WO2010060262A1 - Gene chip and kit for detecting important pathogenic bacterium in aquatic products - Google Patents

Gene chip and kit for detecting important pathogenic bacterium in aquatic products Download PDF

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Publication number
WO2010060262A1
WO2010060262A1 PCT/CN2009/001190 CN2009001190W WO2010060262A1 WO 2010060262 A1 WO2010060262 A1 WO 2010060262A1 CN 2009001190 W CN2009001190 W CN 2009001190W WO 2010060262 A1 WO2010060262 A1 WO 2010060262A1
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Prior art keywords
gene chip
proteus
probe
dna sequence
seq
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PCT/CN2009/001190
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French (fr)
Chinese (zh)
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王磊
冯露
曹勃阳
王敏
刘蕾
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天津生物芯片技术有限责任公司
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Publication of WO2010060262A1 publication Critical patent/WO2010060262A1/en

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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
    • C12Q1/6876—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
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/00029—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor provided with flat sample substrates, e.g. slides
    • G01N2035/00099—Characterised by type of test elements
    • G01N2035/00158—Elements containing microarrays, i.e. "biochip"
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to a gene chip and a kit for detection, and more particularly to a gene chip and a kit for detecting important pathogenic bacteria in aquatic products.
  • Food quality and food safety are related to human health and are therefore highly valued by countries all over the world.
  • Foodborne diseases caused by microorganisms are a major problem in food safety.
  • Foodborne diseases caused by bacterial contamination are the most prominent problems in food safety in China.
  • pathogenic bacteria there are two main types of pathogenic bacteria in aquatic products: one is its own pathogenic bacteria, which are widely distributed in water, such as cold-sourced bacteria, Listeria monocytogenes, thermophilic bacteria. Such as Vibrio parahaemolyticus and Vibrio cholerae; Second, non-self pathogenic bacteria, that is, contaminated pathogenic bacteria in the production process, mainly in the water environment of human and animal intestines and contaminated by human or animal feces, common Including Salmonella, Shigella, Staphylococcus aureus, etc. (Yang Wenge, Sun Cuiling, Pan Yunqi, et al. Rapid detection methods for pathogenic microorganisms in aquatic products. Chinese Journal of Food Science,
  • the detection range of the bacteria detection chip is the following 10 common bacteria: S.
  • Shigella including Shigella flexneri, Shigella sonnei, Shigella flexneri and Shigella dysenteriae A total of 4 species
  • Salmonella Staphylococcus aureus, Vibrio parahaemolyticus, Vibrio cholerae, Listeria monocytogenes, Proteus mirabilis, Proteus panicula and Proteus vulgaris.
  • the most commonly used target molecules for microbial identification are 16S rRNA and 23S rRNA, which have been reported in many countries. Bacterial microorganisms can be identified to species or genera using 16S rRNA and 23S rRNA, but identification of closely related bacterial species or genera is difficult (Bodrossy L, Sessitsch A. 2004. Oligonucleotide microarrays in microbial diagnostics. Current Opinion in Microbiology 7:245-25). At present, the identification of bacteria using the 16S-23S rRNA inter-region as a target molecule has gradually become a research hotspot (Nubel U, Schmidt PM, ReiB E, et al. 2004.
  • FEMS Microbiology Letters 240: 215-223. which has a mutation rate equivalent to ten times that of 16S rRNA or 23S rRNA, so it has higher resolution and can even distinguish bacteria into types, for close relatives.
  • the species distinction has the advantage that 16S rRNA and 23S rRNA are incomparable.
  • both ends are conserved 16S rRNA gene and 23S rRNA gene region. Designing universal primers in conserved regions at both ends can avoid the problem of primer dimers brought by multiple pairs of primers, ranging from 200 bp to 1000 bp.
  • One object of the present invention is to provide a gene chip for detecting important pathogenic bacteria in aquatic products, so as to make up for the time-consuming and labor-intensive defects of traditional common aquatic product pathogenic bacteria detection technology, expand the detection range of pathogenic bacteria, and improve detection sensitivity. And specificity, reduce labor intensity, and shorten the detection cycle.
  • the gene chip for detecting important pathogenic bacteria in aquatic products includes a solid phase carrier and An oligonucleotide probe immobilized on the solid phase support, wherein the oligonucleotide probe immobilized on the solid phase carrier comprises one or more selected from the following sequences:
  • the oligonucleotide probe immobilized on the solid phase carrier has one or more of the DNA sequences shown in SEQ ID NO: 2 - SEQ ID NO: 26.
  • the gene chip of the present invention further comprises a positive control probe, a negative control probe or a fluorescent
  • the positive control probe is selected from a DNA fragment in a bacterial 16S rDNA conservation region or a complementary DNA or RNA sequence thereof.
  • the positive control probe has the DNA sequence set forth in SEQ ID NO: 1.
  • the invention also provides the application of the gene chip, mainly for detecting Shigella, Salmonella, Vibrio parahaemolyticus, Vibrio cholerae, Listeria monocytogenes, Staphylococcus aureus, Streptococcus pyogenes, singularity Use of at least one pathogenic bacteria in Proteus, Oryzae, and Proteus panicula.
  • the detection primer is used.
  • the detection primer has at least one of the DNA sequence shown in SEQ ID NO: 27-SEQ ID NO: 30 or a complementary sequence thereof.
  • the invention also provides a kit comprising the gene chip as described above.
  • the kit of the present invention further comprises a detection primer.
  • the detection primer has at least one of the DNA sequence represented by SEQ ID NO: 27-SEQ ID NO: 30 or a complementary sequence thereof. kind.
  • the invention also provides the application of the above kit, which is mainly for detecting Shigella and Shamen Application of at least one pathogenic bacteria in bacteria, Vibrio parahaemolyticus, Vibrio cholerae, Listeria monocytogenes, Staphylococcus aureus, Streptococcus pyogenes, Proteus mirabilis, Proteus vulgaris, Proteus paniculata .
  • the present invention introduces gene chip technology into the field of important pathogenic bacteria detection in aquatic products for the first time and simultaneously detects 10 types of pathogenic bacteria, and establishes a brand new type which is fast, sensitive, accurate and reproducible.
  • the gene chip for detecting important pathogenic bacteria in aquatic products and the detection method thereof can use the gene chip of the invention to achieve the purpose of detecting important pathogenic bacteria in aquatic products, because of simple operation, high accuracy and high reproducibility, For medical tests, food safety inspections, import and export inspection and quarantine and epidemiological investigations.
  • FIG. 1 is a schematic view showing the appearance of an embodiment of a gene chip of the present invention.
  • Fig. 2 is a schematic view showing the arrangement of a single dot matrix probe on an embodiment of the gene chip of the present invention.
  • Fig. 3A is a result of hybridization when a purulent scaffold is detected using the gene chip of the present invention.
  • Fig. 3B is a result of hybridization when Shigella is detected using the gene chip of the present invention.
  • Fig. 3C shows the results of hybridization when S. aureus was detected using the gene chip of the present invention.
  • I Fig. 3D shows the results of hybridization when Salmonella was detected using the gene chip of the present invention.
  • Fig. 3E shows the results of hybridization when the Listeria monocytogenes were detected using the gene chip of the present invention.
  • Fig. 3F is a result of hybridization when Vibrio parahaemolyticus was detected using the gene chip of the present invention.
  • Fig. 3G shows the results of hybridization when Vibrio cholerae was detected using the gene chip of the present invention.
  • Fig. 3H shows the results of hybridization when the Proteus mirabilis was detected using the gene chip of the present invention.
  • Fig. 31 shows the results of hybridization when the Proteus proteus was detected using the gene chip of the present invention.
  • Fig. 3J is a result of hybridization when the Proteus vulgaris is detected by the gene chip of the present invention.
  • Fig. 4 is a schematic diagram showing the specific probe arrangement pattern of recombining other pathogenic bacteria other than Proteus spp.
  • Fig. 5A shows the results of hybridization when Vibrio parahaemolyticus was detected using the gene chip of the present invention.
  • Fig. 5B is a result of hybridization when S. pyogenes is detected using the gene chip of the present invention.
  • Fig. 5C shows the results of hybridization when Salmonella was detected using the gene chip of the present invention.
  • Fig. 5D shows the results of hybridization when the Proteus vulgaris was detected using the gene chip of the present invention.
  • Fig. 5E is a result of hybridization when the Proteus mirabilis was detected using the gene chip of the present invention.
  • Fig. 5F is a result of hybridization when Shigella is detected using the gene chip of the present invention.
  • Fig. 5G shows the results of hybridization when S. aureus was detected using the gene chip of the present invention.
  • Fig. 5H shows the results of hybridization when the Listeria monocytogenes were detected using the gene chip of the present invention.
  • Proteus In order to meet the needs of specific intraspecies conservation in target sequence analysis, 48 Proteus were selected for Proteus with few ITS resources (including all 4 species of this genus: 22 Proteus mirabilis, Proteus vulgaris 14 The strain, 10 strains of Proteus panicula, and 2 strains of Proteus spp.) were sequenced and 831 ITS sequences were determined.
  • the above-mentioned primers designed from the 16sS rDNA and 23S rDNA sequences were used to amplify the intergenic region, and the PCR product was purified and ligated into the T vector, and then electrotransformed into the DH5a competent state, and the plasmid containing 500 bp to 1000 bp was picked and sequenced. 3700.
  • the sequence was spliced using the Staden Package software to obtain the sequence of the Proteus mirabilis and the common Proteus and its related bacteria.
  • Probe synthesis Extend the 5' end of the probe sequence in Table 1 by 10 T (the extended fluorescent probe sequence shown in Table 1 already contains an extended 10 T) and amidate the probe. Synthetic Company (Beijing Aoke Company) Synthetic, spare.
  • Probe screening The synthesized probe is dissolved and diluted in an appropriate amount, and then the gene chip is prepared on the glass substrate by using a gene chip spotting instrument, and the probe is screened by the hybridization experiment, and finally the gene for preparing the gene is obtained. A specific, sensitive probe required for the chip.
  • probes having a length of 35 bp ⁇ 2 bp and a T m 75 ° C ⁇ 2 ° C were selected, and probes were screened by 360 hybridization experiments, and finally obtained as shown in Table 1.
  • the probe sequence numbered NO. 1 (SEQ ID ⁇ : 1) is selected from 16S rDNA of all bacteria, used as a positive control to detect the presence or absence of bacteria, and the probe numbered NO.
  • the sequence (SEQ ID NO: 2-SEQ ID NO: 22) is selected from common pathogens (Salmon) 16S-23S rDNA intergenic region of Vibrio, Vibrio parahaemolyticus, Vibrio cholerae, Listeria monocytogenes, Staphylococcus aureus, Streptococcus pyogenes, Proteus mirabilis, Proteus vulgaris, Proteus panicula,
  • the four probe sequences of No. 26-NO.29 (SEQ ID NO: 23 - SEQ ID NO: 26) are selected from the ipali gene of Shigella.
  • Table 1 Oligonucleotide probe sequences selected on the gene chip of the present invention and detectable pathogenic bacteria
  • Sequence acquisition Sequence of the same design probe as before.
  • Primer synthesis The primer sequences in Table 2 below were entrusted to the primer synthesis company (Beijing Aoke) for synthesis.
  • Primer screening The synthesized primers were dissolved and diluted in appropriate amount, and the amplification of the 16S-23S rDNA intergenic region and the Shigella/gene sequence detection primers were respectively amplified by PCR reaction. Sex, the other side, two pairs of primers simultaneously amplified the different strains of 10 strains to test the compatibility of the two pairs of primers, and finally obtained the specific and sensitive primers needed for preparing the gene chip of the present invention.
  • an important pathogenic bacteria (Shigella, Salmonella, Vibrio parahaemolyticus, Vibrio cholerae, 10 strains) of the 10 types of aquatic products are selected to be included in both the probe and the 2 pairs of primers.
  • Lysis Probe The probe synthesized in Example 1 was separately dissolved in a 50% DMSO solution, and diluted to a final concentration of 1 ⁇ 8 / ⁇ 1 of the probe.
  • the dot matrix area size is 3 mm x 2 mm
  • the dot pitch of the dot matrix is 250 ⁇
  • matrix: 12x8, 12> ⁇ 250 ⁇ 3 ⁇
  • 8> ⁇ 250 ⁇ 2 ⁇
  • Crosslinking Crosslink 2 times with a cross-linker (uvpcl-2000M ultraciolet Crosslinker) 600J. Put the cross-linked chips back into the clean chip box and set aside.
  • a cross-linker uvpcl-2000M ultraciolet Crosslinker 600J. Put the cross-linked chips back into the clean chip box and set aside.
  • the position indicated by the N0.1 box is the positive control probe for detecting bacteria.
  • the position indicated by the N0.2 box is the fluorescent probe, the position indicated by the N0.3 box is the negative control probe, and the N0.4 box is indicated.
  • Sample processing According to the national standard operation method, use sterile cotton swab, aseptic operation, spread the cockroaches and intestines of fish, shrimp, crab and other aquatic products into the prepared 2YT medium, 37 °C Incubate at 200 rpm overnight with shaking.
  • Amplification of the target sequence 3 ul of the middle layer supernatant extracted by the above genomic extraction method was added as a template to the PCR reaction mixture, and the PCR reaction mixture formulation is shown in Table 3 below. (Note: PCR buffer, MgCl 2 , dNTP mixture in Table 3 - Table 4 below, Taq enzymes were purchased from Sangon) Multiplex PCR Reaction Mix Formula
  • ⁇ -2 and ⁇ -4 in the table are the primers listed in Table 2. Place the reaction tube in the PCR instrument (Biometra) and set the cycle parameters as follows: 94 °C 5 minutes 94 °C 30 seconds 50 °C 30 seconds
  • Hybridization 70 ⁇ l ddH 2 0 was pre-charged into the hybridization cassette (Boao) to maintain humidity.
  • the 12 ⁇ 1 hybridization solution (formulated as shown below) was used to reconstitute the dried product and added to the probe array area of the common pathogen detection gene chip in the intestine prepared in Example 3, and covered with a custom cover sheet (Boao Company) ( Note that there should be no air bubbles between the cover slip and the slide. Close the hybridization cassette and mix for 16 hours in a 40 ° C water bath.
  • Hybrid solution formulation 10% dextran Sulfate; 25% formamide; 0.1% SDS (sodium dodecyl sulfate); 6xSSPE
  • Lotion A l xSSC (sodium chloride - sodium citrate solution); 0.1% SDS
  • Scan Scan with the GenePix personal 4100A BioScanner (AXON instrument) with the following parameters:
  • the gene chips of the present invention are used to detect common intestinal pathogenic bacteria (Shigella, Salmonella, Vibrio parahaemolyticus, Vibrio cholerae, Listeria monocytogenes, gold Hybridization scan results for Staphylococcus aureus, Streptococcus pyogenes, Proteus mirabilis, Proteus vulgaris, Proteus panicularum are shown in Figures 3A-3J.
  • the specificity of the important pathogenic bacteria detection gene chip in the aquatic product prepared in Example 3 was identified as follows:
  • Streptococcus suis 1 Streptococcus agalactiae 1 n 1 Streptococcus faecium 1 d 1 Streptococcus faecalis 1 c 1 Streptococcus porcinus 1 k 1 Streptococcus bovis 1 c 1 Vibrio vulnificus Vibrio vulnficus 1 k 1 Vibrio fluvialis 1 k 1 Vibrio furnissii 1 k 1 Vibrio minicus 1 o 1 Vibrio alginolyticus 1 o 1 Listeria innocua 1 Listeria welshimeri 1 Proteus myxofaciens i 1 , i k 2 a, Institute of Epidemiology and Microbiology (IEM), Chinese Society for Preventive Medicine.
  • IEM Institute of Epidemiology and Microbiology
  • ATCC American Type Culture Collection
  • the detection sensitivity of the gene chip was verified by 153 hybridization experiments.
  • the colonies in 0.1 ng of micro genomic DNA or 10 4 cfu/ml pure bacteria samples ensured stable and good hybridization results of the above 10 pathogenic bacteria.
  • the gene chip of the present invention has high detection sensitivity.
  • Example 7 Simulation experiment on the gene chip Considering that the probability of occurrence of Proteus paniculata in the actual sample is small, the specific probes of the nine pathogenic bacteria other than Proteus spp. are recombined to form the same as shown in Fig. 4. Schematic diagram of the arrangement of single dot matrix probes in the simulated embodiment.
  • Treatment of the experimental strain The monoclonal strain was picked and inserted into the prepared 4 mL 2YT medium, and shake cultured at 37 ° C, 200 rpm overnight. 100 bacteria solution was added to 900 physiological saline, and repeatedly washed to dilute.
  • 3 mg was ground with a mortar, added to 30 mL of sterile physiological saline, soaked in fish, and mixed. Into a diluent. 0.5 mL of physiological saline soaked in fish meat was taken and added to the spare cells collected by centrifugation. The cells were collected by centrifugation at 12,000 rpm for 2 minutes.
  • the concentration of the strain used in the following simulation experiments is the lowest detection concentration that can be achieved by the invention (Table
  • FIGS. 5A to 5H The results of the hybridization scan are shown in FIGS. 5A to 5H, wherein FIG. 5A is a result of hybridization when the Vibrio parahaemolyticus is detected by the gene chip of the present invention; and FIG. 5B is a result of hybridization when the S. pyogenes is detected by the gene chip of the present invention; 5C is a hybridization result when Salmonella is detected by the gene chip of the present invention; FIG. 5D is a hybridization result when the Proteus vulgaris is detected by the gene chip of the present invention; and FIG. 5E is a hybridization when the Proteus mirabilis is detected by the gene chip of the present invention; Fig.
  • Fig. 5F is the result of hybridization when detecting Shigella using the gene chip of the present invention
  • Fig. 5G is the result of hybridization when using the gene chip of the present invention to detect Staphylococcus aureus
  • Fig. 5H is the detection by the gene chip of the present invention Hybridization results when Listeria monocytogenes was added.

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Abstract

Gene chip and kit for detecting important pathogenic bacterium in aquatic products are provided. The gene chip comprises solid phase supports and oligonucleotide probes fixed on the solid phase supports, said oligonucleotide probes comprise one or more sequences selected from: (1) the DNA equence that is selected from the 16S-23S rDNA internal transcribed spacers (ITS) of Salmonella, Vibrio parahaemolyticus, Vibrio cholerae, Listeria monocytogenes, Staphylococcus aureus, Streptococcus pyogenes, Proteus mirabilis, Proteus vulgaris and Proteus Penneri, as well as the ipaH gene of Shigella; (2) the complemental DNA sequence of the selected DNA sequence in (1); and (3) the complemental RNA sequence of the selected DNA sequence in (1) or (2).

Description

检测水产品中重要致病菌的基因芯片和试剂盒 技术领域 本发明涉及一种基因芯片和检测用试剂盒，尤其涉及检测水产品中重 要致病菌的基因芯片和试剂盒。 背景技术 食品质量与食品安全关系人类健康， 因而受到世界各国的高度重视。 微生物引起的食源性疾病是食品安全的主要问题。细菌污染造成的食源性 疾病 （食物中毒） 是我国食品安全中最突出的问题。 据卫生部统计， 2000 年共收到重大食物报告达 150起， 中毒 6237人， 死亡 135 人； 2001年共 发生重大食物中毒事件 185起， 15715人中毒， 116人死亡。 2005年全国 各地上报的食物中毒事件中，微生物食物中毒人数最多， 占总数的 43.0%。 国家食源性疾病监测网个案报告的资料分析表明：微生物性病原占 46.4%， 微生物性食源性疾病暴发中， 副溶血性弧菌导致的疾病暴发占 40.1%, 变 形杆菌占 11.3%, 葡萄球菌肠毒素占 9.4%, 蜡样芽孢杆菌占 8.6%, 沙门 氏菌占 8.1%, 致病性大肠杆菌占 4%。我国食物中毒的高危食品为： 肉类、 粮食、 水产品、 水果蔬菜、 鸡蛋、 豆类、 奶。 这一排序是基于国家食源性 疾病监测网部分资料的分析的动态情况。 其中水产品排名第三， 是重要的 食物中毒高危食品。 水产品致病菌导致的食源性疾病影响的国家范围广、 危急健康人群多， 而且还给国家之间相关的食品贸易带来危机， 这使得水 产品安全问题受到了空前的关注。这些事实也可以充分说明尽管现代科技 已经发展到了相当高的水平，但水产品致病菌导致的疾病不论在发达国家 还是在发展中国家， 都没有很好的被控制， 仍然危害着人民的健康。  TECHNICAL FIELD The present invention relates to a gene chip and a kit for detection, and more particularly to a gene chip and a kit for detecting important pathogenic bacteria in aquatic products. BACKGROUND OF THE INVENTION Food quality and food safety are related to human health and are therefore highly valued by countries all over the world. Foodborne diseases caused by microorganisms are a major problem in food safety. Foodborne diseases caused by bacterial contamination (food poisoning) are the most prominent problems in food safety in China. According to the statistics of the Ministry of Health, in 2000, a total of 150 major food reports were received, 6237 cases were poisoned, and 135 people died. In 2001, there were 185 major food poisoning incidents, 15715 people were poisoned and 116 people died. Among the food poisoning incidents reported in various parts of the country in 2005, the number of microbial food poisonings was the highest, accounting for 43.0% of the total. According to the data analysis of the National Foodborne Disease Surveillance Network case report, microbial pathogens accounted for 46.4%. In microbial foodborne disease outbreaks, 40.1% of the outbreaks caused by Vibrio parahaemolyticus and 11.3% of Proteus spp. Coccidial enterotoxin accounted for 9.4%, Bacillus cereus accounted for 8.6%, Salmonella accounted for 8.1%, and pathogenic E. coli accounted for 4%. The high-risk foods for food poisoning in China are: meat, food, aquatic products, fruits and vegetables, eggs, beans, milk. This ranking is based on the dynamic analysis of some of the data from the National Foodborne Disease Surveillance Network. Among them, aquatic products ranked third, which is an important food poisoning high-risk food. The wide range of food-borne diseases caused by aquatic pathogens, the high number of critically ill people, and the crisis in food trade between countries have brought unprecedented attention to the safety of aquatic products. These facts can also fully demonstrate that although modern technology has developed to a fairly high level, diseases caused by aquatic product pathogens are not well controlled in both developed and developing countries, and still endanger people's health. .
水产品中的致病菌主要有以下两类： 一是其自身原有致病菌， 这类细 菌广泛分布于水中， 如冷源性细菌单核细胞增生李斯特氏菌， 噬热性细菌 如副溶血弧菌和霍乱弧菌等 ； 二是非自身原有致病菌， 即生产过程中被 污染的致病菌，主要存在与人和动物肠道内及被人或动物粪便污染的水环 境中， 常见的包括沙门氏菌、 志贺氏菌、 金黄色葡萄球菌等 （杨文鸽， 孙 翠玲， 潘云娣， 等.水产品中致病微生物的快速检测方法.中国食品学报，There are two main types of pathogenic bacteria in aquatic products: one is its own pathogenic bacteria, which are widely distributed in water, such as cold-sourced bacteria, Listeria monocytogenes, thermophilic bacteria. Such as Vibrio parahaemolyticus and Vibrio cholerae; Second, non-self pathogenic bacteria, that is, contaminated pathogenic bacteria in the production process, mainly in the water environment of human and animal intestines and contaminated by human or animal feces, common Including Salmonella, Shigella, Staphylococcus aureus, etc. (Yang Wenge, Sun Cuiling, Pan Yunqi, et al. Rapid detection methods for pathogenic microorganisms in aquatic products. Chinese Journal of Food Science,
2006， 6 ( 1 )： 402〜406)。 统计了卫生部公布的国内近十年公布的微生物 性食物中毒病例， 综合国家食源性疾病监测网部分资料的分析的动态情 况， 天津出入境检验检疫局提供的调查统计数据， 确定水产品致病菌检测 芯片检测范围为下列常见的 10类菌： 化脓性链球菌， 志贺氏菌 （包含福 氏志贺氏菌、 宋氏志贺氏菌、 鲍氏志贺菌和痢疾志贺氏菌共 4个种）， 沙 门氏菌， 金黄色葡萄球菌， 副溶血弧菌， 霍乱弧菌， 单增李斯特菌， 奇异 变形杆菌， 潘氏变形杆菌和普通变形杆菌。 2006, 6 (1): 402~406). Statistics on the microbiological food poisoning cases published by the Ministry of Health in the past ten years, the analysis of the data of some national foodborne disease surveillance networks, the survey statistics provided by the Tianjin Entry-Exit Inspection and Quarantine Bureau, and the determination of aquatic products The detection range of the bacteria detection chip is the following 10 common bacteria: S. pyogenes, Shigella (including Shigella flexneri, Shigella sonnei, Shigella flexneri and Shigella dysenteriae) A total of 4 species), Salmonella, Staphylococcus aureus, Vibrio parahaemolyticus, Vibrio cholerae, Listeria monocytogenes, Proteus mirabilis, Proteus panicula and Proteus vulgaris.
我国目前对于水产品的病原菌检测、鉴定手段仍停留常规的微生物学 检验水平，通常以分离培养、生化试验及血清学试验为主，具有操作复杂、 手工劳动量大、 检验周期长 （ 6-7天） 、 特异性不强等缺点， 在相当大 的程度上限制了对食源性疾病病源水产品的快速检测鉴定能力， 并且在判 断是否为阳性时只靠实验人员肉眼观察， 没有足够可靠的依据， 降低实验 结果的准确性， 不能达到新鲜水产品贮藏时间短、 需要快速检测的要求。 一些简单的分子生物学的方法如 PCR、 ELISA都有较高的假阳性结果出 现， 并且运用 PCR和 ELISA得出的结果都不能作为最终的检测依据。 日 前丹麦、澳人利亚等一些欧洲和澳洲国家食源性病原菌的检测结果综合了 实时荧光 PCR和 ELISA两种技术在 DNA和蛋白质两种水平上检测的结 果进行判断， 并且实验操作只有 1 h左右。 因此我国有必要建立起水产品 致病菌的高通量、 快速、 准确的监测体系， 以确保在相对较短的时间内正 确判定水产品的安全性， 这是有效地预防和控制食源性疾病的重要前提。 只有这样才能在一定程度上提前消除由于水产品中的致病菌所造成的危 害， 提高我国食源性疾病的检测和控制能力， 为加入 WTO后尽早与其他 国家达成通关协议搭建雄厚的技术平台。  At present, the detection and identification methods of pathogenic bacteria for aquatic products still remain at the routine microbiological test level, usually based on separation culture, biochemical test and serological test, with complicated operation, large manual labor and long test period (6-7). Disadvantages such as lack of specificity, to a considerable extent limit the ability of rapid detection and identification of aquatic products in food-borne diseases, and only rely on the naked eye of the experimenter when judging whether it is positive, not reliable enough According to the method, the accuracy of the experimental results is reduced, and the requirement that the fresh aquatic product has a short storage time and needs to be quickly detected cannot be achieved. Some simple molecular biology methods such as PCR and ELISA have higher false positive results, and the results obtained by PCR and ELISA cannot be used as the final test basis. Recently, the results of food-borne pathogens in some European and Australian countries such as Denmark and Australia have been combined with the results of real-time fluorescent PCR and ELISA detection at the DNA and protein levels, and the experimental operation is only 1 h. about. Therefore, it is necessary to establish a high-throughput, rapid and accurate monitoring system for aquatic product pathogens to ensure the safety of aquatic products is correctly determined in a relatively short period of time, which is effective in preventing and controlling food-borneness. An important prerequisite for the disease. Only in this way can we eliminate the damage caused by pathogenic bacteria in aquatic products to a certain extent, improve the detection and control ability of foodborne diseases in China, and establish a strong technical platform to reach customs clearance agreements with other countries as soon as possible after joining the WTO. .
1997年 7月， Affymetrix, Inc. (Santa Clara, CA) 公司的 Thomas R. 等人发明的第 6,228,575号美国专利公开了以生物芯片技术对微生物进行 定种和表型分析的方法。 从二十世纪九十年代开始， 基因芯片技术作为一 种全新的核酸分析检测技术建立起来， 并随着人类基因组计划的开展而逐 步发展。该技术综合运用了分子生物学、集成电路制造、计算机、半导体、 共聚焦激光扫描、荧光标记等技术，使检测过程具有敏感性高、特异性强、 大规模集成化、 自动化、 操作简便快捷、 效率高等特点， 在基因表达相关 研究和生物制药研究等方面得到普遍应用。 因此， 如果将基因芯片技术用 于细菌的鉴定， 不仅可以大大简化检测手段， 提高检测速度， 而且具有高 准确性、 高灵敏度、 高通量、 可重复性强等诸多优点。 In July 1997, U.S. Patent No. 6,228,575 to Thomas R. et al., issued to Affymetrix, Inc. (Santa Clara, CA), discloses microbiology using biochip technology. Methods for seeding and phenotypic analysis. Since the 1990s, gene chip technology has been established as a new nucleic acid analysis and detection technology, and has evolved with the development of the Human Genome Project. The technology integrates molecular biology, integrated circuit manufacturing, computer, semiconductor, confocal laser scanning, fluorescent labeling and other technologies to make the detection process sensitive, specific, large-scale integration, automation, and easy to operate. High efficiency, widely used in gene expression research and biopharmaceutical research. Therefore, if the gene chip technology is used for the identification of bacteria, not only the detection means can be greatly simplified, the detection speed can be improved, but also the advantages of high accuracy, high sensitivity, high throughput, and high reproducibility are obtained.
目前最常使用的微生物鉴定的靶分子是 16S rRNA和 23S rRNA，国内 外已有很多文献报道。 利用 16S rRNA和 23S rRNA可将细菌微生物鉴定 至种或属，但是对于亲缘关系较近的细菌种或属的鉴定十分困难 (Bodrossy L, Sessitsch A. 2004. Oligonucleotide microarrays in microbial diagnostics. Current Opinion in Microbiology. 7:245-25 )。 目前利用 16S-23S rRNA间区 作为靶分子进行细菌的鉴定已经逐渐成为研究的热点 （Nubel U, Schmidt PM, ReiB E, et al. 2004. Oligonucleotide microarray for identification of Bacillus anthracis based on intergenic transcribed spacers in ribosomal DNA. FEMS Microbiology Letters 240: 215-223. ) , 它的变异速率相当于 16S rRNA或者 23S rRNA的十倍， 因此具有更高的分辨率， 甚至可将细菌区 分到型， 对于亲缘关系较近的种属的区分具有 16S rRNA和 23S rRNA不 可比拟的优势。 同时两端是保守的 16S rRNA基因和 23S rRNA基因区， 可在两端的保守区设计通用引物可避免了多对引物带来的引物二聚体的 问题， 长度在 200 bp- 1000 bp之间， 大小易于操作， 因而靶序列的扩增和 标记过程更加简化， 也更容易控制， 符合操作简便快捷的实际要求。 发明内容 本发明的一个目的是提供一种检测水产品中重要致病菌的基因芯片， 以弥补传统常见水产品致病菌检测技术存在的费时耗力的缺陷，扩展病原 菌检测范围， 提高检测灵敏度和特异性， 降低劳动强度， 缩短检测周期。  The most commonly used target molecules for microbial identification are 16S rRNA and 23S rRNA, which have been reported in many countries. Bacterial microorganisms can be identified to species or genera using 16S rRNA and 23S rRNA, but identification of closely related bacterial species or genera is difficult (Bodrossy L, Sessitsch A. 2004. Oligonucleotide microarrays in microbial diagnostics. Current Opinion in Microbiology 7:245-25). At present, the identification of bacteria using the 16S-23S rRNA inter-region as a target molecule has gradually become a research hotspot (Nubel U, Schmidt PM, ReiB E, et al. 2004. Oligonucleotide microarray for identification of Bacillus anthracis based on intergenic transcribed spacers in ribosomal DNA. FEMS Microbiology Letters 240: 215-223. ) , which has a mutation rate equivalent to ten times that of 16S rRNA or 23S rRNA, so it has higher resolution and can even distinguish bacteria into types, for close relatives. The species distinction has the advantage that 16S rRNA and 23S rRNA are incomparable. At the same time, both ends are conserved 16S rRNA gene and 23S rRNA gene region. Designing universal primers in conserved regions at both ends can avoid the problem of primer dimers brought by multiple pairs of primers, ranging from 200 bp to 1000 bp. The size is easy to operate, so the amplification and labeling process of the target sequence is more simplified and easier to control, meeting the practical requirements of simple and quick operation. SUMMARY OF THE INVENTION One object of the present invention is to provide a gene chip for detecting important pathogenic bacteria in aquatic products, so as to make up for the time-consuming and labor-intensive defects of traditional common aquatic product pathogenic bacteria detection technology, expand the detection range of pathogenic bacteria, and improve detection sensitivity. And specificity, reduce labor intensity, and shorten the detection cycle.
本发明所述的检测水产品中重要致病菌的基因芯片包括固相载体和 固定在该固相载体上的寡聚核苷酸探针，其中所述固定在该固相载体上的 寡聚核苷酸探针包含从以下序列中选取的一种或多种： The gene chip for detecting important pathogenic bacteria in aquatic products according to the present invention includes a solid phase carrier and An oligonucleotide probe immobilized on the solid phase support, wherein the oligonucleotide probe immobilized on the solid phase carrier comprises one or more selected from the following sequences:
( 1 ) 从沙门氏菌、 副溶血弧菌、 霍乱弧菌、 单增李斯特氏菌、 金黄 色葡萄球菌、 化脓性链球菌、 奇异变形杆菌、 普通变形杆菌、 潘氏变形杆 菌的 16S-23S rDNA间区以及志贺氏菌的/ H ( invasion plasmid antigen gene) 毒力基因中选取的 DNA序列；  (1) From 16S-23S rDNA between Salmonella, Vibrio parahaemolyticus, Vibrio cholerae, Listeria monocytogenes, Staphylococcus aureus, Streptococcus pyogenes, Proteus mirabilis, Proteus vulgaris, Proteus paniculata a DNA sequence selected from the virulence gene of the region and the H (invagin plasmid antigen gene);
(2) 上述 （1 ) 中选取的 DNA序列的互补 DNA序列；  (2) a complementary DNA sequence of the DNA sequence selected in the above (1);
(3 ) 上述 （1 ) 或 （2) 中选取的 DNA序列的互补 RNA序列。 本发明的优选实施例中， 固定在该固相载体上的寡聚核苷酸探针具有 SEQ ID NO:2-SEQ ID NO:26所示的 DNA序列中的一种或多种。 本发明所述的基因芯片， 还包含阳性对照探针、 阴性对照探针或荧光  (3) A complementary RNA sequence of the DNA sequence selected in the above (1) or (2). In a preferred embodiment of the invention, the oligonucleotide probe immobilized on the solid phase carrier has one or more of the DNA sequences shown in SEQ ID NO: 2 - SEQ ID NO: 26. The gene chip of the present invention further comprises a positive control probe, a negative control probe or a fluorescent
本发明所述的基因芯片中， 所述阳性对照探针选自细菌 16S rDNA保 守区中的 DNA片段或者其互补的 DNA或 RNA序列。 本发明的优选实施例中， 所述阳性对照探针具有 SEQ ID ΝΟ:1所示的 DNA序列。 本发明还提供所述的基因芯片的应用， 主要为在检测志贺氏菌、 沙门 氏菌、 副溶血弧菌、 霍乱弧菌、 单增李斯特氏菌、 金黄色葡萄球菌、 化脓 性链球菌、 奇异变形杆菌、 普通变形杆菌、 潘氏变形杆菌中至少一种致病 菌的应用。 上述应用中， 包含使用检测引物， 本发明的优选实施例中， 所 述检测引物具有 SEQ ID NO: 27-SEQ ID NO: 30所示的 DNA序列或其互补 序列中的至少一种。 In the gene chip of the present invention, the positive control probe is selected from a DNA fragment in a bacterial 16S rDNA conservation region or a complementary DNA or RNA sequence thereof. In a preferred embodiment of the invention, the positive control probe has the DNA sequence set forth in SEQ ID NO: 1. The invention also provides the application of the gene chip, mainly for detecting Shigella, Salmonella, Vibrio parahaemolyticus, Vibrio cholerae, Listeria monocytogenes, Staphylococcus aureus, Streptococcus pyogenes, singularity Use of at least one pathogenic bacteria in Proteus, Oryzae, and Proteus panicula. In the above application, the detection primer is used. In a preferred embodiment of the present invention, the detection primer has at least one of the DNA sequence shown in SEQ ID NO: 27-SEQ ID NO: 30 or a complementary sequence thereof.
本发明还提供一种试剂盒， 其包含权利上述的基因芯片。  The invention also provides a kit comprising the gene chip as described above.
本发明所述的试剂盒， 还包括检测引物， 本发明的优选实施例中， 所 述检测引物具有 SEQ ID NO: 27-SEQ ID NO: 30所示的 DNA序列或其互补 序列中的至少一种。  The kit of the present invention further comprises a detection primer. In a preferred embodiment of the present invention, the detection primer has at least one of the DNA sequence represented by SEQ ID NO: 27-SEQ ID NO: 30 or a complementary sequence thereof. Kind.
本发明还提供上述的试剂盒的应用， 其主要为在检测志贺氏菌、 沙门 氏菌、 副溶血弧菌、 霍乱弧菌、 单增李斯特氏菌、 金黄色葡萄球菌、 化脓 性链球菌、 奇异变形杆菌、 普通变形杆菌、 潘氏变形杆菌中至少一种致病 菌的应用。 The invention also provides the application of the above kit, which is mainly for detecting Shigella and Shamen Application of at least one pathogenic bacteria in bacteria, Vibrio parahaemolyticus, Vibrio cholerae, Listeria monocytogenes, Staphylococcus aureus, Streptococcus pyogenes, Proteus mirabilis, Proteus vulgaris, Proteus paniculata .
由上述的技术方案可见，本发明首次将基因芯片技术引入水产品中重 要致病菌检测领域同时检测 10类致病菌， 建立了一种快速、 灵敏、 准确 性高、重复性强的全新的用于检测水产品中重要致病菌的基因芯片及其检 测方法， 利用本发明的基因芯片可以达到检测水产品中重要致病菌的目 的， 由于操作简便， 准确性高， 重复性强， 可用于医学检验、 食品安全检 验、 进出口检验检疫和流行病学调查。  It can be seen from the above technical solution that the present invention introduces gene chip technology into the field of important pathogenic bacteria detection in aquatic products for the first time and simultaneously detects 10 types of pathogenic bacteria, and establishes a brand new type which is fast, sensitive, accurate and reproducible. The gene chip for detecting important pathogenic bacteria in aquatic products and the detection method thereof can use the gene chip of the invention to achieve the purpose of detecting important pathogenic bacteria in aquatic products, because of simple operation, high accuracy and high reproducibility, For medical tests, food safety inspections, import and export inspection and quarantine and epidemiological investigations.
为保证本发明的上述和其它目的特征和优点更明显易懂，下面特举较 佳实施例， 并配合说明书附图， 作详细说明如下。 附图说明 图 1 为本发明基因芯片的一个实施例的外形示意图。  The above and other objects and features of the present invention will be more apparent from the following description. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic view showing the appearance of an embodiment of a gene chip of the present invention.
图 2 为本发明基因芯片的一个实施例上单一点阵探针排布规律示意 图。  Fig. 2 is a schematic view showing the arrangement of a single dot matrix probe on an embodiment of the gene chip of the present invention.
图 3A为利用本发明的基因芯片检测化脓性链球时的杂交结果。  Fig. 3A is a result of hybridization when a purulent scaffold is detected using the gene chip of the present invention.
图 3B 为利用本发明的基因芯片检测志贺氏菌时的杂交结果。  Fig. 3B is a result of hybridization when Shigella is detected using the gene chip of the present invention.
图 3C为利用本发明的基因芯片检测金黄色葡萄球菌时的杂交结果。 I 图 3D为利用本发明的基因芯片检测沙门氏菌时的杂交结果。  Fig. 3C shows the results of hybridization when S. aureus was detected using the gene chip of the present invention. I Fig. 3D shows the results of hybridization when Salmonella was detected using the gene chip of the present invention.
图 3E为利用本发明的基因芯片检测单增李斯特氏菌时的杂交结果。 图 3F为利用本发明的基因芯片检测副溶血弧菌时的杂交结果。  Fig. 3E shows the results of hybridization when the Listeria monocytogenes were detected using the gene chip of the present invention. Fig. 3F is a result of hybridization when Vibrio parahaemolyticus was detected using the gene chip of the present invention.
图 3G为利用本发明的基因芯片检测霍乱弧菌时的杂交结果。  Fig. 3G shows the results of hybridization when Vibrio cholerae was detected using the gene chip of the present invention.
图 3H为利用本发明的基因芯片检测奇异变形杆菌时的杂交结果。 图 31为利用本发明的基因芯片检测潘氏变形杆菌时的杂交结果。  Fig. 3H shows the results of hybridization when the Proteus mirabilis was detected using the gene chip of the present invention. Fig. 31 shows the results of hybridization when the Proteus proteus was detected using the gene chip of the present invention.
图 3J为利用本发明的基因芯片检测普通变形杆菌时的杂交结果。  Fig. 3J is a result of hybridization when the Proteus vulgaris is detected by the gene chip of the present invention.
图 4是重新组合除潘氏变形杆菌外的其它致病菌的特异探针排布规律 示意图。  Fig. 4 is a schematic diagram showing the specific probe arrangement pattern of recombining other pathogenic bacteria other than Proteus spp.
图 5A为利用本发明的基因芯片检测副溶血弧菌时的杂交结果。 图 5B 为利用本发明的基因芯片检测化脓链球菌时的杂交结果。 Fig. 5A shows the results of hybridization when Vibrio parahaemolyticus was detected using the gene chip of the present invention. Fig. 5B is a result of hybridization when S. pyogenes is detected using the gene chip of the present invention.
图 5C为利用本发明的基因芯片检测沙门氏菌时的杂交结果。  Fig. 5C shows the results of hybridization when Salmonella was detected using the gene chip of the present invention.
图 5D为利用本发明的基因芯片检测普通变形杆菌时的杂交结果。 图 5E为利用本发明的基因芯片检测奇特变形杆菌时的杂交结果。 图 5F为利用本发明的基因芯片检测志贺氏杆菌时的杂交结果。  Fig. 5D shows the results of hybridization when the Proteus vulgaris was detected using the gene chip of the present invention. Fig. 5E is a result of hybridization when the Proteus mirabilis was detected using the gene chip of the present invention. Fig. 5F is a result of hybridization when Shigella is detected using the gene chip of the present invention.
图 5G为利用本发明的基因芯片检测金黄色葡萄球菌时的杂交结果。 图 5H为利用本发明的基因芯片检测单增李斯特菌时的杂交结果。 具体实施方式  Fig. 5G shows the results of hybridization when S. aureus was detected using the gene chip of the present invention. Fig. 5H shows the results of hybridization when the Listeria monocytogenes were detected using the gene chip of the present invention. detailed description
实施例 1 探针的设计和制备 Example 1 Design and Preparation of Probes
1. 序列获得：  1. Sequence acquisition:
( 1 ) 细菌 16S rDNA序列的获得： 从 GenBank公共数据库下载得到 十种菌的全部 16S rDNA序列。  (1) Acquisition of bacterial 16S rDNA sequence: All 16S rDNA sequences of ten species were downloaded from the GenBank public database.
(2 ) 16S-23S rDNA间区序列的获得：从 GenBank公共数据库下载得 到沙门氏菌、副溶血弧菌、霍乱弧菌、单增李斯特氏菌、金黄色葡萄球菌、 化脓性链球菌、 及它们的近缘菌的所有 16S-23S rDNA间区序列。  (2) Acquisition of the 16S-23S rDNA intergenic region: Salmonella, Vibrio parahaemolyticus, Vibrio cholerae, Listeria monocytogenes, Staphylococcus aureus, Streptococcus pyogenes, and their downloads were obtained from the public database of GenBank. All 16S-23S rDNA intergenic sequences of related bacteria.
为满足靶序列分析时种内保守种间特异的需要，针对 ITS资源极少的 变形杆菌属， 选取了 48株变形杆菌 （包含该属全部 4个种： 奇异变形杆 菌 22株， 普通变形杆菌 14株， 潘氏变形杆菌 10株， 产粘变形杆菌 2株) 进行了间区的测序， 测得 831条 ITS序列。 用上述从 16sS rDNA和 23S rDNA序列设计的引物扩增间区， PCR产物纯化后连接到 T载体上， 后电 转进 DH5a感受态中， 挑取含 500 bp- 1000 bp的质粒测序， 测序仪 ABI 3700。 测得的序列用 Staden Package软件拼接， 从而得到奇异变形杆菌和 普通变形杆菌及其近缘菌的间区序列。  In order to meet the needs of specific intraspecies conservation in target sequence analysis, 48 Proteus were selected for Proteus with few ITS resources (including all 4 species of this genus: 22 Proteus mirabilis, Proteus vulgaris 14 The strain, 10 strains of Proteus panicula, and 2 strains of Proteus spp.) were sequenced and 831 ITS sequences were determined. The above-mentioned primers designed from the 16sS rDNA and 23S rDNA sequences were used to amplify the intergenic region, and the PCR product was purified and ligated into the T vector, and then electrotransformed into the DH5a competent state, and the plasmid containing 500 bp to 1000 bp was picked and sequenced. 3700. The sequence was spliced using the Staden Package software to obtain the sequence of the Proteus mirabilis and the common Proteus and its related bacteria.
( 3 ) 基因序列的获得： 从 GenBank公共数据库下载得到志贺氏 菌四个种的全部 ipaii基因序列。  (3) Acquisition of gene sequences: All ipaii gene sequences of four species of Shigella were downloaded from the GenBank public database.
2. 探针设计：  2. Probe design:
( 1 )细菌的通用探针： 10类菌全部的 16S rDNA序列及其他菌的 16S rDNA序列导入 Glustal X软件中， 选取靠近间区的一段 16S rDNA保守序 列作为探针， 满足长度 35±2bp， Tm75°C±2°C。 (1) Universal probe for bacteria: 16S rDNA sequences of all 10 strains and 16S of other bacteria The rDNA sequence was introduced into the Glustal X software, and a 16S rDNA conserved sequence close to the inter-region was selected as a probe, which satisfies the length of 35±2 bp and T m 75°C±2°C.
(2) 间区探针： 分别将沙门氏菌、 副溶血弧菌、 霍乱弧菌、 单增李 斯特氏菌、 金黄色葡萄球菌、 化脓性链球菌、 奇异变形杆菌、 普通变形杆 菌、 潘氏变形杆菌的所有 16S-23S rDNA间区序列导入 GlustalX软件中， 从而得知该菌的间区序列有几种类型，每种类型选取一条代表序列在公共 数据 NCBI中作 Blastn比对，确定可否作为特异靶点以及特异靶点的位置。 对照 Glustal X比对结果， 选取满足不同株间该区段都保守的性质， 同时 长度 35士 2bp， Tm75。C±2。C。 (2) Inter-zone probe: Salmonella, Vibrio parahaemolyticus, Vibrio cholerae, Listeria monocytogenes, Staphylococcus aureus, Streptococcus pyogenes, Proteus mirabilis, Proteus vulgaris, Proteus paniculata All 16S-23S rDNA intergenic sequences were introduced into GlustalX software, and it was found that there are several types of intergenic sequences in this bacterium. One representative sequence of each type was selected as a Blastn alignment in the public data NCBI to determine whether it can be used as a specific target. The location of the point and the specific target. Compared with the results of Glustal X, the selection was consistent with the conserved nature of this segment, and the length was 35 ± 2 bp, T m 75. C±2. C.
(3) 基因探针： 将上述从 GenBank公共数据库下载得到的志贺 氏菌四个种的 / H基因序列用序列比对软件 Glustal X比对， 找到该基因 的保守区段， 将该保守区段导入 OligoArray2.0软件中， 参数设定如下： -n 20； -130； -L40; -D3000; -t 79； -T 90； -s65°C; -x65°C; -N2; -p33， -P 65； -m GGGGG CCCCC TTTTT AAAAA; -g 15。 运行程序在线设计探 针。 从输出结果中选择长度在 35bp±2bp， Tm值 75°C±2°C的探针。 (3) Gene probe: The above-mentioned four gene/Shi gene sequences of Shigella downloaded from the GenBank public database were aligned with the sequence alignment software Glustal X to find a conserved region of the gene, and the conserved region was found. The segment is imported into the OligoArray 2.0 software, and the parameters are set as follows: -n 20; -130; -L40; -D3000; -t 79; -T 90; -s65°C; -x65°C; -N2; -p33, -P 65; -m GGGGG CCCCC TTTTT AAAAA; -g 15. Run the program to design the probe online. A probe having a length of 35 bp ± 2 bp and a T m value of 75 ° C ± 2 ° C was selected from the output.
3. 探针合成： 将下表 1中的探针序列的 5'端延长 10个 T (表 1所示 的荧光探针序列中已包含了延长的 10个 T) 并氨基化后委托探针合成公 司 （北京奥科公司） 合成， 备用。  3. Probe synthesis: Extend the 5' end of the probe sequence in Table 1 by 10 T (the extended fluorescent probe sequence shown in Table 1 already contains an extended 10 T) and amidate the probe. Synthetic Company (Beijing Aoke Company) Synthetic, spare.
4. 探针筛选:将合成好的探针溶解并适量稀释后用基因芯片点样仪点 在玻璃片基上制成基因芯片， 通过杂交实验进行探针筛选， 最终得到用于 制备本发明基因芯片所需的特异、 灵敏的探针。  4. Probe screening: The synthesized probe is dissolved and diluted in an appropriate amount, and then the gene chip is prepared on the glass substrate by using a gene chip spotting instrument, and the probe is screened by the hybridization experiment, and finally the gene for preparing the gene is obtained. A specific, sensitive probe required for the chip.
在本发明的优选实施例中， 选择了 28 条长度在 35 bp±2 bp、 Tm 75°C±2°C的探针， 并通过 360次杂交实验进行探针筛选， 最终得到如表 1 所示的探针。 其中， 编号为 NO. 1 (SEQ ID ΝΟ:1) 的探针序列选自所有 细菌的 16S rDNA, 作为阳性对照用来检测是否有细菌， 编号为 NO.2的 探针作为荧光探针， 编号为 NO.3的探针为多聚 T片段， 用作阴性对照， 编号为 NO.4的探针为 50%的 DMSO, 用作空白对照， 编号 NO.5-NO.25 的 21条探针序列 （SEQIDNO:2-SEQIDNO:22) 选自常见致病菌 （沙门 氏菌、 副溶血弧菌、 霍乱弧菌、 单增李斯特氏菌、 金黄色葡萄球菌、 化脓 性链球菌、奇异变形杆菌、普通变形杆菌、潘氏变形杆菌）的 16S-23S rDNA 间区， 编号 NO.26-NO.29 ( SEQ ID NO:23-SEQ ID NO:26 ) 的 4条探针序 列选自志贺氏菌的 ipali基因。 In a preferred embodiment of the invention, 28 probes having a length of 35 bp ± 2 bp and a T m 75 ° C ± 2 ° C were selected, and probes were screened by 360 hybridization experiments, and finally obtained as shown in Table 1. The probe shown. Wherein, the probe sequence numbered NO. 1 (SEQ ID ΝΟ: 1) is selected from 16S rDNA of all bacteria, used as a positive control to detect the presence or absence of bacteria, and the probe numbered NO. 2 is used as a fluorescent probe, numbered The probe for NO.3 is a poly T fragment, used as a negative control, the probe numbered NO.4 is 50% DMSO, used as a blank control, 21 probes numbered NO.5-NO.25 The sequence (SEQ ID NO: 2-SEQ ID NO: 22) is selected from common pathogens (Salmon) 16S-23S rDNA intergenic region of Vibrio, Vibrio parahaemolyticus, Vibrio cholerae, Listeria monocytogenes, Staphylococcus aureus, Streptococcus pyogenes, Proteus mirabilis, Proteus vulgaris, Proteus panicula, The four probe sequences of No. 26-NO.29 (SEQ ID NO: 23 - SEQ ID NO: 26) are selected from the ipali gene of Shigella.
表 1 ： 本发明基因芯片上选用的寡核苷酸探针序列及可检测出的致病菌 Table 1: Oligonucleotide probe sequences selected on the gene chip of the present invention and detectable pathogenic bacteria
Figure imgf000010_0001
AGGCACTATGCTTGAAGCATCGCG 单增李斯特
Figure imgf000010_0001
AGGCACTATGCTTGAAGCATCGCG single increase Liszt
NO.12 NO:9 C 氏菌 NO.12 NO: 9 C bacteria
GAGAGGTTAGTACTTCTCAGTATGT 单增李斯特 GAGAGGTTAGTACTTCTCAGTATGT single increase Liszt
NO.13 NO: 10 TTGTTCTT 氏菌 NO.13 NO: 10 TTGTTCTT bacteria
AATGGTTACTTCATTAGAAGTGATT 副溶血弧菌 AATGGTTACTTCATTAGAAGTGATT Vibrio parahaemolyticus
N0.14 NO: 11 AGCTC N0.14 NO: 11 AGCTC
CCGATTAGCTCCACCACTGACTTCC 副溶血弧菌 CCGATTAGCTCCACCACTGACTTCC Vibrio parahaemolyticus
NO.15 NO: 12 T NO.15 NO: 12 T
TTTTCGCTGAGAATGTTTAAAAATG 霍乱弧菌 TTTTCGCTGAGAATGTTTAAAAATG Vibrio cholerae
NO.16 NO: 13 GTT NO.16 NO: 13 GTT
CTTTAAGCGTTTTCGCTGAGAATGT 霍乱弧菌 CTTTAAGCGTTTTCGCTGAGAATGT Vibrio cholerae
NO.17 NO: 14 TT NO.17 NO: 14 TT
GCCTTGCCTAAAGAAAAAGCTTCTT 奇异变形杆 GCCTTGCCTAAAGAAAAAGCTTCTT singular deformation rod
NO.18 NO: 15 ATTATAA 菌 NO.18 NO: 15 ATTATAA bacteria
GAATAACTAAGCTAATTCAAATGA 奇异变形杆 GAATAACTAAGCTAATTCAAATGA singular deformation rod
NO.19 NO: 16 GTTATCTTACT 菌 NO.19 NO: 16 GTTATCTTACT bacteria
CCACCCAGATAGTCTTTGAAAGAG 奇异变形杆 CCACCCAGATAGTCTTTGAAAGAG singular deformation rod
NO.20 NO: 17 ACACTTT 菌 NO.20 NO: 17 ACACTTT bacteria
AAAAGGAGTGGTTATACGGGTATT 奇异变形杆 AAAAGGAGTGGTTATACGGGTATT singular deformation rod
N0.21 NO: 18 AAAACATTA 菌 N0.21 NO: 18 AAAACATTA
AAAGGAACATTTCCGATAAGAAAG 潘氏变形杆 AAAGGAACATTTCCGATAAGAAAG Pan's deformation rod
N0.22 NO: 19 AAAGCTGAGTA 菌 N0.22 NO: 19 AAAGCTGAGTA
GAATAGTTAAGATAATTCGATGAG 潘氏变形杆 GAATAGTTAAGATAATTCGATGAG Pan's deformation rod
N0.23 NO:20 TTATTTTACCT 菌 N0.23 NO: 20 TTATTTTACCT
AGCGCACAGTCAGCGCAACATACA 潘氏 /普通变 AGCGCACAGTCAGCGCAACATACA Pan's / ordinary change
N0.24 NO:21 TTA 形杆菌 N0.24 NO: 21 TTA
CCCAGACGTCATTAAGAAGAAACA 普通变形杆 CCCAGACGTCATTAAGAAGAAACA ordinary deformation rod
N0.25 NO:22 TCT 菌 N0.25 NO: 22 TCT bacteria
N0.26 NO:23 GATAATGATACCGGCGCTCTGCTCT 志贺氏菌 CC N0.26 NO: 23 GATAATGATACCGGCGCTCTGCTCT Shigella CC
AGATAGAAGTCTACCTGGCCTTCCA 志贺氏菌  AGATAGAAGTCTACCTGGCCTTCCA Shigella
N0.27 NO:24 GACCA  N0.27 NO: 24 GACCA
AGGAAATGCGTTTCTATGGCGTGTC 志贺氏菌  AGGAAATGCGTTTCTATGGCGTGTC Shigella
N0.28 NO:25 G  N0.28 NO: 25 G
ACCATGGCATGCTGTACTGAAGCGT 志贺氏菌  ACCATGGCATGCTGTACTGAAGCGT Shigella
N0.29 NO:26 AC  N0.29 NO: 26 AC
实施例 2 引物的设计和制备 Example 2 Design and preparation of primers
1. 序列获得： 同前设计探针的序列。  1. Sequence acquisition: Sequence of the same design probe as before.
2. 设计引物：  2. Design primers:
( 1 ) 扩增间区序列引物的设计： 从公共数据库 NCBI 中下载得到的 十种细菌的 16S rDNA用序列比对软件 Glustal X比对后，选取靠近间区的 一段 16S rDNA保守序列作为上游引物， 长度符合 Tm值 50°C±5°C、 长度 17bp士 2bp、 Hairpin: NONE. Dimer: NONE. False Priming: NONE. Cross Dimer: NONE, 且包含细菌通用探针序列在内。 (1) Design of primers for the amplified intergenic region: 16S rDNA of ten bacteria downloaded from the public database NCBI was aligned with the sequence alignment software Glustal X, and a 16S rDNA conserved sequence close to the intergenic region was selected as the upstream primer. The length is in accordance with the T m value of 50 ° C ± 5 ° C, the length of 17 b ± 2 bp, Hairpin: NONE. Dimer: NONE. False Priming: NONE. Cross Dimer: NONE, and contains the bacterial universal probe sequence.
(2)扩增 基因序列引物的设计：将上述从 GenBank公共数据库 下载得到的志贺氏菌四个种的 /^H基因序列用序列比对软件 Glustal X比 对， 找到该基因的保守区段， 将该保守区段导入引物设计软件 Primer Premier 5.0软件中， 相应参数设定如下： Search For: PCR Primers, Search types: Both. Search Ranges: Sense Primer 1 to 672, Anti-sense Primer 1 to 672, PCR Product Size: 100 bp to 1000 bp. Primer Length: 20bp±2bp. Search Mode: Automatic 从输出结果中选取 Tm值 50°C±5°C、 长度 17bp±2bp、 Hairpin: NONE, Dimer: NONE, False Priming: NONE、 Cross Dimer: NONE且包含 探针序列在内的引物。  (2) Design of primers for amplified gene sequences: The above-mentioned /H gene sequences of four species of Shigella downloaded from the GenBank public database were aligned with the sequence alignment software Glustal X to find a conserved segment of the gene. The conservative section was introduced into the primer design software Primer Premier 5.0 software, and the corresponding parameters were set as follows: Search For: PCR Primers, Search types: Both. Search Ranges: Sense Primer 1 to 672, Anti-sense Primer 1 to 672, PCR Product Size: 100 bp to 1000 bp. Primer Length: 20bp±2bp. Search Mode: Automatic Select Tm value 50°C±5°C, length 17bp±2bp, Hairpin: NONE, Dimer: NONE, False Priming: NONE, Cross Dimer: NONE and primers containing the probe sequence.
3. 引物合成： 将下表 2中的引物序列委托引物合成公司 （北京奥科） 合成， 备用。  3. Primer synthesis: The primer sequences in Table 2 below were entrusted to the primer synthesis company (Beijing Aoke) for synthesis.
4. 引物筛选： 将合成好的引物溶解并适量稀释， 一方面通过 PCR反 应分别扩增 16S-23S rDNA间区和志贺氏菌/ 基因序列检测引物的扩增 性， 另一发面， 两对引物同时对 10类菌的不同菌株进行扩增检测两对引 物的相容性，最终得到用于制备本发明基因芯片所需的特异、灵敏的引物。 4. Primer screening: The synthesized primers were dissolved and diluted in appropriate amount, and the amplification of the 16S-23S rDNA intergenic region and the Shigella/gene sequence detection primers were respectively amplified by PCR reaction. Sex, the other side, two pairs of primers simultaneously amplified the different strains of 10 strains to test the compatibility of the two pairs of primers, and finally obtained the specific and sensitive primers needed for preparing the gene chip of the present invention.
在本发明的优选实施例中，选取了既包含探针又适合同时使用 2对引 物扩增 10类水产品中重要致病菌 （志贺氏菌、 沙门氏菌、 副溶血弧菌、 霍乱弧菌、 单增李斯特氏菌、 金黄色葡萄球菌、 化脓性链球菌、 奇异变形 杆菌、 普通变形杆菌、 潘氏变形杆菌） DNA的引物 4条， 为适应同时两 对引物的 PCR, 经生物信息学初筛并通过大量 PCR实验筛选， 筛选出如 表 2所示的适用引物。  In a preferred embodiment of the present invention, an important pathogenic bacteria (Shigella, Salmonella, Vibrio parahaemolyticus, Vibrio cholerae, 10 strains) of the 10 types of aquatic products are selected to be included in both the probe and the 2 pairs of primers. 4 primers for Listeria monocytogenes, Staphylococcus aureus, Streptococcus pyogenes, Proteus mirabilis, Proteus vulgaris, Proteus paniculata, to adapt to simultaneous PCR of two pairs of primers, bioinformatics Screening and screening by a large number of PCR experiments, screening suitable primers as shown in Table 2.
表 2 用于肠道中 10类常见致病菌检测 DNA的 PCR扩增的引物序列  Table 2 PCR-amplified primer sequences for detection of 10 common pathogens in the intestine
Figure imgf000013_0001
Figure imgf000013_0001
实施例 3 基因芯片制备——芯片点样 Example 3 Gene chip preparation - chip spotting
1. 溶解探针： 将实施例 1中合成的探针分别溶解于 50 %DMSO溶液 中， 稀释使探针的终浓度达到 1 μ8/μ1。 1. Lysis Probe: The probe synthesized in Example 1 was separately dissolved in a 50% DMSO solution, and diluted to a final concentration of 1 μ 8 /μ1 of the probe.
2. 加板： 将溶解好的探针加入 384孔板的相应位置， 每孔 10μ1。 2. Add plate: Add the dissolved probe to the corresponding position of the 384-well plate, 10μ1 per well.
3. 点样： 将如图 1所示的 57.5mmx25.5mmx lmm (长 χ宽 χ高） 的洁 净的醛基化玻片（CapitalBio Corp., China)放到芯片点样仪（ Spotarray 72 ) 的载物台上， 使用 SpotArray的控制软件 （Telechem smp3 stealty pin) , 运 行程序， 按图 2所示的排布方式点在醛基化的玻片上 4.5mmx4.5mm的点 样区内， 构成中低密度 DNA微矩阵， 玻片上的六个点阵区内阵列排布规 律相同。点阵区域尺寸 3mmx2mm，该点阵内点间距 250 μηι,矩阵： 12x8， 12><250μηι=3ηιηι, 8><250μηι=2ηιηι,标准片基尺寸： 75.5mmx25.5mmx lmm。 3. Spotting: Place a clean aldehydeized slide (CapitalBio Corp., China) of 57.5mm x 25.5mmx lmm (long χ χ χ high) as shown in Figure 1 on the chip spotter (Spotar 72) On the stage, use SpotArray's control software (Telechem smp3 stealty pin), run the program, point 4.5mmx4.5mm on the aldolized slide according to the arrangement shown in Figure 2. In the sample area, the medium-low density DNA micro-matrix is formed, and the array arrangement in the six dot matrix areas on the slide is the same. The dot matrix area size is 3 mm x 2 mm, the dot pitch of the dot matrix is 250 μηι, matrix: 12x8, 12><250μηι=3ηιηι, 8><250μηι=2ηιηι, standard base size: 75.5mmx25.5mmx lmm.
4. 干燥： 将点好的芯片室温下过夜干燥， 然后在 45°C烘箱干燥 2小 时。  4. Drying: The spotted chips were dried overnight at room temperature and then dried in an oven at 45 ° C for 2 hours.
5. 交联： 用交联仪 （uvpcl-2000M ultraciolet Crosslinker) 600J交联 2 次。 将交联好的芯片放回洁净芯片盒中， 备用。  5. Crosslinking: Crosslink 2 times with a cross-linker (uvpcl-2000M ultraciolet Crosslinker) 600J. Put the cross-linked chips back into the clean chip box and set aside.
由图 2可见， 每个点样区内为 12 (行） x8 (列） 个探针点。 N0.1框 区示意的位置为检测细菌的正对照探针， N0.2 框区示意的位置为荧光探 针， N0.3框区示意的位置为负对照探针， N0.4框区示意的是空白对照， 其它为各致病菌的特异探针 （对应于表 1中的相应探针编号）。  As can be seen from Figure 2, there are 12 (rows) x8 (columns) probe points in each sample area. The position indicated by the N0.1 box is the positive control probe for detecting bacteria. The position indicated by the N0.2 box is the fluorescent probe, the position indicated by the N0.3 box is the negative control probe, and the N0.4 box is indicated. A blank control, the other specific probe for each pathogen (corresponding to the corresponding probe number in Table 1).
实施例 4 利用基因芯片快速检测水产品中重要致病菌 Example 4 Rapid detection of important pathogenic bacteria in aquatic products using gene chips
1. 样品处理：按照国标的操作方法，用灭菌棉签，无菌操作，涂抹鱼、 虾、 蟹等水产品的腮和肠道等部位， 放入配制好的 2YT培养基中， 37°C， 200rpm过夜振荡培养。  1. Sample processing: According to the national standard operation method, use sterile cotton swab, aseptic operation, spread the cockroaches and intestines of fish, shrimp, crab and other aquatic products into the prepared 2YT medium, 37 °C Incubate at 200 rpm overnight with shaking.
2. 提取基因组： 1ml过夜培养的样品 8000rpm离心 5分钟沉淀可能 存在的致病菌菌体， 弃上清（尽量空干）。 向沉淀中加入 lOOul去离子水重 悬， 8000rpm， 离心 5分钟， 去除上清。 加入 lOOul的裂解液（配方如下）， 100°C沸水浴 15分钟， 12000rpm离心 3分钟，上清即为粗提的 DNA模板。  2. Extraction of the genome: 1 ml of the overnight cultured sample was centrifuged at 8000 rpm for 5 minutes to precipitate the pathogenic bacteria that may be present, and the supernatant was discarded (as dry as possible). The supernatant was resuspended in lOOul of deionized water, centrifuged at 8000 rpm for 5 minutes, and the supernatant was removed. Add lOOul of the lysate (formulated as follows), centrifuge at 100 °C for 15 minutes, centrifuge at 12000 rpm for 3 minutes, and the supernatant is the crude DNA template.
附： 裂解液配方：  Attachment: Lysate formulation:
lxPCR缓冲液 （含 Mg+) lxPCR buffer (including Mg + )
0.5%的 NP 40  0.5% NP 40
0.5%的 Tween 20  0.5% of Tween 20
3. 扩增靶序列：取上述基因组提取方法提取的 3ul中层上清作为模板 加入 PCR反应混合液中， PCR反应混合液配方如下表 3所示。（注： 以下 表 3-表 4中的 PCR缓冲液、 MgCl2、dNTP混合物， Taq酶均购自 Sangon 公司） Multiplex PCR反应混合液配方
Figure imgf000015_0001
3. Amplification of the target sequence: 3 ul of the middle layer supernatant extracted by the above genomic extraction method was added as a template to the PCR reaction mixture, and the PCR reaction mixture formulation is shown in Table 3 below. (Note: PCR buffer, MgCl 2 , dNTP mixture in Table 3 - Table 4 below, Taq enzymes were purchased from Sangon) Multiplex PCR Reaction Mix Formula
Figure imgf000015_0001
ddH20 - 36 ddH 2 0 - 36
lOxPCR缓冲液 10x 5  lOxPCR buffer 10x 5
MgCl2 25mM 5 MgCl 2 25mM 5
dNTP混合物 lOmM 0.5  dNTP mixture lOmM 0.5
P-l和 P-2 ΙΟμΜ 各 1  P-l and P-2 ΙΟμΜ each 1
P-3和 P-4 ΙΟμΜ 各 0·3  P-3 and P-4 ΙΟμΜ each 0·3
Taq酶 5υ/μ1 0.5  Taq enzyme 5υ/μ1 0.5
注： 表中 P-1至 Ρ-2以及 Ρ-3和 Ρ-4为表 2中所列的引物。  Note: P-1 to Ρ-2 and Ρ-3 and Ρ-4 in the table are the primers listed in Table 2.
将反应管放入 PCR仪 （Biometra) 中， 设定的循环参数如下： 94 °C 5分钟  Place the reaction tube in the PCR instrument (Biometra) and set the cycle parameters as follows: 94 °C 5 minutes
94 °C 30秒  94 °C 30 seconds
50 °C 30秒  50 °C 30 seconds
72 °C 1分钟 回到第二步， 共 35个循环  72 °C 1 minute Back to the second step, a total of 35 cycles
72 °C 5分钟  72 °C 5 minutes
4°C 20分钟  4 ° C 20 minutes
3. 纯化： 将上述获得的 PCR扩增产物用纯化柱 （MILIPORE公司） 纯化， 具体步骤如下：  3. Purification: The PCR amplification product obtained above was purified by a purification column (MILIPORE). The specific steps are as follows:
( 1 ) 将 PCR产物转移至纯化柱中， 加水补足至 400μ1。  (1) Transfer the PCR product to the purification column and add water to 400 μl.
(2) 25°C、 6000rpm离心 15分钟， 丢弃收集管。  (2) Centrifuge at 25 ° C, 6000 rpm for 15 minutes, discard the collection tube.
(3 ) 将纯化柱转移到新的 1.5ml 的离心管中， 加入 25μ1 的超纯水 (MilliQ), 37°C放置 5分钟。  (3) Transfer the purification column to a new 1.5 ml centrifuge tube, add 25 μl of ultrapure water (MilliQ), and place at 37 ° C for 5 minutes.
(4)将纯化柱倒置放在 1.5ml的离心管上， 6000rpm离心 2分钟， 收 集产物。  (4) The purification column was placed upside down on a 1.5 ml centrifuge tube and centrifuged at 6000 rpm for 2 minutes to collect the product.
4. 标记靶序列： 取 12μ1纯化产物， 加入标记混合液中， 标记反应混 合液配方如下表 4所示。 标记混合液配方 4. Labeling the target sequence: 12 μl of the purified product was added to the labeled mixture, and the reaction mixture was labeled as shown in Table 4 below. Marking mixture formula
加样量  Sample loading
成分 浓度  Ingredient concentration
(μΐ)  (μΐ)
dd¾0 - 9.3  Dd3⁄40 - 9.3
lOxPCR缓冲液 10x 3  lOxPCR buffer 10x 3
MgCl2 25mM 3 MgCl 2 25mM 3
dNTP混合物 lOmM 0.3  dNTP mixture lOmM 0.3
P-2和 P-4 ΙΟμΜ 各 0·6  P-2 and P-4 ΙΟμΜ each 0·6
Cy3-dUTP 25nM 0.3  Cy3-dUTP 25nM 0.3
Taq酶 5υ/μ1 0.3  Taq enzyme 5υ/μ1 0.3
注： 表中 Ρ-2和 Ρ-4为表 2中所列的引物。 将反应管放入 PCR仪 （Biometra) 中， 设定的循环参数如下： 94 °C 5分钟 94 °C 30秒 50 °C 30秒  Note: Ρ-2 and Ρ-4 in the table are the primers listed in Table 2. Place the reaction tube in the PCR instrument (Biometra) and set the cycle parameters as follows: 94 °C 5 minutes 94 °C 30 seconds 50 °C 30 seconds
72 °C 1分钟 回到第二步， 共 35个循环 72 °C 5分钟 4°C 20分钟 72 °C 1 minute Back to the second step, a total of 35 cycles 72 °C 5 minutes 4 °C 20 minutes
5. 烘干： 将标记产物置 65 °C烘箱烘干。 5. Drying: The labeled product is oven dried at 65 °C.
6. 杂交： 向杂交盒 （博奥公司） 内预加入 70μ1 ddH20以保持湿度。6. Hybridization: 70 μl ddH 2 0 was pre-charged into the hybridization cassette (Boao) to maintain humidity.
12μ1杂交液（配方如下所示）回溶烘干产物并加在实施例三中制备的肠道 中常见致病菌检测基因芯片的探针阵列区域，盖上定制的盖片（博奥公司） (注意盖片和载玻片之间不能有气泡）， 盖紧杂交盒， 40°C水浴锅中杂交 16小时。 The 12μ1 hybridization solution (formulated as shown below) was used to reconstitute the dried product and added to the probe array area of the common pathogen detection gene chip in the intestine prepared in Example 3, and covered with a custom cover sheet (Boao Company) ( Note that there should be no air bubbles between the cover slip and the slide. Close the hybridization cassette and mix for 16 hours in a 40 ° C water bath.
7. 洗涤： 杂交到时， 取出杂交盒， 去除盖片， 将基因芯片依次在洗液 A中洗涤 3分钟， 洗液 B中洗涤 3分钟， 洗液 C中洗涤 90秒， 空气中风 干。 杂交液配方： 10%硫酸葡聚糖 （dextran Sulfate )； 25%甲酰胺 (formamide); 0.1% SDS (十二垸基硫酸钠） ； 6xSSPE 7. Washing: When hybridizing, remove the hybridization cassette, remove the cover slip, and wash the gene chip in Wash A for 3 minutes, Wash B for 3 minutes, Wash C for 90 seconds, and air dry. Hybrid solution formulation: 10% dextran Sulfate; 25% formamide; 0.1% SDS (sodium dodecyl sulfate); 6xSSPE
洗液 A: l xSSC (氯化钠 -柠檬酸钠溶液）； 0.1% SDS  Lotion A: l xSSC (sodium chloride - sodium citrate solution); 0.1% SDS
洗液 B: 0.05xSSC  Lotion B: 0.05xSSC
洗液 C: 95%乙醇  Lotion C: 95% ethanol
8. 扫描： 用 GenePix personal 4100A 生物芯片扫描仪 （AXON instrument) 扫描， 所用参数如下：  8. Scan: Scan with the GenePix personal 4100A BioScanner (AXON instrument) with the following parameters:
软件及版本： GenePix Pro 6.0  Software and version: GenePix Pro 6.0
official name: 575DF35  Official name: 575DF35
PMT Gain: 550 扫描分辨率： ΙΟμηι  PMT Gain: 550 Scanning Resolution: ΙΟμηι
扫描结果存为 JPG、 TIF、 GPR格式 用本发明的基因芯片分别检测常见的肠道中致病菌（志贺氏菌、 沙门 氏菌、 副溶血弧菌、 霍乱弧菌、 单增李斯特氏菌、 金黄色葡萄球菌、 化脓 性链球菌、 奇异变形杆菌、 普通变形杆菌、 潘氏变形杆菌） 时的杂交扫描 结果如图 3A-3J所示。  Scanning results are stored in JPG, TIF, GPR format. The gene chips of the present invention are used to detect common intestinal pathogenic bacteria (Shigella, Salmonella, Vibrio parahaemolyticus, Vibrio cholerae, Listeria monocytogenes, gold Hybridization scan results for Staphylococcus aureus, Streptococcus pyogenes, Proteus mirabilis, Proteus vulgaris, Proteus panicularum are shown in Figures 3A-3J.
9. 分析判读： 由于此芯片检测的目标菌有 10种， 探针 28 条， 属于 低密度芯片， 检测结果可由肉眼判断。 根据扫描出的杂交图像， 以正对照 探针的位置作为图像坐标， 判断出现荧光信号的特异探针的位置， 对照点 阵排布图判断出致病菌。 若只有正对照探针有信号， 则不存在此 10种致  9. Analysis and interpretation: Since there are 10 target bacteria detected by this chip and 28 probes, which are low-density chips, the test results can be judged by the naked eye. Based on the scanned hybridization image, the position of the positive control probe is used as the image coordinate to determine the position of the specific probe in which the fluorescent signal appears, and the pathogen is determined by the dot matrix arrangement. If only the positive control probe has a signal, there is no such 10
实施例 5 对基因芯片进行特异性鉴定和灵敏度检测 Example 5 Specific identification and sensitivity detection of gene chips
对实施例 3中制备的水产品中重要致病菌检测基因芯片的特异性进行 鉴定如下：  The specificity of the important pathogenic bacteria detection gene chip in the aquatic product prepared in Example 3 was identified as follows:
总计用 197株致病菌的代表性菌株和检出株以及他们的近缘菌株来鉴 定实施例 3中制备的水产品中重要致病菌检测基因芯片的特异性。在该特 异性鉴定试验中， 使用的所有菌株情况见表 5。 利用本发明的基因芯片和 上述检测方法进行杂交检测， 均显示了正确的杂交结果， 这说明本发明的 基因芯片具有良好的特异性。 A total of 197 strains of pathogenic bacteria and the detected strains and their related strains were used to identify the specificity of the important pathogenicity detecting gene chip in the aquatic product prepared in Example 3. In this special In the heterosexual identification test, the conditions of all the strains used are shown in Table 5. Hybridization detection using the gene chip of the present invention and the above detection method showed correct hybridization results, indicating that the gene chip of the present invention has good specificity.
表 5 : 特异性试验用到的菌株  Table 5: Strain for specificity test
菌株 细菌名称 拉丁文 不同来源的菌株数目  Strain Bacterial Name Latin Number of strains from different sources
总数 志贺氏菌 Shigella 17a, lb 18 沙门氏菌 Salmonella 14b， 3P, 2q， 4s, Ϋ 24 金黄色葡萄球 Staphylococcus aureus lc， 4d， 3r, 8s, 2q， 4U， \ 2P 25 囷 Total Shigella Shigella 17 a , l b 18 Salmonella 14 b , 3 P , 2 q , 4 s , Ϋ 24 Staphylococcus aureus l c , 4 d , 3 r , 8 s , 2 q , 4 U , \ 2 P 25 囷
化脓性链球菌 Streptococcus pyogenes 3d, Γ 4 副溶血弧菌 Vibrio parahaemolyticus 1°, 3C， l le 15 霍乱弧菌 Vibrio cholerae 3b, 6。 9 单增李斯特氏 Listeria monocytogenes 2d,lf, 3V 8 囷 Streptococcus pyogenes 3 d , Γ 4 Vibrio parahaemolyticus 1°, 3 C , ll e 15 Vibrio cholerae 3 b , 6. 9 Single-increasing Listeria monocytogenes 2 d , l f , 3 V 8 囷
奇异变形杆菌 Proteus mirabilis 2g， 4h，l。， 61， 3P, 2q， 2s, lw, 22 Proteus mirabilis 2 g , 4 h , l. , 6 1 , 3 P , 2 q , 2 s , l w , 22
丄  丄
普通变形杆菌 Proteus vulgaris ld， 3g, lh， 101, Γ 16 潘氏变形杆菌 Proteus penneri 131, 2j 15 大肠杆菌 Escherichia coli ld， 4b 5 阴沟肠杆菌 Enterobacter cloacae lc， ld， 2° 4 产气肠杆菌 Enterobacter aerogenes lc， ld， 1。 3 团聚肠杆菌 Enterobacter agglomerans 1° 1 日沟维肠杆菌 Enterobacter gergoviae 1J 1 沃氏葡萄球菌 Staphylococcus warneri lk 1 腐生葡萄球菌 Staphylococcus simulans lr 1 腐生葡萄球菌 Staphylococcus Proteus vulgaris l d , 3 g , l h , 10 1 , Γ 16 Proteus penneri 13 1 , 2 j 15 Escherichia coli l d , 4 b 5 Enterobacter cloacae l c , l d , 2° 4 Enterobacter aerogenes l c , l d , 1. 3 Enterobacter agglomerans 1° 1 Enterobacter gergoviae 1 J 1 Staphylococcus warneri l k 1 Staphylococcus simulans l r 1 Staphylococcus Staphylococcus
丄  丄
saprophyticus  Saprophyticus
表皮葡萄球菌 Staphylococcus i丄d  Staphylococcus epidermidis Staphylococcus i丄d
epidermidis  Epidermidis
松鼠葡萄球菌 Staphylococcus sciuri 1J 1 缓慢葡萄球菌 Staphylococcus lentus 1J 1 小牛葡萄球菌 Staphylococcus vitulinus 1J 1 山羊葡萄球菌 Staphylococcus caprae 1 头状葡萄球菌 Staphylococcus capitis ) 1 肺炎链球菌 Streptococcus penumoniae ld lk l1 Staphylococcus sciuri 1 J 1 Staphylococcus lentus 1 J 1 Staphylococcus vitulinus 1 J 1 Staphylococcus caprae 1 Staphylococcus capitis 1 Streptococcus penumoniae l d l k l 1
唾液链球菌 Streptococcus salivarius 1 c 1 Streptococcus salivarius 1 c 1
1 m  1 m
猪链球菌 Streptococcus suis 1 无乳链球菌 Streptococcus agalactiae 1 n 1 屎链球菌 Streptococcus faecium 1 d 1 粪链球菌 Streptococcus faecalis 1 c 1 豕链球菌 Streptococcus porcinus 1 k 1 牛链球菌 Streptococcus bovis 1 c 1 创伤弧菌 Vibrio vulnficus 1 k 1 河流弧菌 Vibrio fluvialis 1 k 1 弗氏弧菌 Vibrio furnissii 1 k 1 拟态弧菌 Vibrio minicus 1 o 1 溶藻弧菌 Vibrio alginolyticus 1 o 1 无害李斯特菌 Listeria innocua ) 1 威氏李斯特菌 Listeria welshimeri ) 1 产粘变形杆菌 Proteus myxofaciens i1, ik 2 a, 流行病学微生物学研究所 （IEM) , 中国预防医学研究会。 Streptococcus suis 1 Streptococcus agalactiae 1 n 1 Streptococcus faecium 1 d 1 Streptococcus faecalis 1 c 1 Streptococcus porcinus 1 k 1 Streptococcus bovis 1 c 1 Vibrio vulnificus Vibrio vulnficus 1 k 1 Vibrio fluvialis 1 k 1 Vibrio furnissii 1 k 1 Vibrio minicus 1 o 1 Vibrio alginolyticus 1 o 1 Listeria innocua 1 Listeria welshimeri 1 Proteus myxofaciens i 1 , i k 2 a, Institute of Epidemiology and Microbiology (IEM), Chinese Society for Preventive Medicine.
b, 澳大利亚医学与兽医学研究所 (IMVS )。 b, Australian Institute of Medicine and Veterinary Medicine (IMVS).
c, 中科院微生物研究所 （As) 。 c, Institute of Microbiology, Chinese Academy of Sciences (As).
d, 中国医学微生物菌种保藏中心 CMCC：)。 d, China Medical Microbial Culture Collection Center CMCC:).
e, 台湾苏州大学。 e, Suzhou University, Taiwan.
f, 中国农业微生物菌种保藏中心 (ACCC：)。 f, China Agricultural Microorganisms Collection (ACCC:).
g, 瑞典哥德堡大学培养物保藏中心 (CCUG：)。 g, Culture Collection of the University of Gothenburg, Sweden (CCUG:).
h, 捷克典型菌种保藏中心 (Prk：)。 h, Czech Collection of Typical Cultures (Prk:).
i, 波兰罗兹大学微生物学和免疫学研究所。 i, Institute of Microbiology and Immunology, University of Lodz, Poland.
j, 捷克微生物保藏中心 (CCM)。 j, Czech Collection of Microorganisms (CCM).
k, 美国典型菌种保藏中心 (ATCC：)。 k, American Type Culture Collection (ATCC:).
1, 澳大利亚新南威尔士州疾病感染和微生物中心 (CIDM)。  1, New South Wales Department of Disease Infection and Microbiology (CIDM), Australia.
m, 比利时菌种中心 （LMG)。 m, Belgian strain center (LMG).
n, 中国兽医学菌种国家保藏中心 (CVCC：)。 o, 来自天津出入境检验检疫局的检出株。 n, China National Collection of Veterinary Species (CVCC:). o, the detected strain from the Tianjin Entry-Exit Inspection and Quarantine Bureau.
P, 来自于天津中医药大学的临床菌株。 P, a clinical strain from Tianjin University of Traditional Chinese Medicine.
q, 来自于天津人民医院的临床菌株。 q, a clinical strain from Tianjin People's Hospital.
r, 来自于天津一中心医院的临床菌株。 r, a clinical strain from a central hospital in Tianjin.
s, 来自于天津三中心医院的临床菌株。 s, a clinical strain from Tianjin Sanzhong Hospital.
t, 来自于天津儿童医院的临床菌株。 t, a clinical strain from Tianjin Children's Hospital.
U, 来自于天津南开医院的临床菌株。  U, a clinical strain from Tianjin Nankai Hospital.
V, 来自于天津血液研究所的临床菌株。  V, a clinical strain from the Tianjin Blood Institute.
W,来自于天津胸科医院的临床菌株。  W, a clinical strain from Tianjin Chest Hospital.
X, 来自于天津医科大学第二附属医院的临床菌株。  X, a clinical strain from the Second Affiliated Hospital of Tianjin Medical University.
对实施例 3中制备的水产品中重要致病菌检测基因芯片的灵敏度进行 检测如下：  The sensitivity of the important pathogenicity detecting gene chip in the aquatic product prepared in Example 3 was examined as follows:
该基因芯片的检测灵敏度经过 153次杂交实验的验证， 0.1 ng的微量 基因组 DNA或 104 cfu/ml纯菌样品中菌落就可保证上述 10种致病菌具有 稳定、良好的杂交结果，这说明本发明的基因芯片具有很高的检测灵敏度。 实施例 6 对基因芯片进行双盲实验 The detection sensitivity of the gene chip was verified by 153 hybridization experiments. The colonies in 0.1 ng of micro genomic DNA or 10 4 cfu/ml pure bacteria samples ensured stable and good hybridization results of the above 10 pathogenic bacteria. The gene chip of the present invention has high detection sensitivity. Example 6 Double-blind experiment on gene chip
总计用 22株致病菌的代表性菌株， 在只知菌株编号不知菌株名称的 情况下进行盲测， 在该特异性鉴定试验中， 使用的所有菌株情况见表。 利 用本发明的基因芯片和上述检测方法进行杂交检测，均显示了正确的杂交 结果， 这说明本发明的基因芯片具有良好的特异性。 A total of 22 representative strains of pathogenic bacteria were used for blind measurement in the case where only the strain number was unknown to the strain name, and all the strains used in the specificity identification test are shown in the table. Hybridization detection using the gene chip of the present invention and the above detection method all showed correct hybridization results, indicating that the gene chip of the present invention has good specificity.
表 6: 双盲实验所用菌株 Table 6: Strains used in double-blind experiments
Figure imgf000021_0001
Figure imgf000021_0001
实施例 7 对基因芯片进行模拟实验 考虑潘氏变形杆菌在实际样品中出现的几率很小， 故重新组合除潘氏 变形杆菌外的其它 9种致病菌的特异探针形成图 4所示的模拟实施例单一 点阵探针排布规律示意图。 实验菌株处理： 挑单克隆菌株， 接入到配制好的 4mL 2YT培养基中， 37°C， 200rpm过夜振荡培养。取 100 菌液加入到 900 生理盐水中， 反复吹洗进行稀释。 按照同样方法， 依次制备 10倍递增稀释液， 每递增 稀释一次， 稀释至 10° cfu/mL 菌体浓度。 取不同稀释度的 2mL 菌液， 12000rpm离心 2分钟， 收集菌体， 备用。 模拟样品处理： 参考国家标准操作方法， 采取检样的部位为背脊， 先 用流水将鱼体体表冲洗， 去鳞， 在无菌条件下， 用 75%酒精棉球擦净鲫鱼 背部，用无菌剪刀沿鲫鱼脊背切开 5cm，再切开两端背脊分别向两侧翻开， 用无菌剪刀剪取鱼肉 3mg用研钵研碎， 加入到 30mL灭菌生理盐水中，浸 泡鱼肉， 混匀成稀释液。 取 0.5mL浸泡过鱼肉的生理盐水， 加入到离心 收集的备用菌体中。 12000rpm离心 2 分钟， 收集菌体。 Example 7 Simulation experiment on the gene chip Considering that the probability of occurrence of Proteus paniculata in the actual sample is small, the specific probes of the nine pathogenic bacteria other than Proteus spp. are recombined to form the same as shown in Fig. 4. Schematic diagram of the arrangement of single dot matrix probes in the simulated embodiment. Treatment of the experimental strain: The monoclonal strain was picked and inserted into the prepared 4 mL 2YT medium, and shake cultured at 37 ° C, 200 rpm overnight. 100 bacteria solution was added to 900 physiological saline, and repeatedly washed to dilute. In the same manner, 10-fold incremental dilutions were prepared in sequence, and diluted to 10 ° cfu/mL bacterial concentration for each incremental dilution. Take 2 mL of different dilutions of the bacteria solution, centrifuge at 12000 rpm for 2 minutes, collect the cells, and set aside. Simulated sample processing: Refer to the national standard operating method, take the sample part as the back ridge, first rinse the fish body surface with running water, remove the scale, under sterile conditions, wipe the back of the squid with 75% alcohol cotton ball, use no The scissors were cut 5 cm along the back of the squid, and then the ridges of both ends were cut open to the sides. The fish was cut with sterile scissors. 3 mg was ground with a mortar, added to 30 mL of sterile physiological saline, soaked in fish, and mixed. Into a diluent. 0.5 mL of physiological saline soaked in fish meat was taken and added to the spare cells collected by centrifugation. The cells were collected by centrifugation at 12,000 rpm for 2 minutes.
提取基因组以下步骤参考实施例 4。  Extraction of genomes The following steps are referred to in Example 4.
用本发明的基因芯片分别检测常见的肠道中致病菌（志贺氏菌、 沙门 氏菌、 副溶血弧菌、 单增李斯特氏菌、 金黄色葡萄球菌、 化脓性链球菌、 奇异变形杆菌、 普通变形杆菌） 时的模拟实验。  Using the gene chip of the present invention to detect common intestinal pathogenic bacteria (Shigella, Salmonella, Vibrio parahaemolyticus, Listeria monocytogenes, Staphylococcus aureus, Streptococcus pyogenes, Proteus mirabilis, common Simulation experiment when Proteus).
菌体最低检测浓度的确定：  Determination of the minimum detection concentration of the bacteria:
以下模拟实验所用菌株浓度是该项发明所能达到的最低检测浓度（表 The concentration of the strain used in the following simulation experiments is the lowest detection concentration that can be achieved by the invention (Table
7) 7)
Figure imgf000022_0001
杂交扫描结果如图 5A-5H所示， 其中， 图 5A为利用本发明的基因 芯片检测副溶血弧菌时的杂交结果； 图 5B 为利用本发明的基因芯片检测 化脓链球菌时的杂交结果； 图 5C为利用本发明的基因芯片检测沙门氏菌 时的杂交结果； 图 5D为利用本发明的基因芯片检测普通变形杆菌时的杂 交结果；图 5E为利用本发明的基因芯片检测奇特变形杆菌时的杂交结果; 图 5F为利用本发明的基因芯片检测志贺氏杆菌时的杂交结果；图 5G为利 用本发明的基因芯片检测金黄色葡萄球菌时的杂交结果； 图 5H为利用本 发明的基因芯片检测单增李斯特菌时的杂交结果。
Figure imgf000022_0001
The results of the hybridization scan are shown in FIGS. 5A to 5H, wherein FIG. 5A is a result of hybridization when the Vibrio parahaemolyticus is detected by the gene chip of the present invention; and FIG. 5B is a result of hybridization when the S. pyogenes is detected by the gene chip of the present invention; 5C is a hybridization result when Salmonella is detected by the gene chip of the present invention; FIG. 5D is a hybridization result when the Proteus vulgaris is detected by the gene chip of the present invention; and FIG. 5E is a hybridization when the Proteus mirabilis is detected by the gene chip of the present invention; Fig. 5F is the result of hybridization when detecting Shigella using the gene chip of the present invention; Fig. 5G is the result of hybridization when using the gene chip of the present invention to detect Staphylococcus aureus; Fig. 5H is the detection by the gene chip of the present invention Hybridization results when Listeria monocytogenes was added.
根据本发明的技术方案及其较佳实施例的描述，任何本领域的技术人 员， 在不脱离本发明的精神和范围内， 可以做出各种可能的等同改变或替 换， 而所有这些改变或替换都应属于本发明的权利要求的保护范围。  In view of the technical solutions of the present invention and the description of the preferred embodiments thereof, any person skilled in the art can make various possible equivalent changes or substitutions without departing from the spirit and scope of the invention, and all such changes or Substitutions are intended to fall within the scope of the claims of the present invention.

Claims

权 利 要 求 书 Claim
1. 一种检测水产品中致病菌的基因芯片，包括固相载体和固定在该固 相载体上的寡聚核苷酸探针，其特征在于所述固定在该固相载体上的寡聚 核苷酸探针包含从以下序列中选取的一种或多种： A gene chip for detecting a pathogenic bacteria in an aquatic product, comprising a solid phase carrier and an oligonucleotide probe immobilized on the solid phase carrier, characterized in that the oligonucleotide immobilized on the solid phase carrier The polynucleotide probe comprises one or more selected from the following sequences:
( 1 ) 从沙门氏菌、 副溶血弧菌、 霍乱弧菌、 单增李斯特氏菌、 金黄 色葡萄球菌、 化脓性链球菌、 奇异变形杆菌、 普通变形杆菌、 潘氏变形杆 菌的 16S-23 S rDNA间区以及志贺氏菌的 φ Η毒力基因中选取的 DNA序 列；  (1) 16S-23 S rDNA from Salmonella, Vibrio parahaemolyticus, Vibrio cholerae, Listeria monocytogenes, Staphylococcus aureus, Streptococcus pyogenes, Proteus mirabilis, Proteus vulgaris, Proteus paniculata The intervening region and the DNA sequence selected from the φ Η virulence gene of Shigella;
(2) 上述 （1 ) 中选取的 DNA序列的互补 DNA序列；  (2) a complementary DNA sequence of the DNA sequence selected in the above (1);
(3 ) 上述 （1 ) 或 （2) 中选取的 DNA序列的互补 RNA序列。  (3) A complementary RNA sequence of the DNA sequence selected in the above (1) or (2).
2. 根据权利要求 1所述的基因芯片， 其特征在于所述固定在该固相载 体上的寡聚核苷酸探针具有 SEQ ID NO:2-SEQ ID NO:26所示的 DNA序列 中的一种或多种。 The gene chip according to claim 1, wherein the oligonucleotide probe immobilized on the solid phase carrier has the DNA sequence shown in SEQ ID NO: 2 - SEQ ID NO: 26. One or more.
3. 根据权利要求 1或 2所述的基因芯片，其特征在于还包含阳性对照探 针、 阴性对照探针或荧光探针。  The gene chip according to claim 1 or 2, further comprising a positive control probe, a negative control probe or a fluorescent probe.
4. 根据权利要求 3所述的基因芯片， 其特征在于所述阳性对照探针选 自细菌 16S rDNA保守区中的 DNA片段或者其互补的 DNA或 RNA序列。  4. The gene chip according to claim 3, wherein the positive control probe is selected from a DNA fragment in a conserved region of the bacterial 16S rDNA or a complementary DNA or RNA sequence thereof.
5. 根据权利要求 4所述的基因芯片， 其特征在于所述阳性对照探针具 有 SEQ ID NO: 1所示的 DNA序列。  The gene chip according to claim 4, wherein the positive control probe has the DNA sequence shown in SEQ ID NO: 1.
6. 权利要求 5所述的基因芯片的应用， 其特征在于在检测志贺氏菌、 沙门氏菌、 副溶血弧菌、 霍乱弧菌、 单增李斯特氏菌、 金黄色葡萄球菌、 化脓性链球菌、 奇异变形杆菌、 普通变形杆菌、 潘氏变形杆菌中至少一种 致病菌的应用。  The use of the gene chip according to claim 5, characterized in that the detection of Shigella, Salmonella, Vibrio parahaemolyticus, Vibrio cholerae, Listeria monocytogenes, Staphylococcus aureus, Streptococcus pyogenes The use of at least one pathogenic bacteria in Proteus mirabilis, Proteus vulgaris, and Proteus panicula.
7. 根据权利要求 6所述的基因芯片的应用，其特征在于包含使用检测 引物，其中所述检测引物具有 SEQ ID NO: 27-SEQ ID NO: 30所示的 DNA 序列或其互补序列中的至少一种。 7. The use of the gene chip according to claim 6, comprising the use of a detection primer, wherein the detection primer has the DNA sequence shown in SEQ ID NO: 27-SEQ ID NO: 30 or a complement thereof At least one.
8.一种试剂盒， 其特征在于包含权利要求 1所述的基因芯片。 A kit comprising the gene chip of claim 1.
9. 根据权利要求 8所述的试剂盒，其特征在于还包括检测引物，其中 所述检测引物具有 SEQ ID NO: 27-SEQ ID NO: 30所示的 DNA序列或其 互补序列中的至少一种。  The kit according to claim 8, further comprising a detection primer, wherein the detection primer has at least one of a DNA sequence represented by SEQ ID NO: 27-SEQ ID NO: 30 or a complement thereof Kind.
10. 权利要求 8或 9所述的试剂盒的应用， 其特征在于在检测志贺氏 菌、 沙门氏菌、 副溶血弧菌、 霍乱弧菌、 单增李斯特氏菌、 金黄色葡萄球 菌、 化脓性链球菌、 奇异变形杆菌、 普通变形杆菌、 潘氏变形杆菌中至少 一种致病菌的应用。  The use of the kit according to claim 8 or 9, characterized in that the detection of Shigella, Salmonella, Vibrio parahaemolyticus, Vibrio cholerae, Listeria monocytogenes, Staphylococcus aureus, suppurative Use of at least one pathogenic bacteria in Streptococcus, Proteus mirabilis, Proteus vulgaris, and Proteus panicula.
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