the species level. The term "printing", as used herein, refers to the immobilization of genomes of a variety of lactic acid bacteria onto a microarray, and is used interchangeably with the term "spotting". Genomic DNA of one lactic acid bacterial species may be spotted at less than 5 positions, preferably 1 to 4 positions, onto the microarray of the present invention. The term "detection", as used herein, means to detect the presence of lactic acid bacteria in a sample and identify the types of bacteria when present in the sample. In another aspect, the present invention relates to a method of detecting lactic acid bacteria, comprising hybridizing labeled DNA derived from a sample containing lactic acid bacteria to the genomic microarray of lactic acid bacteria and detecting the presence of a hybridization signal . In detail, the method comprises hybridizing labeled DNA derived from a sample containing lactic acid bacteria to the genomic microarray of lactic acid bacteria; washing the microarray; detecting the presence of a hybridization signal; and identifying lactic acid bacteria at detected positions. The sample containing lactic acid bacteria includes all types of samples having the potential to contain lactic acid bacteria, for example, derived from environments, human and animal gastrointestinal tracts, water and foods. Examples of the samples include kimchi, yogurt, fermented milk, cheese, soy sauce, soybean paste, milk, butter, breads, silage, probiotics, sour milk, refined rice wine and ensilage. Also, when one or more strains or species of lactic acid bacteria are present in a single sample one or more strains or species of lactic acid bacteria may be detected. The term "hybridization", as used herein, refers to the base pairing between two complementary nucleic acid strands. Hybridization and hybridization strength are determined by the extent of complementarity between two nucleic acid strands, Tm of a formed hybrid, stringency of reaction conditions and GC content of nucleic acids. Genomic DNA from lactic acid bacteria may be extracted according to a known technique (Yoon, J. H. et.al., 1996, Identification of Saccharomonospora strains by the use of genomic DNA fragments, and rRNA gene probes.

Int. J. Syst. Bacteriol. 46:502-505). The present inventors obtained a plurality of lactic acid bacteria, which belong to the genera Carnobacterium, Lactobacillus, Streptococcus, Leuconostoc, Pediococcus,

Lactococcus, Bifidobacterium, Enterococcus, Oenococcus,

Weissella or Propionibacterium, through direct isolation in our lab or from a depositary authority, for example, Korean

Collection for Type Cultures (KCTC) , cultured the bacteria in suitable media under suitable culture conditions for each species, and extracted nucleic acids from the resulting bacterial cultures . Thus, in a preferred aspect, the lactic acid bacteria of the detection method include the genera Carnobacterium, Lactobacillus, Lactococcus, Streptococcus, Enterococcus,

Oenococcus, Leuconostoc, Pediococcus, Bifidobacterium,

Weissella and Propionibacterium. In detail, genomic DNA of each of the lactic acid bacteria listed in Table 1, below, which have been directly isolated in our lab or obtained from KCTC, are used as probes. In Table 1, along with serial numbers of probes, the serial number, KCTC number, Latin name, known G+C content, Genbank accession number and position on the microarray prepared in the present invention, for each bacterium, are summarized. The amount of genomes of lactic acid bacteria, spotted onto the microarray, is determined within the limited concentration range having good sensitivity by performing sensitivity experiments according to concentrations prior to application to the microarray, and for example, may range from about 100 to 200 ng/ l.

TABLE 1

^* Lactic acid bacteria isolated in our lab ^** Strains to be used as internal controls Anaerobic thermophilic isolates to be used as negative controls 16S rDNA of representative lactic acid bacteria and Escherichia coli to be used as a control for comparison with a conventional cDNA chip f ^"< Thus, in a more preferred aspect, in the detection method, the lactic acid bacteria include the lactic acid bacteria listed in Table 1. In the detection method using the genomic microarray of lactic acid bacteria according to the present invention, genomic DNA probes from lactic acid bacteria may be printed at less than 5 positions, and preferably 1 to 4 positions, onto the microarray. Since the present method of detecting lactic acid bacteria using the genomic microarray of lactic acid bacteria provides high sensitivity for microarray hybridization, it is able to identify and/or detect lactic acid bacteria even with a small amount of genomic DNA in a sample, preferably even with an amount less than 2.5 ng. In the present method for detecting the presence of lactic acid bacteria in a sample and identifying the kinds of lactic acid bacteria, DNA hybridized with genomic DNA spotted on a microarray may be prepared according to a typical DNA extraction method. A DNA sample as prepared above may be labeled to determine whether it hybridizes with a probe spotted on the microarray. DNA labeling may be achieved using a known technique. For example, genomic DNA or a portion thereof is mixed with random hexamers, a Cy3 or Cy5 fluorescent dye, etc., and the thus obtained genomic DNA or a portion thereof, labeled with the fluorescent substance, is denatured into a single-stranded form and is used for the hybridization reaction. The Cy3 or Cy5 fluorescent dye (Amersham Pharmacia, U.K.) is used for labeling genomic

DNA. Genomic DNA is labeled to determine gene hybridization using a certain difference in color of fluorescence. Cy3- dUTP or Cy5-dUTP is used herein, but Cy3-dCTP or Cy5-dCTP is also available. Hybridization of the microarray with DNA obtained from a sample and washing of the hybridized microarray may be performed under suitable conditions according to a general method with varying denaturing agents, temperatures, salt concentrations, and the like. Typically, as a denaturing agent in a hybridization buffer, formamide or dimethyl sulfoxide may be used. Sensitivity of hybridization generally increases as the concentration of the denaturing agent increases. 10 to 70%, preferably 30 to 50%, of formamide may be used herein. Hybridization temperature and salt concentrations in a washing solution may also be determined in a suitable range by taking hybridization sensitivity into consideration. Hybridization may be carried out at 15°C to 75°C, preferably 35°C to 55°C. Also, washing of bhe hybridized microarray may be done at a salt concentration of 0 to lx SSC, preferably 0.01 to O.lx SSC. Detection methods of hybridization between a sample and a probe may vary according to the methods of labeling DNA isolated from the sample. Typically, hybridization may be measured by washing a hybridized microarray to remove non-hybridized genes and analyzing hybridized genes with a high-resolution fluorescence scanner such as a laser fluorescence analyzer to detect and identify lactic acid bacteria. Laser induced fluorescence measurement employing fluorescent dyes is currently the most used detection method, and is advantageous in terms of overcoming detection limits caused by fluorescence and having relatively low background noise levels. A CCD or confocal laser may be used as a detection unit at a condensing part. In a detailed aspect, when nucleic acids derived from a sample are labeled with Cy3 or Cy5, whose maximum wavelengths of absorption/emission are 550/570 nm and 649/670 nm, respectively, and which fluoresce green and red, respectively, the detection is done by comparing intensities of fluorescence signals. Also, fluorescence signals and fluorescence signal intensities may be quantitatively measured via numerical computation. Microarray hybridization results may be obtained as images by scanning the surface of a slide glass with a fluorescence scanner. Since these images are output in BMP or TIFF formats from the scanner, the fluorescent signal intensity of each spot in these images is necessary for numerical computation using image processing software, which is supplied with the scanner, or other software. In a detailed embodiment of the present invention, genomic DNA derived from internal reference strains and negative control strains was spotted, along with 16S rDNA of representative lactic acid bacteria and Escherichia coli for comparison of the present genomic microarray with a conventional cDNA chip, onto the same microarray and evaluated for the degree of hybridization in order to determine the resolution of the present microarray for each bacterial strain. As internal reference strains, Thermoactinomyces intermedius, Thermoactinomyces sp. , Bacillus subtilis and Escherichia coli were selected, and were represented by probe numbers 144 to 147, respectively. As negative control strains, anaerobic thermophiles, for examples, Geobacillus sp. and a newly isolated anaerobic thermophile, were selected, and were represented by probe numbers 148 and 149, respectively. Also, in order to compare the microarray of the present invention and a conventional cDNA chip, 16S rDNA of Weissella confusa, Leuconostoc citreum, Lactobacillus sakei, Lactococcus plantarum and Escherichia coli were spotted (probe numbers 150 to 154, respectively) . As shown in FIG. 2, when only the Escherichia coli genome was allowed to react with the microarray, only hybridization signals for the E. coli genome were found with no cross-hybridization, while hybridization signals for genomic DNA of the negative control strains and other lactic acid bacteria were not observed. These results indicate that the microarray of the present invention has high resolution. In addition, as shown in FIGS. 3 and 4, when genomes from two species of lactic acid bacteria were tested for hybridization to the microarray of the present invention, they hybridized only to their complementary genomic probes without cross-hybridization, but produced signals for all 16S rDNA from four species of lactic acid bacteria, resulting in a high frequency of cross-hybridization. These results indicate that genomic DNA probes of the present invention hybridize specifically with their complementary genomic DNA, but that 16S rDNA probes react in a nonspecific manner. In a further aspect, the present invention relates to a method of preparing a genomic microarray of lactic acid bacteria, comprising extracting genomic DNA from a variety of lactic acid bacteria and spotting the multiple genomic DNA onto specific positions of a microarray support. In a preferred aspect, the lactic acid bacteria include the genera Carnobacterium, Lactobacillus, Lactococcus, Streptococcus, Enterococcus, Oenococcus , Leuconostoc, Pediococcus, Bifidobacterium, Weissella and Propionibacterium. Microarrays may be prepared by conventionally known methods, including microspotting using pins, microdropping using the inkjet principle, and electronic addressing using electric current, and preferably, the pin microarray technique, especially using slotted pins like a quill pen. Solid supports for microarray preparation are not particularly limited as long as they are available in hybridization, and typically include slide glasses, silicon chips, and nitrocellulose or nylon membranes. The support surface is any capable of immobilizing single-stranded or double-stranded nucleic acids by covalent or non-covalent bonding. Preferably, the support surface has a hydrophilic or hydrophobic functional group, whose examples include, but are not particularly limited to, hydroxyl, amino, thiol, aldehyde, carboxyl and acyl groups. These functional groups may be obtained with surface features of the support itself, and may be also introduced by surface treatment. Such surface-treated products are exemplified by glasses treated with a commercially available silane coupling agent such as aminoalkylsilane or with polycations such as polylysine or polyethyleneamine . Also, some slide glasses treated as described above are commercially available on the market. The genomic DNA of the present invention or a substantial portion thereof may be spotted onto a slide glass as mentioned above using a microarray machine, and UV light may be radiated onto the slide glass to immobilize probes on the glass through cross-linking, followed by long-term incubation under suitably controlled humidity. A better understanding of the present invention may be obtained through the following examples which are set forth to illustrate, but are not to be construed as the limit of the present invention.

EXAMPLE 1: Microarray construction and post-processing Genomic DNA was extracted from 154 bacterial strains to be used as probes, including lactic acid bacteria, E. coli and Bacillus subtilis and listed in Table 1 (Yoon, J.H. et. al., 1996, Identification of Saccharomonospora strains by the use of genomic DNA fragments and rRNA gene probes. Int. J. Syst. Bacteriol. 46:502-505). Herein, four sets for each genomic DNA sample, that is, a total of 616 (154x4) probe samples, were prepared. The genomic DNA was then treated with RNase A. Then, the genomic DNA was diluted in a diluting solution containing 50% dimethyl sulfoxide as a DNA denaturing reagent to a final concentration of 200 ng/ml .

10 μl of each sample was transferred into a 384-well microplate for printing. DNA probe .samples were spotted onto 25x75 mm silicon-coated glass slides (Corning^®, UltraGAPS™) with a single pin using a robotic printer, and their positions are given in Table 1. 154 different probes were printed in quadruplets. The DNA on the microarrays was more tightly fixed by UN cross-linking at 120 mJ, and the slides were allowed to react with 0.17 M succinic anhydride dissolved in a mixture of 240 ml l-methyl-2-pyrrolidinone and 10.7 ml 1 M boric acid so as to inactivate residues unbound to DΝA. Immediately after inactivation, DΝA was denatured by boiling for 2 min. The microarrays were briefly immersed in pre-cooled 95% ethanol, air-dried at room temperature and stored in the dark. To evaluate the quality of printing, a slide printed according to the same procedure was stained for 30 min in a PicoGreen solution, which had been 1:200 diluted in lx TE buffer. The slides were then washed sequentially in lx TE, 0.5x TE and sterile water for 1 min each, and were scanned with an Axon GenePix 4000B Microarray Scanner. The scanning results are given in FIG. 1.

EXAMPLE 2: Preparation of fluorescently labeled DNA Genomic DNA was extracted from two members of lactic acid bacteria, Enterococcus mundtii and Weissella confusa, and Escherichia coli as a reference strain. DNA samples to be reacted in Example 1 were prepared according to a direct labeling procedure. 1 μg of genomic DNA was denatured by boiling for 2 min and immediately chilled on ice. Each 40-μl fluorescence labeling reaction mixture contained the denatured genomic DNA, 1.5 g of random hexamers, lx EcoPol buffer (5 mM dATP, dCTP and dGTP; 2.5 mM dUTP; 5 mM Cy3-dUTP or Cy5-dUTP) , 2.5 mM dithiothreitol, and 10 U of the large Klenow fragment of DNA polymerase I. The reaction mixture was incubated at 37°C for 2 hrs, boiled in a heating block for 3 min, and immediately chilled on ice. The fluorescently labeled DNA was purified with a QIAquick PCR purification column, concentrated in a Speedvac at 40°C for 1 hr 30 min, and resuspended in 10 μl of distilled water.

EXAMPLE 3: Microarray hybridization In order to determine the specificity of microarray hybridization for each bacterial strain, microbial genomes were mixed at various concentrations and hybridized on a microarray. Microarray hybridizations were carried out in triplicate (a total of twelve replicates per gene probe) . Fluorescently labeled DNA was denatured by boiling for 2 min, deposited onto coverslips by pipetting and added to a hybridization solution. The hybridization solution contained 3x SSC (lx SSC contained 150 mM NaCl and 15 mM trisodium citrate) , lg of unlabeled herring sperm DNA and 0.3% SDS in a total standard volume of 7.5 μl. 50% formamide was used as a denaturing agent. The microarray slide, maintained at a reaction temperature of 37°C, was placed printed side down on the coverslip, and then placed into a waterproof hybridization chamber. Hybridization reactions were carried out at a temperature ranging from room temperature to 55°C for 12 to 15 hrs. After hybridization, the slides were washed with lx SSC, 0.2% SDS and O.lx SSC, and 0.2% SDS for 5 min each, finally washed with O.lx SSC for 30 sec, and dried in the dark. EXAMPLE 4 : Microarray scanning and quantitative analysis of hybridization signals

Hybridized microarrays were scanned initially at a resolution of 50 μm to obtain a quick display image and then at 5 μm using a scanning laser confocal fluorescence microscope (ScanArray 5000 System) . The emitted fluorescence signals were detected by a photomultiplier tube (PMT) at 532 nm for Cy3 or 635 nm for Cy5. For more accurate and sensitive analysis of samples, the laser power and PMT gain were both 100%. For all other microarray experiments, the laser power was 95% and the PMT gain was 90%. The scanned images were saved as 16-bit TIFF files and analyzed by quantifying the pixel density (intensity) of each hybridization spot using the Axon GenePix Software 4.1. The data from the software were then exported to Excel for further processing. The local background signal was subtracted automatically from the hybridization signal of each separate spot. Fluorescence intensity values for the negative controls were averaged and then subtracted from the final values for each hybridization signal. Statistical analysis was performed using SigmaPlot 5.0 software (Jandel Scientific). The results are given in FIGS. 2 to 4. As shown in FIG. 2, when only the Escherichia coli genome was allowed to react with the microarray, only hybridization signals for the E. coli genome were found, with no cross-hybridization. As shown in FIG. 3, when only the Enterococcus mundtii genome was hybridized to the microarray, only hybridization signals for the E. mundtii genome were produced on the array, with no cross- hybridization. Simultaneously, hybridization signals were observed for printed 16S rDNA of Lactobacillus sakei, Weissella confusa, Lactococcus plantarum and Leuconostoc citreum. These results demonstrate that 16S rDNA-based cDNA chips have reduced specificity for detection of lactic acid bacteria due to high cross-hybridization. Thus, these results indicate that the genomic microarray of the present invention has high specificity. Also, as shown in FIG. 4, when only the Weissella confusa genome was hybridized to the microarray, only hybridization signals for the W. confusa genome were observed on the array, with no cross- hybridization. In this case, like the case of FIG. 3, hybridization signals were observed for 16S rDNA of the four members of lactic acid bacteria.

Industrial Applicability

The microbial genome microarray of the present invention using lactic acid bacteria has many advantages because it uses whole genomes of lactic acid bacteria, as follows. Unlike conventional oligomer microarrays, the present genomic microarray is capable of remedying low sensitivity occurring when oligomers present in trace amounts in the environment are detected, and has excellent accuracy. Also, the present genomic microarray has high resolution allowing bacterial identification even at the subspecies level, and requires much shorter detection time. The present genomic microarray provides additional advantages of being cost-effective because it does not need cost-, time- and labor-consuming probe preparation essential for oligomer microarray construction, and of allowing direct detection of lactic acid bacteria in environmental samples without DNA amplification due to its high sensitivity and high specificity. The present microarray and method using the microarray, for detecting and/or identifying lactic acid bacteria, allow rapid, accurate and economic large-scale performance of distribution studies of lactic acid bacteria in clinically important human and animal gastrointestinal tracts or industrially important food items, such as yogurts or kimchi, from very small samples.

Claims

1. A genomic microarray of lactic acid bacteria for detecting lactic acid bacteria, which is printed with genomic DNA probes from lactic acid bacteria comprising the genera Carnobacterium, Lactobacillus, Lactococcus,

Streptococcus, Enterococcus, Oenococcus, Leuconostoc, Pediococcus, Bifidobacterium, Weissella and

Propioniba cterium.

2 . The genomic microarray of lactic acid bacteria according to claim 1, wherein the lactic acid bacteria are selected from the group consisting of bacteria listed in the following table.

Bifidobacterium angulatum Bifidobacterium bifidum Bifidobacterium cor neforme Bifidobacterium magnum Bifidobacterium minimum Bifidobacterium saeculare Carnobacterium divergens Carnobacterium piscicola Enterococcus cecorum Enterococcus durans Enterococcus faecium Enterococcus fiavescens Enterococcus haemoperoxidus Enterococcus hirae Enterococcus malodoratus Enterococcus moraviensis Enterococcus mundtii Enterococcus phoeniculicola Enterococcus saccharofyticus Enterococcus solitarius Enterococcus xilllorum Lactobacillus acidophilus Lactobacillus agilis Lactobacillus aiimentarius Lactobacillus amylophilus Lactobacillus amylovorus Lactobacillus animalis Lactobacillus brevis Lactobacillus casei Lactobacillus coryniformis subsp. coryniformis

Lactobacillus coryniformis subsp. torquens Lactobacillus crispatus Lactobacillus curvatus Lactobacillus cypricasei

Lactobacillus delbrueckii subsp. bulgaricus Lactobacillus delbrueckii subsp. delbrueckii Lactobacillus delbrueckii subsp. lactis Lactobacillus diolivorans Lactobacillus equi Lactobacillus farciminis Lactobacillus fer entum Lactobacillus fructivorans Lactobacillus fuchuensis Lactobacillus graminis Lactobacillus helveticus Lactobacillus ingluviei Lactobacillus jensenii Lactobacillus johnsonii Lactobacillus kefiri Lactobacillus kimchi Lactobacillus malefermentans Lactobacillus mali Lactobacillus maltaromicus Lactobacillus urinus Lactobacillus oris Lactobacillus parabuchneri Lactobacillus paracasei subsp. paracasei Lactobacillus paracasei subsp. tolerans Lactobacillus pentosus Lactobacillus plantarum Lactobacillus reuteri Lactobacillus rhamnosus Lactobacillus ruminis Lactobacillus sakei subsp. carnosus Lactobacillus sakei subsp. sakei Lactobacillus salivarius subsp. salicinius Lactobacillus sanfranciscensis Lactobacillus sharpeae Lactobacillus suebicus Lactobacillus vaccinostercus Lactobacillus vaginatis Lactobacillus versmoldensis Lactobacillus vitulinus Lactobacillus zeae Lactococcus garvieae Lactococcus lactis subsp. hordniae Lactococcus lactis subsp. lactis Lactococcus piscium Lactococcus plantarum Lactococcus rafftnolactis Leuconostoc argentinum Leuconostoc carnosum Leuconostoc citreum Leuconostoc citreum Leuconostoc fallax Leuconostoc fructosum Leuconostoc gasicomitatum Leuconostoc gelidum Leuconostoc inhae Leuconostoc lactis

Leuconostoc mesenteroides subsp. cremoris Leuconostoc mesenteroides subsp. dextranicum Leuconostoc mesenteroides subsp. mesenteroides Leuconostoc pseudomesenteroides Oenococcus oeni Pediococcus acidilactici Pediococcus claussenii Pediococcus damnosus Pediococcus dextrinicus Pediococcus inopinatus Pediococcus parvulus Pediococcus pentosaceus Pediococcus urinaeequi Propionibacterium freudenreichii subsp. freudenreichii Streptococcus alactolyticus Streptococcus anginosus Streptococcus australis Streptococcus criceti Streptococcus downei Streptococcus entericus

Streptococcus equi subsp. zooepidemicus Streptococcus ferus Streptococcus gordonii Streptococcus gordonii Streptococcus hyointestinalis Streptococcus iniae Streptococcus mitis Streptococcus mutans Streptococcus pyogenes Streptococcus ratti Streptococcus sanguinis Streptococcus sobrinus Streptococcus suis Streptococcus thermophilus Streptococcus vestibularis Weissella cibaria Weissella confusa Weissella halotolerans Weissella hanii Weissella hellenica Weissella kandleri Weissella kimchii Weissella minor Weissella paramesenteroides Weissella soli Weissella thailandensis Weissella viridescens Lactobacillus sakei Leuconostoc carnosum Leuconostoc gasicomitatum Leuconostoc gasicomitatum Weissella koreensis Leuconostoc gelidum

3. A method of detecting lactic acid bacteria, comprising hybridizing labeled DNA derived from a sample containing lactic acid bacteria to the genomic microarray of lactic acid bacteria according to claim 1 and detecting the presence of a hybridization signal.

4. The method according to claim 3, wherein the lactic acid bacteria are selected from the group consisting of bacteria listed in the following table.

Bifidobacterium angulalum Bifidobacterium bifidum Bifidobacterium coryneforme Bifidobacterium magnum Bifidobacterium minimum Bifidobacterium saeculare Carnobacterium divergens Carnobacterium piscicola Enterococcus cecorum Enterococcus durans Enterococcus faecium Enterococcus ficrvescens Enterococcus haemoperoxidus Enterococcus hirae Enterococcus malodoratus Enterococcus moraviensls Enterococcus mundtii Enterococcus phoeniculicola Enterococcus saccharolyticus Enterococcus solitarius Enterococcus villorum Lactobacillus acidophilus Lactobacillus agilis Lactobacillus alimentarius Lactobacillus amylophilus Lactobacillus amylovorus Lactobacillus animalis Lactobacillus brevis Lactobacillus casei Lactobacillus coryniformis subsp. coryniformis Lactobacillus coryniformis subsp. torquens Lactobacillus crispatus Lactobacillus curvatus Lactobacillus cypric s i

Lactobacillus delbrueckii subsp. bulgaricus Lactobacillus delbrueckii subsp. delbrueckii Lactobacillus delbrueckii subsp. lactis Lactobacillus diolivorans Lactobacillus equi Lactobacillus farciminis Lactobacillus fermentum — Lactobacillus fructivorans Lactobacillus fuchuensis Lactobacillus graminis Lactobacillus helveticus Lactobacillus inglitviei Lactobacillus jensenii Lactobacillus johnsonii Lactobacillus kefiri Lactobacillus kimchi Lactobacillus malefermenta s Lactobacillus mali Lactobacillus maltaramicus Lactobacillus murinus Lactobacillus oris Lactobacillus parabuchneri Lactobacillus paracasei subsp. paracasei Lactobacillus paracasei subsp. tolerans Lactobacillus pentosus Lactobacillus plantarum Lactobacillus reuteri Lactobacillus rhamnosus Lactobacillus ruminis Lactobacillus sakei subsp. carnosus Lactobacillus sakei subsp. sakei Lactobacillus salivarius subsp. salicinius Lactobacillus sanfranciscensis Lactobacillus sharpeae Lactobacillus suebicus Lactobacillus vaccinostercus Lactobacillus vaginalis Lactobacillus versmoldensis Lactobacillus vitulinus Lactobacillus zeae Lactococcus garvieae Lactococcus lactis subsp. hordniae Lactococcus lactis subsp. lactis Lactococcus piscium Lactococcus plantarum Lactococcus rafjfϊnolactis Leuconostoc argentinum Leuconostoc carnosum Leuconostoc citreum Leuconostoc citreum Leuconostoc fallax Leuconostoc fructosum Leuconostoc gasicomitatum Leuconostoc gelidum Leuconostoc inhae Leuconostoc lactis

Leuconostoc mesenteroides subsp. cremoris Leuconostoc mesenteroides subsp. dextranicum Leuconostoc mesenteroides subsp. mesenteroides Leuconostoc pseudomesenteroides Oenococcus oeni Pediococcus acidilactici Pediococcus claussenii Pediococcus damnosus Pediococcus dextrinicus Pediococcus inopinatus Pediococcus parvulus Pediococcus pentosaceus Pediococcus urinaeequi Propionibacterium freudenreichii subsp. freudenreichii Streptococcus alactolyticus Streptococcus anginosus Streptococcus australis Streptococcus criceli Streptococcus downei Streptococcus entericus Streptococcus equi subsp. zooepidemicus Streptococcus ferus Streptococcus gordonii Streptococcus gordonii Streptococcus hyointestinalis Streptococcus iniae Streptococcus mitis Streptococcus mutans Streptococcus pyogenes Streptococcus ratti Streptococcus sanguinis Streptococcus _spbrinus_ Streptococcus suis Streptococcus thermophilus Streptococcus vestibularis Weissella cibaria Weissella confusa Weissella halotolerans Weissella hanii Weissella hellenica Weissella kandleri Weissella kimchii Weissella minor Weissella paramesenteroides Weissella soli Weissella thailandensis Weissella viridescens Lactobacillus sakei Leuconostoc carnosum Leuconostoc gasicomitatum Leuconostoc gasicomitatum Weissella koree sis Leuconostoc gelidum

5. The method according to claim 3 or 4, wherein the sample containing lactic acid bacteria is selected from among kimchi, yogurt, fermented milk, cheese, soy sauce, soybean paste, milk, butter, breads, silage, probiotics, sour milk, refined rice wine and ensilage.