WO2010060262A1 - Puce à adn et kit pour la détection d'une bactérie pathogène importante dans des produits aquatiques - Google Patents

Puce à adn et kit pour la détection d'une bactérie pathogène importante dans des produits aquatiques Download PDF

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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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gene chip
proteus
probe
dna sequence
seq
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PCT/CN2009/001190
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English (en)
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/fr

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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

L'invention porte sur une puce à ADN et un kit pour la détection d'une bactérie pathogène importante dans des produits aquatiques. La puce à ADN comprend des supports en phase solide et des sondes oligonucléotidiques fixées sur les supports en phase solide, lesdites sondes oligonucléotidiques comprenant une ou plusieurs séquences choisies parmi : (1) la séquence d'ADN qui est choisie parmi les espaceurs transcrits internes (ITS) d'ADNr 16S-23S de Salmonella, de Vibrio parahaemolyticus, de Vibrio cholerae, de Listeria monocytogenes, de Staphylococcus aureus, de Streptococcus pyogenes, de Proteus mirabilis, de Proteus vulgaris et de Proteus Penneri, ainsi que le gène ipaH de Shigella; (2) la séquence d'ADN complémentaire de la séquence d'ADN choisie en (1); et (3) la séquence d'ARN complémentaire de la séquence d'ADN choisie en (1) ou (2).
PCT/CN2009/001190 2008-11-03 2009-10-26 Puce à adn et kit pour la détection d'une bactérie pathogène importante dans des produits aquatiques WO2010060262A1 (fr)

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CN2008101723981A CN101724686B (zh) 2008-11-03 2008-11-03 检测水产品中重要致病菌的基因芯片和试剂盒
CN200810172398.1 2008-11-03

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CN102140507A (zh) * 2010-12-20 2011-08-03 南开大学 用于感染性腹泻的检测型基因芯片及检测用试剂盒
CN105133040A (zh) * 2015-06-30 2015-12-09 宁波大学 一种检测海洋致病弧菌的基因芯片及其制备方法与检测方法
CN109852674A (zh) * 2019-01-23 2019-06-07 浙江工商大学 基于随机扩增标记和原位合成微流体芯片的水产病原微生物检测方法
CN112011448A (zh) * 2020-07-20 2020-12-01 深圳市刚竹医疗科技有限公司 微流控芯片与试剂盒及其应用方法
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CN103540668A (zh) * 2013-10-22 2014-01-29 宁波大学 检测10种海域致病菌的基因芯片
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CN102140507A (zh) * 2010-12-20 2011-08-03 南开大学 用于感染性腹泻的检测型基因芯片及检测用试剂盒
CN102140515A (zh) * 2011-01-04 2011-08-03 天津生物芯片技术有限责任公司 用于检测水产品中重要致病菌的核苷酸及其应用
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CN109852674A (zh) * 2019-01-23 2019-06-07 浙江工商大学 基于随机扩增标记和原位合成微流体芯片的水产病原微生物检测方法
CN112011448A (zh) * 2020-07-20 2020-12-01 深圳市刚竹医疗科技有限公司 微流控芯片与试剂盒及其应用方法
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