WO2005103294A1 - Micro-reseau genomique pour detecter des bacteries d'acide lactique et methode pour diagnostiquer des bacteries d'acide lactique faisant appel a ce micro-reseau - Google Patents

Micro-reseau genomique pour detecter des bacteries d'acide lactique et methode pour diagnostiquer des bacteries d'acide lactique faisant appel a ce micro-reseau Download PDF

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WO2005103294A1
WO2005103294A1 PCT/KR2005/000569 KR2005000569W WO2005103294A1 WO 2005103294 A1 WO2005103294 A1 WO 2005103294A1 KR 2005000569 W KR2005000569 W KR 2005000569W WO 2005103294 A1 WO2005103294 A1 WO 2005103294A1
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lactobacillus
streptococcus
leuconostoc
lactic acid
subsp
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Yong-Ha Park
Jin-Woo Bae
Sung-Keun Rhee
Ja Ryeong Park
Byung-Chun Kim
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Korea Research Institute of Bioscience and Biotechnology KRIBB
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Korea Research Institute of Bioscience and Biotechnology KRIBB
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms

Definitions

  • the present invention relates to a genomic microarray for detecting lactic acid bacteria and a method of detecting lactic acid bacteria using the same.
  • 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
  • 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.
  • important factors in the manufacture of fermented dairy products are starter selection and inhibition of acidity increase during storage.
  • 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.
  • Lactic acid bacteria as probiotics 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.
  • identifying lactic acid bacteria include cultivating lactic acid bacteria in selective media for several days and observing grown bacteria.
  • 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.
  • 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 .
  • TTGE temporary temperature gradient gel electophoresis
  • DGGE Denaturing Gradient Gel Electrophoresis
  • 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.
  • 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.
  • 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.
  • One strategy for microbial population analysis includes constructing a phylogenetic framework based on nucleotide sequence information of SS ⁇ (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.
  • this method has a significant drawback of having very low resolution.
  • oligo arrays 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.
  • RSGP Reverse Sample Genome Probing
  • 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.
  • RSGP analysis is a representative macroarray method of over 100 cm 2 , which requires a massive amount of environmental DNA every time probes are prepared.
  • 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.
  • resolution is determined by similarity between microbial genomes.
  • a process and conditions of microarray preparation should be changed according to target analytes .
  • target analytes 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.
  • 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 .
  • 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.
  • the present invention relates to a genomic microarray for detecting lactic acid bacteria, which is printed with genomic DNA of lactic acid bacteria.
  • lactic acid bacteria 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.
  • lactic acid bacteria include the genera Carnobacterium, Lactobacillus , Lactococcus r Streptococcus, Enterococcus, Oenococcus, Leuconostoc Pediococcus , Bifidobacterium, - Weissella, and
  • 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 .
  • 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 .
  • the biochip particularly indicates a nucleic acid chip, and the nucleic acid refers to particularly genomic DNA.
  • 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/cm 2 , more preferably over 100 /c ⁇ X, 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.
  • 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.
  • detection means to detect the presence of lactic acid bacteria in a sample and identify the types of bacteria when present in the sample.
  • 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 .
  • 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.
  • hybridization 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.
  • the present inventors obtained a plurality of lactic acid bacteria, which belong to the genera Carnobacterium, Lactobacillus, Streptococcus, Leuconostoc, Pediococcus,
  • Lactococcus Bifidobacterium, Enterococcus, Oenococcus,
  • the lactic acid bacteria of the detection method include the genera Carnobacterium, Lactobacillus, Lactococcus, Streptococcus, Enterococcus,
  • 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.
  • the lactic acid bacteria include the lactic acid bacteria listed in Table 1.
  • genomic DNA probes from lactic acid bacteria may be printed at less than 5 positions, and preferably 1 to 4 positions, onto the microarray.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Thermoactinomyces intermedius, Thermoactinomyces sp. , Bacillus subtilis and Escherichia coli were selected, and were represented by probe numbers 144 to 147, respectively.
  • negative control strains anaerobic thermophiles, for examples, Geobacillus sp.
  • microarray of the present invention has high resolution.
  • 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.
  • genomic DNA probes of the present invention hybridize specifically with their complementary genomic DNA, but that 16S rDNA probes react in a nonspecific manner.
  • 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.
  • 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.
  • 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 .
  • silane coupling agent such as aminoalkylsilane
  • polycations such as polylysine or polyethyleneamine
  • 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.
  • 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.
  • 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).
  • 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.
  • 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 .
  • DNA probe .samples were spotted onto 25x75 mm silicon-coated glass slides (Corning ® , UltraGAPSTM) 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 D ⁇ A.
  • 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 ⁇ g of genomic DNA was denatured by boiling for 2 min and immediately chilled on ice.
  • Each 40- ⁇ l 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 ⁇ l of distilled water.
  • EXAMPLE 3 Microarray hybridization
  • 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 ⁇ l.
  • Hybridized microarrays were scanned initially at a resolution of 50 ⁇ m to obtain a quick display image and then at 5 ⁇ m 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.
  • PMT photomultiplier tube
  • the laser power and PMT gain were both 100%.
  • 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 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.

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Abstract

L'invention concerne un micro-réseau génomique pour détecter des bactéries d'acide lactique au moyen de techniques de micro-réseau. Le micro-réseau génomique de l'invention permet de détecter la présence de bactéries d'acide lactique dans un échantillon, ou d'identifier ces bactéries sur au moins un niveau d'espèce, lorsque l'ADN génomique extrait directement de l'échantillon est hybridé, en tant que sondes, sur le micro-réseau génomique présentant l'ADN génomique provenant des bactéries d'acide lactique. L'invention concerne également une méthode de détection de bactéries d'acide lactique faisant appel au micro-réseau génomique des bactéries d'acide lactique. Le micro-réseau génomique de bactéries d'acide lactique permet une détection et une identification rapide des bactéries d'acide lactique présentes dans une variété d'échantillons, notamment des échantillons provenant de voies gastro-intestinales humaines et animales, d'échantillons alimentaires et d'échantillons environnementaux, les bactéries d'acide lactique comprenant des bactéries de production d'acide, par exemple Carnobacterium, Lactobacillus, Lactococcus, Streptoccccus, Enterococcus, Oenococcus, Leuconostoc, Pediococcus, Bifidobacterium et Weissella, et de l'acide propionique ou des bactéries de fermentation d'acide citrique, par exemple, Propionibacterium.
PCT/KR2005/000569 2004-04-24 2005-03-02 Micro-reseau genomique pour detecter des bacteries d'acide lactique et methode pour diagnostiquer des bacteries d'acide lactique faisant appel a ce micro-reseau Ceased WO2005103294A1 (fr)

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Cited By (6)

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EP2096180A1 (fr) * 2008-02-27 2009-09-02 Institut National de la Recherche Agronomique (INRA) Combinaison de gènes marqueurs pour caractériser une souche de Lactobacillus sakei
WO2011023675A1 (fr) * 2009-08-27 2011-03-03 Institut National De La Recherche Agronomique (Inra) Association de marqueurs adn pour la caractérisation et la cartographie peptidique d'un isolat de lactobacillus sakei
CN112029885A (zh) * 2020-09-29 2020-12-04 广东省微生物研究所（广东省微生物分析检测中心） 用于鉴定瑞士乳杆菌、发酵乳杆菌和嗜酸乳杆菌的分子标记、检测引物和检测方法
CN114540234A (zh) * 2022-03-02 2022-05-27 西南民族大学 一种戊糖乳杆菌及其在抗冻融青贮中的应用
CN116836860A (zh) * 2023-06-28 2023-10-03 云南大学 泰国魏斯氏菌baz54菌株及其在降解生物胺中的应用
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EP2096180A1 (fr) * 2008-02-27 2009-09-02 Institut National de la Recherche Agronomique (INRA) Combinaison de gènes marqueurs pour caractériser une souche de Lactobacillus sakei
WO2009106491A1 (fr) * 2008-02-27 2009-09-03 Institut National De La Recherche Agronomique (Inra) Combinaison de gènes marqueurs pour la caractérisation d'une souche lactobacillus sakei
JP2011524158A (ja) * 2008-02-27 2011-09-01 アンスティテュ ナシオナル ドゥ ラ ルシェルシュ アグロノミック （インラ） Ｌａｃｔｏｂａｃｉｌｌｕｓｓａｋｅｉ株を特徴付けするためのマーカー遺伝子の組み合わせ
US8329402B2 (en) 2008-02-27 2012-12-11 Institut National De La Recherche Agronomique Combination of marker genes for characterizing a Lactobacillus sakei strain
WO2011023675A1 (fr) * 2009-08-27 2011-03-03 Institut National De La Recherche Agronomique (Inra) Association de marqueurs adn pour la caractérisation et la cartographie peptidique d'un isolat de lactobacillus sakei
EP2292800A1 (fr) * 2009-08-27 2011-03-09 Institut National De La Recherche Agronomique (INRA) Combinaison de marqueurs d'ADN pour caractériser et cartographier un isolat de Lactobacillus sakei
CN112029885A (zh) * 2020-09-29 2020-12-04 广东省微生物研究所（广东省微生物分析检测中心） 用于鉴定瑞士乳杆菌、发酵乳杆菌和嗜酸乳杆菌的分子标记、检测引物和检测方法
CN112029885B (zh) * 2020-09-29 2022-06-07 广东省微生物研究所（广东省微生物分析检测中心） 用于鉴定瑞士乳杆菌、发酵乳杆菌和嗜酸乳杆菌的分子标记、检测引物和检测方法
US20240150815A1 (en) * 2021-02-26 2024-05-09 Yokogawa Electric Corporation Measurement method and measurement system
EP4299713A4 (fr) * 2021-02-26 2025-01-22 Yokogawa Electric Corporation Procédé de mesure et système de mesure
CN114540234A (zh) * 2022-03-02 2022-05-27 西南民族大学 一种戊糖乳杆菌及其在抗冻融青贮中的应用
CN114540234B (zh) * 2022-03-02 2023-05-16 西南民族大学 一种戊糖乳杆菌及其在抗冻融青贮中的应用
CN116836860A (zh) * 2023-06-28 2023-10-03 云南大学 泰国魏斯氏菌baz54菌株及其在降解生物胺中的应用

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