WO2022143115A1 - 眼科临床微生物快速检测用探针、芯片、引物、试剂盒和应用 - Google Patents

眼科临床微生物快速检测用探针、芯片、引物、试剂盒和应用 Download PDF

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WO2022143115A1
WO2022143115A1 PCT/CN2021/137383 CN2021137383W WO2022143115A1 WO 2022143115 A1 WO2022143115 A1 WO 2022143115A1 CN 2021137383 W CN2021137383 W CN 2021137383W WO 2022143115 A1 WO2022143115 A1 WO 2022143115A1
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probes
seq
primers
primer
detection
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WO2022143115A9 (zh
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黄钰森
张碧凝
李文凤
刘晴
任志超
陈华波
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山东第一医科大学附属眼科研究所
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Priority to US17/929,875 priority Critical patent/US11891670B2/en
Publication of WO2022143115A9 publication Critical patent/WO2022143115A9/zh

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    • 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
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • 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 invention relates to the technical field of microorganism detection, in particular to probes, chips, primers, kits and applications for rapid detection of ophthalmic clinical microorganisms.
  • bacterial culture and smear staining were used to identify ophthalmic infectious bacteria, but there were problems of long identification time and low positive rate. Eye infections progress rapidly, and early and timely diagnosis is particularly critical in order to save patients' vision and reduce damage in a timely manner. Relying solely on bacterial morphological judgment may also lead to misjudgment, unable to characterize the pathogenic bacteria, and can only make a suspected diagnosis. Most ophthalmic infections are mixed infections with complex flora, usually the result of the combined action of multiple pathogenic bacteria. Traditional culture methods can usually only identify a single bacterial species, which has limited guiding significance for later treatment.
  • 16S rDNA is a sequence encoding rRNA on bacterial chromosomes, and the detection of bacteria by 16S rDNA molecular sequencing technology is highly specific. 16S rDNA sequencing has a 100% positive rate for identifying pathogenic bacteria, and can qualitatively and quantitatively identify pathogenic bacteria, and the identified major pathogenic bacteria groups provide a reference for clinical combination drugs.
  • 16S rDNA sequencing technology relies on large-scale sequencing platforms and equipment, and it cannot be applied on a large scale in hospitals at present, so this technology still relies on the sequencing services provided by third-party companies, and the process is cumbersome and the cycle of returning results is long. Sexual help.
  • the purpose of the present invention is to provide probes, chips and primers for rapid detection of ophthalmic clinical microorganisms.
  • the invention combines molecular diagnostic sequencing technology and chip technology, and invents a rapid detection chip that can directly detect common microorganisms in ophthalmology in a clinical pathogen laboratory, with strong specificity and high positive rate for detecting microorganisms in clinical samples of ophthalmology.
  • the detection accuracy is improved, and the detection speed is improved, making the product suitable for clinical large-scale promotion and use.
  • the present invention provides probes for rapid detection of ophthalmic clinical microorganisms.
  • the probes include probes for detecting Staphylococcus epidermidis, probes for detecting Staphylococcus aureus, probes for detecting Staphylococcus hemolyticus, and probes for detecting Pseudomonas aeruginosa.
  • Probes probes for the detection of Staphylococcus human, probes for the detection of Serratia marcescens, probes for the detection of Escherichia coli, probes for the detection of Bacillus subtilis and probes for the detection of Enterobacter cloacae;
  • the probes of the Staphylococcus epidermidis include probes whose nucleotide sequences are shown in SEQ ID NO. 1 to 20 respectively;
  • the probes of the Staphylococcus aureus include probes whose nucleotide sequences are shown in SEQ ID NO. 21-40 respectively;
  • the probes of the Staphylococcus hemolytica include probes whose nucleotide sequences are shown in SEQ ID NO.41-60 respectively;
  • the Pseudomonas aeruginosa probes include probes whose nucleotide sequences are shown in SEQ ID NO. 61-80 respectively;
  • the probes of the human Staphylococcus include probes whose nucleotide sequences are respectively shown in SEQ ID NO.81 ⁇ 100;
  • the probes of the Serratia marcescens include probes whose nucleotide sequences are shown in SEQ ID NO.101-120 respectively;
  • the Escherichia coli probes include probes whose nucleotide sequences are shown in SEQ ID NO. 121-140 respectively;
  • Bacillus subtilis probes include probes whose nucleotide sequences are shown in SEQ ID NO. 141-147 respectively;
  • the probes for Enterobacter cloacae include probes whose nucleotide sequences are shown in SEQ ID NOs. 148-156 respectively.
  • the present invention also provides a rapid detection chip for ophthalmic clinical microorganisms, and the chip contains the probe described in the above technical solution.
  • the present invention also provides primers for rapid detection of ophthalmic clinical microorganisms
  • the primers include primers for detecting Staphylococcus epidermidis, primers for detecting Staphylococcus aureus, primers for detecting Staphylococcus hemolyticus, primers for detecting Pseudomonas aeruginosa, primers for detecting human Primers for Staphylococcus, primers for detection of Serratia marcescens, primers for detection of Escherichia coli, primers for detection of Bacillus subtilis and primers for detection of Enterobacter cloacae;
  • the primers of the Staphylococcus epidermidis include a forward primer whose nucleotide sequence is shown in SEQ ID NO.157 and a reverse primer whose nucleotide sequence is shown in SEQ ID NO.158;
  • the primer nucleotide sequence of described Staphylococcus aureus is as the forward primer shown in SEQ ID NO.159 and the nucleotide sequence as the reverse primer shown in SEQ ID NO.160;
  • the primer nucleotide sequence of described Staphylococcus hemolytica is as the forward primer shown in SEQ ID NO.161 and the nucleotide sequence as the reverse primer shown in SEQ ID NO.162;
  • the primer nucleotide sequence of described Pseudomonas aeruginosa is as the forward primer shown in SEQ ID NO.163 and the nucleotide sequence as the reverse primer shown in SEQ ID NO.164;
  • the primer nucleotide sequence of described Staphylococcus human is the forward primer shown in SEQ ID NO.165, and the nucleotide sequence is the reverse primer shown in SEQ ID NO.166;
  • the primer nucleotide sequence of the Serratia marcescens is as the forward primer shown in SEQ ID NO.167 and the nucleotide sequence as the reverse primer shown in SEQ ID NO.168;
  • the primer nucleotide sequence of described Escherichia coli is the forward primer shown in SEQ ID NO.169 and the reverse primer shown in nucleotide sequence as SEQ ID NO.170;
  • the primer nucleotide sequence of described Bacillus subtilis is as the forward primer shown in SEQ ID NO.171 and the nucleotide sequence as the reverse primer shown in SEQ ID NO.172;
  • the nucleotide sequence of the primers of Enterobacter cloacae is the forward primer shown in SEQ ID NO.173 and the reverse primer whose nucleotide sequence is shown in SEQ ID NO.174.
  • the 5' ends of the forward primers in the primers are all labeled with fluorescent groups.
  • the fluorescent group includes CY3.
  • the present invention also provides a primer-probe set for rapid detection of ophthalmic clinical microorganisms, wherein the primer-probe set includes the probe described in the above technical solution and the primer described in the above technical solution.
  • the present invention also provides a rapid detection kit for ophthalmic clinical microorganisms, the kit includes the chip described in the above technical solution and the primer described in the above technical solution.
  • the present invention provides a probe for rapid detection of ophthalmic clinical microorganisms.
  • the present invention compares the whole genome of 9 most common pathogenic bacteria in ophthalmic infectious diseases, finds out the gene sequences with intra-species homology >95% and interspecies homology ⁇ 75%, and constructs hybridization probes for the selected sequences , and constructed PCR primers with fluorescent groups, the present invention subsequently synthesizes hybridization probes on the gene chip, performs PCR on pathogenic bacteria with fluorescently labeled PCR primers to obtain fluorescently labeled products, and hybridizes the products with the hybridization probes , can achieve rapid identification of pathogenic bacteria.
  • the method of the present invention is simple to operate, takes a relatively short time, and can obtain results within 24 hours;
  • the method of the present invention can detect the existence of all bacteria to be tested in the sample in one experiment;
  • the method of the present invention is highly targeted.
  • the specific primers only amplify the target gene of the target species, without generating a large amount of useless data, and even if the primers have non-specific amplification, in the follow-up. In hybridization experiments, it can also be excluded by specific probes, which will not interfere with the final result;
  • the factors that limit the number of strains that can be detected in one experiment are the number of probes that can be accommodated on a chip and whether the known gene sequence or partial sequence specificity of the strain to be tested is sufficient.
  • the inventive method can be extended to detect a wider variety of bacteria present in a sample if desired.
  • Fig. 1 is the hybridization signal diagram that utilizes the chip of the present invention to detect Staphylococcus epidermidis provided by the present invention
  • Fig. 2 is the hybridization signal diagram that utilizes the chip of the present invention to detect Staphylococcus aureus provided by the present invention
  • Fig. 3 is the hybridization signal diagram that utilizes the chip of the present invention to detect Staphylococcus hemolytica provided by the present invention
  • Fig. 4 is the hybridization signal diagram that utilizes the chip of the present invention to detect Pseudomonas aeruginosa provided by the present invention
  • Fig. 5 is the hybridization signal diagram of utilizing the chip of the present invention to detect Staphylococcus human provided by the present invention
  • Fig. 6 is the hybridization signal diagram that utilizes the chip of the present invention to detect Serratia marcescens provided by the present invention
  • Fig. 7 is the hybridization signal diagram of utilizing the chip of the present invention to detect Escherichia coli provided by the present invention
  • Fig. 8 is the hybridization signal diagram that utilizes the chip of the present invention to detect Bacillus subtilis provided by the present invention
  • Fig. 9 is the hybridization signal diagram of detecting Enterobacter cloacae using the chip of the present invention provided by the present invention.
  • the present invention provides probes for rapid detection of ophthalmic clinical microorganisms.
  • the probes include a probe for detecting Staphylococcus epidermidis, a probe for detecting Staphylococcus aureus, a probe for detecting Staphylococcus hemolytica, and a probe for detecting Pseudomonas aeruginosa.
  • the probes of the Staphylococcus epidermidis include probes whose nucleotide sequences are shown in SEQ ID NO. 1 to 20 respectively;
  • the probes of the Staphylococcus aureus include probes whose nucleotide sequences are shown in SEQ ID NO. 21-40 respectively;
  • the probes of the Staphylococcus hemolytica include probes whose nucleotide sequences are shown in SEQ ID NO.41-60 respectively;
  • the Pseudomonas aeruginosa probes include probes whose nucleotide sequences are shown in SEQ ID NO. 61-80 respectively;
  • the probes of the human Staphylococcus include probes whose nucleotide sequences are respectively shown in SEQ ID NO.81 ⁇ 100;
  • the probes of the Serratia marcescens include probes whose nucleotide sequences are shown in SEQ ID NO.101-120 respectively;
  • the Escherichia coli probes include probes whose nucleotide sequences are shown in SEQ ID NO. 121-140 respectively;
  • Bacillus subtilis probes include probes whose nucleotide sequences are shown in SEQ ID NO. 141-147 respectively;
  • the probes for Enterobacter cloacae include probes whose nucleotide sequences are shown in SEQ ID NOs. 148-156 respectively.
  • the present invention collects clinically cultivated strains, performs 16S rDNA sequencing to determine strain information, and then performs genome sequence retrieval for strains. After finding the conserved sequence of a single strain, compare the conserved sequences of all strains in pairs to find out all similar sequences and exclude them. Continue to compare with the NCBI database to obtain strain-specific conserved gene fragments (internal identical). Gene sequences with homology >95% and interspecies homology ⁇ 75%) were used to design probes.
  • the present invention uses Tiling array to design probes on LCS chips. Tiling array probe design was performed on the PCR products of each of the above strains, and multiple probe sequences were designed for the regions with mutations. A total of 2117 probes were designed, and 555 probes were available.
  • the probes were hybridized with the fluorescent PCR products of each strain, and the probe with the strongest hybridization signal was selected.
  • the hybridization signals of other strains were compared, and the probes with the best specificity were selected as the final 86 probes used on the pathogenic bacteria detection chip.
  • the probe of the present invention has the strongest hybridization signal value and the best specificity with the target bacterial species, and produces no signal with non-target bacterial species or produces a signal value close to the background signal.
  • the present invention also provides an ophthalmic clinical microorganism rapid detection chip, which contains the probe described in the above technical solution.
  • the chip hybridization method of the present invention preferably includes the following steps: using LCS_beads magnetic beads to purify the fluorescent PCR product, removing excess primers and impurities, and using an ASP-3700 microspectrophotometer to measure the absorbance at 260 nm and 550 nm to determine the fluorescence incorporation density. The optimum hybridization was when the fluorescence incorporation density was between 20 and 50.
  • the present invention also provides primers for rapid detection of ophthalmic clinical microorganisms
  • the primers include primers for detecting Staphylococcus epidermidis, primers for detecting Staphylococcus aureus, primers for detecting Staphylococcus hemolyticus, primers for detecting Pseudomonas aeruginosa, primers for detecting human Primers for Staphylococcus, primers for detecting Serratia marcescens, primers for detecting Escherichia coli, primers for detecting Bacillus subtilis and primers for detecting Enterobacter cloacae; as shown in Table 2.
  • the primers of the Staphylococcus epidermidis include a forward primer whose nucleotide sequence is shown in SEQ ID NO.157 and a reverse primer whose nucleotide sequence is shown in SEQ ID NO.158;
  • the primer nucleotide sequence of the Staphylococcus aureus is the forward primer shown in SEQ ID NO.159 and the reverse primer shown in the nucleotide sequence of SEQ ID NO.160;
  • the primer nucleotide sequence of described Staphylococcus hemolytica is as the forward primer shown in SEQ ID NO.161 and the nucleotide sequence as the reverse primer shown in SEQ ID NO.162;
  • the primer nucleotide sequence of described Pseudomonas aeruginosa is as the forward primer shown in SEQ ID NO.163 and the nucleotide sequence as the reverse primer shown in SEQ ID NO.164;
  • the primer nucleotide sequence of described Staphylococcus human is the forward primer shown in SEQ ID NO.165, and the nucleotide sequence is the reverse primer shown in SEQ ID NO.166;
  • the primer nucleotide sequence of the Serratia marcescens is as the forward primer shown in SEQ ID NO.167 and the nucleotide sequence as the reverse primer shown in SEQ ID NO.168;
  • the primer nucleotide sequence of described Escherichia coli is the forward primer shown in SEQ ID NO.169 and the reverse primer shown in nucleotide sequence as SEQ ID NO.170;
  • the primer nucleotide sequence of described Bacillus subtilis is as the forward primer shown in SEQ ID NO.171 and the nucleotide sequence as the reverse primer shown in SEQ ID NO.172;
  • the nucleotide sequence of the primers of Enterobacter cloacae is the forward primer shown in SEQ ID NO.173 and the reverse primer whose nucleotide sequence is shown in SEQ ID NO.174.
  • the present invention collects clinically cultivated strains, performs 16S rDNA sequencing to determine strain information, and then performs genome sequence retrieval for strains. After finding the conserved sequence of a single strain, compare the conserved sequences of all strains in pairs to find out all similar sequences and exclude them. Continue to compare with the NCBI database to obtain strain-specific conserved gene fragments (internal identical). Gene sequences with homology >95% and interspecies homology ⁇ 75%) were used to design primers. A batch of primers were designed with primer5, and the designed primers were screened with DNAstat to obtain primer combinations.
  • the 5' ends of the forward primers in the primers are preferably all labeled with fluorescent groups.
  • the fluorescent group preferably includes CY3.
  • a CY3 fluorescent group is added to the 5' end of the forward primer to obtain a strain-specific fluorescent primer.
  • the present invention also provides a primer-probe set for rapid detection of ophthalmic clinical microorganisms, wherein the primer-probe set includes the probe described in the above technical solution and the primer described in the above technical solution.
  • the present invention also provides a rapid detection kit for ophthalmic clinical microorganisms, the kit includes the chip described in the above technical solution and the primer described in the above technical solution.
  • the probes, chips, primers and kits for rapid detection of ophthalmic clinical microorganisms according to the present invention are described in further detail below with reference to specific examples.
  • the technical solutions of the present invention include but are not limited to the following examples.
  • the primers screened and verified in Example 2 have high specificity and can generate readable fluorescent signals after hybridization.
  • the number of probes that can be accommodated in one LCS chip is: 3968 (31*128).
  • the present invention performs Tiling array probe design on the PCR products of each bacteria, that is, the isometric displacement method is adopted,
  • a sequence of a certain length in the present invention, the length is 25 bp
  • the target sequence a sequence of a certain length (in the present invention, the length is 25 bp) is selected from the beginning to the end to form a probe combination, wherein the adjacent probe sequences differ by one base until the entire target sequence is covered.
  • the NCBI blast results of the PCR product sequence, for the segment with mutations, design multiple probe sequences according to the mutated bases. If a probe covers too many mutations (more than 3 mutations in 25bp), this is discarded. probe.
  • the probe is suitable for hybridization needs to be screened by performing hybridization experiments, and the successfully amplified fluorescent products are selected for hybridization reaction with the chip. Data was extracted after the chip was scanned. The obtained data are analyzed, and probes with good hybridization signal and strong specificity are screened.
  • the fluorescent product obtained by the amplification of the primers screened in Example 2 was subjected to a hybridization reaction with the probe screened in Example 3, and data was extracted after chip scanning. Analyze the signals of these probes on the hybridization chip of other strains, and select the probe with the best specificity (that is, the signal value on the hybridization chip of other strains is relatively weak, close to the negative control probe) as the pathogenic bacteria detection chip
  • the final detection probe as shown in Table 1, has a total of 146 probes.
  • the fluorescence-incorporating PCR products were purified with LCS_beads magnetic beads, and after removal of excess primers and impurities, the absorbance at 260 nm and 550 nm was measured with an ASP-3700 microspectrophotometer to determine the fluorescence infiltration density (FOI).
  • An FOI of 20-50 is optimal for hybridization.
  • Fluorescence incorporation density FOI Fluorescence incorporation pmol ⁇ (324.5/cDNA nmol)
  • A absorbance of Cy3 at 550nm or Cy5 at 650nm
  • nuclease-free water preheated at 95°C, cycle and wash at the highest speed for 5-6 minutes;
  • Stripping buffer composition 0.3mM EDTA, 50% formamide, pH 6.6-6.8, this buffer can wash away the nucleic acid sequence that has been hybridized on the probe.
  • the scanner scans the cleaned chip.
  • the temperature of the chip pedestal during the above system cleaning and chip cleaning process is 40 °C.
  • This step is to wash away possible impurities and nucleic acid sequences that may be hybridized on the chip, so as to obtain a relatively clean and uniform background, ready for hybridization.
  • Hybridization buffer (Hybridization solution HB) was washed for 10 minutes at the binding speed (500 ⁇ l/min); Hybridization buffer (Hybridization solution HB) components: 6X SSPE, 25% Formamide, pH 6.6 ⁇ 6.8, Hybridization buffer (Hybridization solution HB) HB) provides a suitable pH environment and salt ion concentration for the hybridization reaction, in which Formamide can reduce the Tm value of the DNA double-strand, so that the sample can carry out the hybridization reaction at 30 °C.
  • Blocking buffer (blocking solution BSA) was washed for 5-6 minutes at the binding speed;
  • Blocking buffer (blocking solution BSA): 148 ⁇ L of hybridization solution HB, 2 ⁇ L of 100 ⁇ heat-treated BSA, BSA in this buffer can block the probe-free part of the chip to reduce the background signal of the chip.
  • Hybridization sample preparation 200 ng of purified PCR product + equal volume of Hybridization buffer (Hybridization solution HB, final volume of 50 ⁇ l), denatured at 95°C for 5 min and then quickly placed on ice for 3 min, the prepared sample was added to the Blocking buffer, Hybridization was performed by cycling at binding speed for 16 hours after mixing.
  • Hybridization buffer Hybridization solution HB, final volume of 50 ⁇ l
  • the hybridization time is set to 16 hours in order to make the hybridization reaction time long enough to reach the reaction equilibrium, which can be appropriately adjusted in actual operation.
  • the temperature of the chip stand during sample hybridization is 40°C.
  • Hybridization buffer Hybridization solution HB
  • wash buffer (washing liquid WB) was circulated at 40°C for 20 min at the cleaning speed.
  • Wash buffer 500 ⁇ L HB, 500 ⁇ L Nuclease-free water, 20 ⁇ L 10% SDS, note that the Wash buffer should be prepared and used immediately.
  • Figure 1 Schematic diagram of hybridization signal and FOI value determination after chip scanning.
  • the order of hybridization signal intensity is white>red>yellow>green>blue.
  • FIG. 2 Hybridization signal diagram of Staphylococcus epidermidis. It can be seen that a specific hybridization signal is generated after scanning the chip, indicating that the chip can be used for screening and detection of Staphylococcus epidermidis in practical clinical applications;
  • Figure 3 The hybridization signal diagram of Staphylococcus aureus, it can be seen that a specific hybridization signal is generated after scanning the chip, indicating that the chip can be used for screening and detection of Staphylococcus aureus in practical clinical applications;
  • FIG. 3 Hybridization signal diagram of Staphylococcus hemolytica. It can be seen that a specific hybridization signal is generated after scanning the chip, indicating that the chip can be used for screening and detection of Staphylococcus hemolytica in practical clinical applications;
  • Figure 4 The hybridization signal diagram of Pseudomonas aeruginosa, it can be seen that after scanning the chip, a specific hybridization signal is generated for the strain, indicating that the chip can be used for screening and detection of Pseudomonas aeruginosa in practical clinical applications;
  • Figure 5 The hybridization signal diagram of Staphylococcus human, it can be seen that the hybridization signal specific to the strain is generated after scanning the chip, indicating that the chip can be used for screening and detection of Staphylococcus human in practical clinical application;
  • FIG. 6 Hybridization signal diagram of Serratia marcescens, it can be seen that a specific hybridization signal is generated after scanning the chip, indicating that the chip can be used for screening and detection of Serratia marcescens in practical clinical applications ;
  • Figure 7 The hybridization signal diagram of Escherichia coli, it can be seen that the hybridization signal specific to the strain is generated after scanning the chip, indicating that the chip can be used for screening and detection of Escherichia coli in clinical practice;
  • FIG. 8 Hybridization signal diagram of Bacillus subtilis, it can be seen that a specific hybridization signal is generated after scanning the chip, indicating that the chip can be used for screening and detection of Bacillus subtilis in practical clinical applications;
  • Figure 9 The hybridization signal diagram of Enterobacter cloacae, it can be seen that the hybridization signal specific to the bacterial species is generated after scanning the chip, indicating that the chip can be used for screening and detection of Enterobacter cloacae in practical clinical application.

Abstract

本发明涉及一种眼科临床微生物快速检测用探针、芯片、引物、试剂盒和应用，属于临床微生物检测技术领域。本发明所述探针包括分别检测表皮葡萄球菌、金黄色葡萄球菌、溶血葡萄球菌、铜绿假单胞菌、人葡萄球菌、粘质沙雷氏菌、大肠埃希氏菌、枯草芽孢杆菌和阴沟肠杆菌的探针。本发明还公开了对目标菌种进行扩增的引物，包含可以扩增出目标菌种种内同源性>95％，种间同源性<75％的基因序列片段的引物。还公开了在芯片上合成杂交探针的方法、杂交反应的方法、扫描检测的方法。

Description

眼科临床微生物快速检测用探针、芯片、引物、试剂盒和应用 技术领域
本发明涉及微生物检测技术领域，具体涉及眼科临床微生物快速检测用探针、芯片、引物、试剂盒和应用。
背景技术
以往眼科感染菌的确定采用细菌培养和涂片染色法，但存在鉴定时间长和阳性率低的问题。眼部感染进展迅速，为了及时挽救患者视力以及减轻损害，早期及时诊断尤为关键。单纯依靠细菌形态学判断也可能造成误判，无法定性感染病原菌，只能作出疑似诊断。大部分眼科感染案例为混合型感染,菌群组成复杂，通常是多种致病菌共同作用的结果。传统培养方式通常只能鉴别出单一菌种，对于后期治疗的指导意义有限。16S rDNA是细菌染色体上编码rRNA的序列，利用16S rDNA分子测序技术检测细菌具有高度的特异性。16S rDNA测序对于鉴别病原菌具有100％的阳性率，并且可以将致病菌定性定量，鉴定出的主要致病菌群为临床联合用药提供参考。但16S rDNA测序技术依赖大型测序平台和设备，目前尚不能在医院临床上大规模应用，使得本技术仍然依赖于第三方公司提供的测序服务，流程繁琐返回结果周期长，无法对日常诊断提供时效性的帮助。
发明内容
本发明的目的在于提供眼科临床微生物快速检测用探针、芯片、引物。本发明将分子诊断测序技术与芯片技术相结合，发明出一种可以在临床病原实验室直接进行眼科常见微生物检测的快速检测芯片，特异性强，检测眼科临床样品中的微生物阳性率高，既提高了检测的准确性，又提高了检测速度，使本产品适宜于临床大规模推广使用。
本发明提供了眼科临床微生物快速检测用探针，所述探针包括检测表皮葡萄球菌的探针、检测金黄色葡萄球菌的探针、检测溶血葡萄球菌的探针、检测铜绿假单胞菌的探针、检测人葡萄球菌的探针、检测粘质沙雷氏菌的探针、检测大肠埃希氏菌的探针、检测枯草芽孢杆菌的探针和检测阴沟肠杆菌的探针；
所述表皮葡萄球菌的探针包括核苷酸序列分别如SEQ ID NO.1～20所示的探针；
所述金黄色葡萄球菌的探针包括核苷酸序列分别如SEQ ID NO.21～40所示的探针；
所述溶血葡萄球菌的探针包括核苷酸序列分别如SEQ ID NO.41～60所示的探针；
所述铜绿假单胞菌的探针包括核苷酸序列分别如SEQ ID NO.61～80所示的探针；
所述人葡萄球菌的探针包括核苷酸序列分别如SEQ ID NO.81～100所示的探针；
所述粘质沙雷氏菌的探针包括核苷酸序列分别如SEQ ID NO.101～120所示的探针；
所述大肠埃希氏菌的探针包括核苷酸序列分别如SEQ ID NO.121～140所示的探针；
所述枯草芽孢杆菌的探针包括核苷酸序列分别如SEQ ID NO.141～147所示的探针；
所述阴沟肠杆菌的探针包括核苷酸序列分别如SEQ ID NO.148～156所示的探针。
本发明还提供了眼科临床微生物快速检测芯片，所述芯片上含有上述技术方案所述的探针。
本发明还提供了眼科临床微生物快速检测用引物，所述引物包括检测表皮葡萄球菌的引物、检测金黄色葡萄球菌的引物、检测溶血葡萄球菌的引物、检测铜绿假单胞菌的引物、检测人葡萄球菌的引物、检测粘质沙雷氏菌的引物、检测大肠埃希氏菌的引物、检测枯草芽孢杆菌的引物和检测阴沟肠杆菌的引物；
所述表皮葡萄球菌的引物包括核苷酸序列如SEQ ID NO.157所示的正向引物和核苷酸序列如SEQ ID NO.158所示的反向引物；
所述金黄色葡萄球菌的引物核苷酸序列如SEQ ID NO.159所示的正向 引物和核苷酸序列如SEQ ID NO.160所示的反向引物；
所述溶血葡萄球菌的引物核苷酸序列如SEQ ID NO.161所示的正向引物和核苷酸序列如SEQ ID NO.162所示的反向引物；
所述铜绿假单胞菌的引物核苷酸序列如SEQ ID NO.163所示的正向引物和核苷酸序列如SEQ ID NO.164所示的反向引物；
所述人葡萄球菌的引物核苷酸序列如SEQ ID NO.165所示的正向引物、核苷酸序列如SEQ ID NO.166所示的反向引物；
所述粘质沙雷氏菌的引物核苷酸序列如SEQ ID NO.167所示的正向引物和核苷酸序列如SEQ ID NO.168所示的反向引物；
所述大肠埃希氏菌的引物核苷酸序列如SEQ ID NO.169所示的正向引物和核苷酸序列如SEQ ID NO.170所示的反向引物；
所述枯草芽孢杆菌的引物核苷酸序列如SEQ ID NO.171所示的正向引物和核苷酸序列如SEQ ID NO.172所示的反向引物；
所述阴沟肠杆菌的引物核苷酸序列如SEQ ID NO.173所示的正向引物和核苷酸序列如SEQ ID NO.174所示的反向引物。
优选的是，所述引物中的正向引物的5’端均进行荧光基团标记。
优选的是，所述荧光基团包括CY3。
本发明还提供了一种眼科临床微生物快速检测用引物探针组，所述引物探针组包括上述技术方案所述的探针和上述技术方案所述的引物。
本发明还提供了一种眼科临床微生物快速检测试剂盒，所述试剂盒包括上述技术方案所述芯片和上述技术方案所述的引物。
本发明提供了眼科临床微生物快速检测用探针。本发明对眼科感染疾病中最常见的9种病原菌进行全基因组比对，找出内同源性>95％，种间同源性<75％的基因序列，针对筛选出的序列构建杂交探针，并构建了带有荧光基团的PCR引物，本发明后续在基因芯片上合成杂交探针，用荧光标记的PCR引物对致病菌进行PCR得到荧光标记产物，将产物与杂交探针进行杂交，可以实现快速鉴别病原菌。
利用本发明探针制备得到的芯片，具有以下优点：
检测快速：相对于需要数天时间的传统细菌培养检测等方式，本发明方法操作简单，花费时间相对较短，24h内即可得到结果；
高通量：相对于普通的一代测序和qPCR检测方式，本发明方法一次实验即可检测所有待测细菌在样本中的存在情况；
判读简单：相对于二代测序等方式，本发明方法的判读十分简单，通过芯片扫描图中探针的二维位置对应的菌种信息即可立刻确认该菌种在样本中存在的情况，无需复杂的数据分析过程；
特异性：相对于近期发展宏基因组测序等方式，本发明方法针对性强，特异性引物仅扩增目标物种目标基因，不产生大量的无用数据，且即使引物存在非特异性扩增，在后续的杂交实验中于也可通过特异性探针排除，对最终结果不产生干扰；
可拓展性：限制一次实验能检测菌种数量的因素为一张芯片可容纳的探针数量和待测菌种已知的基因序列或者部分序列特异性是否足够，满足这两个条件下，本发明方法可进行拓展，在有需要的情况下检测样本中存在的更多种类的细菌。
附图说明
图1为本发明提供的利用本发明芯片检测表皮葡萄球菌的杂交信号图；
图2为本发明提供的利用本发明芯片检测金黄色葡萄球菌的杂交信号图；
图3为本发明提供的利用本发明芯片检测溶血葡萄球菌的杂交信号图；
图4为本发明提供的利用本发明芯片检测铜绿假单胞菌的杂交信号图；
图5为本发明提供的利用本发明芯片检测人葡萄球菌的杂交信号图；
图6为本发明提供的利用本发明芯片检测粘质沙雷氏菌的杂交信号图；
图7为本发明提供的利用本发明芯片检测大肠埃希氏菌的杂交信号图；
图8为本发明提供的利用本发明芯片检测枯草芽孢杆菌的杂交信号图；
图9为本发明提供的利用本发明芯片检测阴沟肠杆菌的杂交信号图。
具体实施方式
本发明提供了眼科临床微生物快速检测用探针，所述探针包括检测表皮葡萄球菌的探针、检测金黄色葡萄球菌的探针、检测溶血葡萄球菌的探针、 检测铜绿假单胞菌的探针、检测人葡萄球菌的探针、检测粘质沙雷氏菌的探针、检测大肠埃希氏菌的探针、检测枯草芽孢杆菌的探针和检测阴沟肠杆菌的探针；如表1所示。
表1探针序列表
Figure PCTCN2021137383-appb-000001
Figure PCTCN2021137383-appb-000002
Figure PCTCN2021137383-appb-000003
Figure PCTCN2021137383-appb-000004
所述表皮葡萄球菌的探针包括核苷酸序列分别如SEQ ID NO.1～20所示的探针；
所述金黄色葡萄球菌的探针包括核苷酸序列分别如SEQ ID NO.21～40所示的探针；
所述溶血葡萄球菌的探针包括核苷酸序列分别如SEQ ID NO.41～60所示的探针；
所述铜绿假单胞菌的探针包括核苷酸序列分别如SEQ ID NO.61～80所示的探针；
所述人葡萄球菌的探针包括核苷酸序列分别如SEQ ID NO.81～100所示的探针；
所述粘质沙雷氏菌的探针包括核苷酸序列分别如SEQ ID NO.101～120所示的探针；
所述大肠埃希氏菌的探针包括核苷酸序列分别如SEQ ID NO.121～140所示的探针；
所述枯草芽孢杆菌的探针包括核苷酸序列分别如SEQ ID NO.141～147所示的探针；
所述阴沟肠杆菌的探针包括核苷酸序列分别如SEQ ID NO.148～156所示的探针。
本发明收集临床培养的菌株，进行16S rDNA测序确定菌种信息后，对菌种进行基因组序列检索。在找到单一菌种保守序列后，再将所有菌种的保守序列进行两两比对，找出所有相似序列并排除在外，继续通过与NCBI数据库比对，得到菌株的特异保守基因片段(内同源性>95％，种间同源性<75％的基因序列)用于设计探针。本发明采用Tiling array在LCS芯片上进行探针设计。对上述各个菌种PCR产物进行Tiling array探针设计，对于存在突变的区域设计多条探针序列。共设计2117条探针，可用探针555条。将探针与各个菌种荧光PCR产物进行杂交，选取杂交信号最强的探针。再与其他菌种杂交信号进行比对，选择特异性最好的探针作为致病菌检测芯片上最终使用的探针86条。本发明所述探针与目标菌种杂交信号值最强、特异性最好，与非目标菌种不产生信号或产生信号值接近于背景信号。
本发明还提供了眼科临床微生物快速检测芯片，所述芯片上含有上述技 术方案所述的探针。本发明芯片杂交方法优选包括以下步骤：荧光PCR产物使用LCS_beads磁珠进行纯化，去除多余引物和杂质，利用ASP-3700微量分光光度计测量260nm和550nm吸光值，确定荧光掺入密度。当荧光掺入密度值在20～50时最适宜杂交。
本发明还提供了眼科临床微生物快速检测用引物，所述引物包括检测表皮葡萄球菌的引物、检测金黄色葡萄球菌的引物、检测溶血葡萄球菌的引物、检测铜绿假单胞菌的引物、检测人葡萄球菌的引物、检测粘质沙雷氏菌的引物、检测大肠埃希氏菌的引物、检测枯草芽孢杆菌的引物和检测阴沟肠杆菌的引物；如表2所示。
所述表皮葡萄球菌的引物包括核苷酸序列如SEQ ID NO.157所示的正向引物和核苷酸序列如SEQ ID NO.158所示的反向引物；
所述金黄色葡萄球菌的引物核苷酸序列如SEQ ID NO.159所示的正向引物和核苷酸序列如SEQ ID NO.160所示的反向引物；
所述溶血葡萄球菌的引物核苷酸序列如SEQ ID NO.161所示的正向引物和核苷酸序列如SEQ ID NO.162所示的反向引物；
所述铜绿假单胞菌的引物核苷酸序列如SEQ ID NO.163所示的正向引物和核苷酸序列如SEQ ID NO.164所示的反向引物；
所述人葡萄球菌的引物核苷酸序列如SEQ ID NO.165所示的正向引物、核苷酸序列如SEQ ID NO.166所示的反向引物；
所述粘质沙雷氏菌的引物核苷酸序列如SEQ ID NO.167所示的正向引物和核苷酸序列如SEQ ID NO.168所示的反向引物；
所述大肠埃希氏菌的引物核苷酸序列如SEQ ID NO.169所示的正向引物和核苷酸序列如SEQ ID NO.170所示的反向引物；
所述枯草芽孢杆菌的引物核苷酸序列如SEQ ID NO.171所示的正向引物和核苷酸序列如SEQ ID NO.172所示的反向引物；
所述阴沟肠杆菌的引物核苷酸序列如SEQ ID NO.173所示的正向引物和核苷酸序列如SEQ ID NO.174所示的反向引物。
表2眼科临床微生物快速检测用引物
Figure PCTCN2021137383-appb-000005
本发明收集临床培养的菌株，进行16S rDNA测序确定菌种信息后，对菌种进行基因组序列检索。在找到单一菌种保守序列后，再将所有菌种的保守序列进行两两比对，找出所有相似序列并排除在外，继续通过与NCBI数据库比对，得到菌株的特异保守基因片段(内同源性>95％，种间同源性<75％的基因序列)用于设计引物。用primer5进行设计出一批引物，用DNAstat对设计的引物进行筛选，得到引物组合。
在本发明中，所述引物中的正向引物的5’端优选均进行荧光基团标记。在本发明中，所述荧光基团优选包括CY3。本发明在正向引物5’端加CY3荧光基团，得到菌株特异性的荧光引物。
本发明还提供了一种眼科临床微生物快速检测用引物探针组，所述引物探针组包括上述技术方案所述的探针和上述技术方案所述的引物。
本发明还提供了一种眼科临床微生物快速检测试剂盒，所述试剂盒包括上述技术方案所述芯片和上述技术方案所述的引物。
下面结合具体实施例对本发明所述的眼科临床微生物快速检测用探针、芯片、引物和试剂盒做进一步详细的介绍，本发明的技术方案包括但不限于以下实施例。
实施例1
引物筛选
通过全基因组比对对菌种特异DNA序列进行筛选。下载NCBI中9种病原菌所有基因组序列，在待检微生物的基因序列中找到种内同源性>95％(大部分能达到97％以上)，种间同源性<75％(大部分低于70％或者种间基本无同源性)的基因序列区段。各菌种筛选出的用于筛选引物的序列所在基因汇总表如表3所示：
表3各菌种筛选出的用于筛选引物的序列所在基因汇总表
Figure PCTCN2021137383-appb-000006
实施例2
引物设计和PCR验证
Figure PCTCN2021137383-appb-000007
首先对选出的各个菌种DNA区段以primer5进行单独的引物设计，筛选出一批引物，最后以DNAstat软件对筛选出的引物进行primer select，找出较为合适的引物组合。合成引物，在正向引物的5’端加上一个荧光基团(CY3)， 这样PCR产物就带上荧光，后续用于杂交本发明芯片后通过扫描仪扫描，即可分辨是否成功杂交探针。
经实施例2筛选并验证出的引物(如表2所示)特异性高，杂交后可产生可读荧光信号。
实施例3
探针设计和筛选
一张LCS芯片可容纳探针数为:3968(31*128)，为了在最大范围的进行探针筛选，本发明对各个细菌的PCR产物进行Tiling array探针设计，即采用等长位移法，按照靶序列从头到尾选取一定长度(本发明中选择长度为25bp)序列形成探针组合，其中相邻探针序列相差一个碱基，直至覆盖整个靶序列。根据对PCR产物序列NCBI blast结果对于存在突变的区段，根据突变的碱基设计多条探针序列，如果某一探针涵盖的突变数目太多(25bp中超过3个突变)则放弃这一探针。探针是否适合杂交则需要通过进行杂交实验进行筛选，选择成功扩增带荧光的产物与芯片进行杂交反应。芯片扫描后提取数据。分析得到的数据，筛选杂交信号好，特异性强的探针。
实施例4
PCR产物纯化和荧光渗入密度计算
利用实施例2筛选得到的引物扩增所得带荧光的产物，与实施例3筛选得到的探针进行杂交反应，芯片扫描后提取数据。分析这些探针在其他菌种杂交芯片上的信号情况，选出特异性最好(即在其他菌种杂交芯片上信号值比较弱，接近阴性对照探针)的探针作为致病菌检测芯片最终的检测探针，如表1所示，共146条。
荧光掺入PCR产物以LCS_beads磁珠进行纯化，去除多余的引物和杂质后，以ASP-3700微量分光光度计进行测量260nm和550nm处的吸光值以确定荧光渗入密度(FOI)。FOI在20-50最适宜杂交。
荧光渗入密度的计算:
荧光掺入密度FOI＝荧光渗入pmol×(324.5/cDNA nmol)
样品中Cy3或Cy5的量(pmol)＝(A/E)×(1/W)x(Z)×df×10 6
A＝Cy3在550nm或者Cy5在650nm的吸光值)
E＝消光系数:Cy3＝150000，Cy5＝250000
Z＝样品体积的微升数
W＝光路＝0.1cm
df＝稀释倍数
本实验中:FOI＝(Cy3在550nm的吸光值/150000)×10×10 6×((324.5/(Cy3在260nm的吸光值×50))
表4荧光PCR产物中荧光渗入密度
Figure PCTCN2021137383-appb-000008
荧光PCR产物 A550 A260 FOI
Staphylococcus haemolyticus(溶血葡萄球菌) 0.009 0.192 20.28125
Enterobacter cloacae(阴沟肠杆菌) 0.01 0.313 13.82322
Staphylococcus epidermidis(表面葡萄球菌) 0.024 0.187 55.52941
Staphylococcus aureus(金黄色葡萄球菌) 0.015 0.205 31.65854
Staphylococcus hominis(人葡萄球菌) 0.02 0.323 26.79051
Pseudomonas aeruginosa(铜绿假单胞菌) 0.012 0.241 21.54357
Escherichia coli(大肠埃希氏菌) 0.02 0.265 32.65409
Serratia marcescens(粘质沙雷氏菌) 0.011 0.247 19.26856
bacillus subtilis(枯草芽孢杆菌) 0.011 0.335 14.20697
根据表4可知，本发明设计的引物和探针达到了杂交标准，可行，可推广做成实物。
实施例5
芯片杂交和扫描
芯片杂交流程
A清洗系统
连接通路，换上废旧芯片，用以下溶液进行清洗系统：
1ml于95℃预热的1％SDS，在最高速下循环清洗20min；
排除1％SDS于废液管后，用3ml无核酸酶水进行清洗；
1ml于95℃预热的无核酸酶水，在最高速下循环清洗5～6min；
排除无核酸酶水于废液管后，用3ml无核酸酶水进行清洗。
B芯片清洗
更换新的芯片，用1ml Stripping buffer(解吸缓冲液SP)于结合速度 下循环清洗20min(注意转换进液方向以排除芯片中气泡)；
Stripping buffer成分：0.3mM EDTA，50％甲酰胺，pH 6.6～6.8，该缓冲液可以清洗掉已经杂交在探针上的核酸序列。
扫描仪对清洗后的芯片进行扫描。
备注：以上系统清洗和芯片清洗过程中芯片台座温度均为40℃。
这一步骤是为了将芯片上可能存在的杂质以及可能杂交上的核酸序列清洗掉，得到比较干净均一的背景，准备杂交。
C样本杂交
(1)1ml Hybridization buffer(杂交液HB)于结合速度(500μl/min)下循环清洗10min；Hybridization buffer(杂交液HB)成分：6X SSPE，25％Formamide，pH 6.6～6.8，Hybridization buffer(杂交液HB)为杂交反应提供一个适宜的pH环境和盐离子浓度，其中的Formamide可以降低DNA双链的Tm值，使样品在30℃时进行杂交反应。
(2)1ml Blocking buffer(封闭液BSA)于结合速度下循环清洗5～6min；
Blocking buffer(封闭液BSA)：148μL杂交液HB，2μL of 100×heat-treated BSA，该缓冲液中的BSA可以封闭芯片上无探针部分，以降低芯片背景信号。
(3)杂交样品制备：200ng纯化后的PCR产物+等体积Hybridization buffer(杂交液HB，最终体积50μl)，95℃变性5min后迅速置于冰上3min，将制备的样本加入到Blocking buffer中，混匀后于于结合速度下循环运行16小时进行杂交。
设定杂交时间为16小时是为了使杂交反应时间足够长，达到反应平衡，实际操作中可以适当调整。
备注：样本杂交过程芯片台座温度为40℃。
D杂交后清洗
(1)1ml Hybridization buffer(杂交液HB)于清洗速度(100μl/min)下循环清洗20min(芯片台座温度32℃)；
(2)1ml Wash buffer(洗液WB)于清洗速度下，在40℃下循环清洗20 min。
Wash buffer(洗液WB)：500μL HB，500μL Nuclease-free water，20μL 10％SDS，注意，Wash buffer要现配现用。
E芯片扫描
根据GenePix 4000B的说明设定相关的光电倍增管(PMT＝300-400)、聚焦距离(focal position＝100-150)和扫描波长(532nm)等对芯片进行扫描。杂交图要结合点阵上探针的位置信息解读，每个菌种探针位置在芯片上是固定的，杂交后的探针有荧光产生，就说明样本中含有该位置探针对应的菌种。
图1：芯片扫描后杂交信号示意图以及FOI值测定。杂交信号强度顺序为白色>红色>黄色>绿色>蓝色。
图2：表皮葡萄球菌杂交信号图，可见对芯片扫描后对该菌种有特异性的杂交信号产生，说明该芯片可以在临床实际应用中对表皮葡萄球菌进行筛选检测；
图3：金黄色葡萄球菌杂交信号图，可见对芯片扫描后对该菌种有特异性的杂交信号产生，说明该芯片可以在临床实际应用中对金黄葡萄球菌进行筛选检测；
图3：溶血葡萄球菌的杂交信号图，可见对芯片扫描后对该菌种有特异性的杂交信号产生，说明该芯片可以在临床实际应用中对溶血葡萄球菌进行筛选检测；
图4：铜绿假单胞菌的杂交信号图，可见对芯片扫描后对该菌种有特异性的杂交信号产生，说明该芯片可以在临床实际应用中对铜绿假单胞菌进行筛选检测；
图5：人葡萄球菌的杂交信号图，可见对芯片扫描后对该菌种有特异性的杂交信号产生，说明该芯片可以在临床实际应用中对人葡萄球菌进行筛选检测；
图6：粘质沙雷氏菌的杂交信号图，可见对芯片扫描后对该菌种有特异性的杂交信号产生，说明该芯片可以在临床实际应用中对粘质沙雷氏菌进行筛选检测；
图7：大肠埃希氏菌的杂交信号图，可见对芯片扫描后对该菌种有特异 性的杂交信号产生，说明该芯片可以在临床实际应用中对大肠埃希氏菌进行筛选检测；
图8：枯草芽孢杆菌的杂交信号图，可见对芯片扫描后对该菌种有特异性的杂交信号产生，说明该芯片可以在临床实际应用中对枯草芽孢杆菌进行筛选检测；
图9：阴沟肠杆菌的杂交信号图，可见对芯片扫描后对该菌种有特异性的杂交信号产生，说明该芯片可以在临床实际应用中对阴沟肠杆菌进行筛选检测。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (9)

  1. 眼科临床微生物快速检测用探针，其特征在于，所述探针包括检测表皮葡萄球菌的探针、检测金黄色葡萄球菌的探针、检测溶血葡萄球菌的探针、检测铜绿假单胞菌的探针、检测人葡萄球菌的探针、检测粘质沙雷氏菌的探针、检测大肠埃希氏菌的探针、检测枯草芽孢杆菌的探针和检测阴沟肠杆菌的探针；
    所述表皮葡萄球菌的探针包括核苷酸序列分别如SEQ ID NO.1～20所示的探针；
    所述金黄色葡萄球菌的探针包括核苷酸序列分别如SEQ ID NO.21～40所示的探针；
    所述溶血葡萄球菌的探针包括核苷酸序列分别如SEQ ID NO.41～60所示的探针；
    所述铜绿假单胞菌的探针包括核苷酸序列分别如SEQ ID NO.61～80所示的探针；
    所述人葡萄球菌的探针包括核苷酸序列分别如SEQ ID NO.81～100所示的探针；
    所述粘质沙雷氏菌的探针包括核苷酸序列分别如SEQ ID NO.101～120所示的探针；
    所述大肠埃希氏菌的探针包括核苷酸序列分别如SEQ ID NO.121～140所示的探针；
    所述枯草芽孢杆菌的探针包括核苷酸序列分别如SEQ ID NO.141～147所示的探针；
    所述阴沟肠杆菌的探针包括核苷酸序列分别如SEQ ID NO.148～156所示的探针。
  2. 眼科临床微生物快速检测芯片，其特征在于，所述芯片上含有权利要求1所述的探针。
  3. 眼科临床微生物快速检测用引物，其特征在于，所述引物包括检测表皮葡萄球菌的引物、检测金黄色葡萄球菌的引物、检测溶血葡萄球菌的引物、检测铜绿假单胞菌的引物、检测人葡萄球菌的引物、检测粘质沙雷氏菌 的引物、检测大肠埃希氏菌的引物、检测枯草芽孢杆菌的引物和检测阴沟肠杆菌的引物；
    所述表皮葡萄球菌的引物包括核苷酸序列如SEQ ID NO.157所示的正向引物和核苷酸序列如SEQ ID NO.158所示的反向引物；
    所述金黄色葡萄球菌的引物核苷酸序列如SEQ ID NO.159所示的正向引物和核苷酸序列如SEQ ID NO.160所示的反向引物；
    所述溶血葡萄球菌的引物核苷酸序列如SEQ ID NO.161所示的正向引物和核苷酸序列如SEQ ID NO.162所示的反向引物；
    所述铜绿假单胞菌的引物核苷酸序列如SEQ ID NO.163所示的正向引物和核苷酸序列如SEQ ID NO.164所示的反向引物；
    所述人葡萄球菌的引物核苷酸序列如SEQ ID NO.165所示的正向引物、核苷酸序列如SEQ ID NO.166所示的反向引物；
    所述粘质沙雷氏菌的引物核苷酸序列如SEQ ID NO.167所示的正向引物和核苷酸序列如SEQ ID NO.168所示的反向引物；
    所述大肠埃希氏菌的引物核苷酸序列如SEQ ID NO.169所示的正向引物和核苷酸序列如SEQ ID NO.170所示的反向引物；
    所述枯草芽孢杆菌的引物核苷酸序列如SEQ ID NO.171所示的正向引物和核苷酸序列如SEQ ID NO.172所示的反向引物；
    所述阴沟肠杆菌的引物核苷酸序列如SEQ ID NO.173所示的正向引物和核苷酸序列如SEQ ID NO.174所示的反向引物。
  4. 根据权利要求3所述的引物，其特征在于，所述引物中的正向引物的5’端均进行荧光基团标记。
  5. 根据权利要求4所述的引物，其特征在于，所述荧光基团包括CY3。
  6. 一种眼科临床微生物快速检测用引物探针组，其特征在于，所述引物探针组包括权利要求1所述的探针和权利要求3～5任一项所述的引物。
  7. 一种眼科临床微生物快速检测试剂盒，其特征在于，所述试剂盒包括权利要求2所述芯片和权利要求3～5任一项所述的引物。
  8. 权利要求1所述探针或权利要求2所述芯片或权利要求3～5任一项所述引物或权利要求7所述的试剂盒在检测眼科临床微生物中的应用。
  9. 根据权利要求8所述的应用，其特征在于，所述眼科临床微生物包括表皮葡萄球菌、金黄色葡萄球菌、溶血葡萄球菌、铜绿假单胞菌、人葡萄球菌、粘质沙雷氏菌、大肠埃希氏菌、枯草芽孢杆菌和阴沟肠杆菌。
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