CN109975542A - A kind of Biomolecule detection kit and biomolecule detecting method - Google Patents
A kind of Biomolecule detection kit and biomolecule detecting method Download PDFInfo
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- CN109975542A CN109975542A CN201910133491.XA CN201910133491A CN109975542A CN 109975542 A CN109975542 A CN 109975542A CN 201910133491 A CN201910133491 A CN 201910133491A CN 109975542 A CN109975542 A CN 109975542A
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6816—Hybridisation assays characterised by the detection means
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
- G01N33/56916—Enterobacteria, e.g. shigella, salmonella, klebsiella, serratia
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
- G01N33/56938—Staphylococcus
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
- G01N2333/21—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Pseudomonadaceae (F)
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
- G01N2333/24—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
- G01N2333/245—Escherichia (G)
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
- G01N2333/24—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
- G01N2333/25—Shigella (G)
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
- G01N2333/305—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Micrococcaceae (F)
- G01N2333/31—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Micrococcaceae (F) from Staphylococcus (G)
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- Hematology (AREA)
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- General Health & Medical Sciences (AREA)
- Virology (AREA)
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- Proteomics, Peptides & Aminoacids (AREA)
- Genetics & Genomics (AREA)
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Abstract
The invention belongs to the technical field of biological medicine more particularly to a kind of Biomolecule detection kit and biomolecule detecting methods.The present invention provides a kind of Biomolecule detection kits, it include: nucleic acid double chain heterozygote, nucleic acid signal probe developing body and exonucleaseⅲ, wherein, nucleic acid double chain heterozygote includes aptamer and nucleic acid promoter chain, and the nucleic acid signal probe developing body includes that colour developing medium and nucleic acid signal probe are single-stranded.The present invention is single-stranded by designing suitable aptamer, nucleic acid promoter chain and nucleic acid signal probe, specific binding using aptamer to biomolecule, coloration occurs by colour developing media aggregates, can naked eyes quick visualization detect biomolecule, it can also be in the single-stranded modification fluorophor of nucleic acid signal probe, enzyme-chain cascade reaction is triggered in colour developing medium interface, the amplification of the fluorescence signal of reaction solution is detected, realizes and the fast super sensitivity detection of biomolecule is analyzed.
Description
Technical field
The invention belongs to the technical field of biological medicine more particularly to a kind of Biomolecule detection kit and biologies point
Sub- detection method.
Background technique
Pathogenetic bacteria is one kind of causal organism molecule, and the pathogenetic bacteria being present in food, water and environment can cause
The outburst of a variety of diseases, it bacterium infection and can lead to many severe complications.Such as some common pathogenic bacteria: Salmonella
Bacterium (food poisoning), helicobacter pylori (gastritis and hepatitis), NEISSERIA GONORRHOEAE (sexually transmitted disease), Neisseria (meningitis), gold
Staphylococcus aureus (burn, cellulitis, abscess, wound infection, poisonous substance syndrome, pneumonia and food poisoning) and streptococcus
(pneumonia, meningitis, ear infection and pharyngitis) etc..Every year have more than 300,000,000 it is serious or even fatal as caused by bacterium infection
Disease, death toll is more than 2,000,000 people, especially causes blood infection by bacterium and pyemia is leads to the high pathogenicity rate in the whole world
And the main reason for death rate, the number that pyemia influences in the world every year are 18,000,000 people, the death rate is up to 30-
40%.By taking the U.S. as an example, related disease economic implication caused by the blood infection as caused by bacterium is up to 17,000,000,000 dollars.
Simultaneously with the enhancing of bacterial drug resistance, especially there is the methicillin-resistant staphylococcus aureus of the title of " superbacteria "
(MRSA) the problem of appearance, antibiotic resistance has become a threat Global Health, is estimated to the year two thousand fifty unless taking
Action, otherwise drug resistance (AMR) causes dead number that can increase to 10,000,000 people every year, produces to the year two thousand fifty to global economy
Raw cumulative cost is up to 100 trillion dollars.On this basis, to the year two thousand fifty, what death toll can be surprising reaches every three seconds just
People's death is had, medical expense can reach a Wan Meiyuan per capita.Therefore need to develop a kind of high sensitivity, specificity it is good,
Quickly and accurately typing of bacteria detection and quantitative analysis method, its correspondence guiding treatment bacterium infection and clinical antibacterials
Exploitation is of great significance.
Traditional typing of bacteria detection method includes biomolecule cultivation, immunological method, molecular biology method etc..
Method for cultivation of bacteria is the standard method of Bacteria Detection, but detection process is cumbersome, takes a long time (24-48 hours), is not suitable for
It is detected in the fast typing of bacterium.Immunological method is to pass through immune response identification target point based on antigen-antibody reaction
Son, this method has many advantages, such as that high sensitivity, specificity are good, detection speed is fast, but obtains in actual sample detection corresponding
Antibody takes a long time, moreover, its specificity is interfered vulnerable to from antibody, influences its detection sensitivity.Molecular biology method
Be based on the nucleic acid detection assay of bacterium, by the molecular probe that is pre-designed to the DNA of particular sequence in bacterium or
MiRNA nucleic acid molecules etc. carry out specific recognition analysis, such as enzyme-linked immunosorbent assay (ELISA), DNA microarray (DNA
Microarrays) and polymerase chain reaction (PCR), these method high sensitivities, specificity are good, can reach trace level detection,
It is of great significance for low content Bacteria Detection.But early period, design of primers was complex, retrieving is cumbersome, to technology people
Member's is more demanding.These conventional method of detecting bacterium are in sensitivity, specificity, detection speed, consuming and bacteria quantified
Testing and analyzing aspect has certain limitation, generally requires especially for various pathogens that may be present in same sample
Sample treatment and detection are carried out respectively, and which increase workload and detection times.In conclusion being deposited in existing clinical bacteria detection
Process it is cumbersome, take a long time, it is strongly professional, at high cost, vulnerable to interference and the technical problems such as poor specificity.Therefore urgent
Need to establish method that is sensitiveer, quick and typing of bacteria detection and quantitative analysis being carried out simultaneously.
Summary of the invention
In view of this, the present invention for technical problem present in the detection of existing clinical bacteria, provide it is a kind of it is easy to detect,
Time saving, specific and high sensitivity Biomolecule detection kit.
The present invention provides a kind of Biomolecule detection kits, comprising:
Nucleic acid double chain heterozygote, nucleic acid signal probe developing body and exonucleaseⅲ；
Wherein, nucleic acid double chain heterozygote includes aptamer and nucleic acid promoter chain, the aptamer and the core
The same end of acid promoter chain is additionally provided with protection sequence, the non-protected sequence of the aptamer and the nucleic acid promoter chain
Non-protected sequence-specific combines；It is described to protect aptamer described in sequence protection and the nucleic acid promoter chain not by the core
Sour III digestion of excision enzyme, the aptamer specific recognition and in conjunction with the biomolecule, and the aptamer with
The associativity of the biomolecule is higher than the associativity of the aptamer and the nucleic acid promoter chain；
The nucleic acid signal probe developing body includes that colour developing medium and nucleic acid signal probe are single-stranded, the nucleic acid signal probe
In conjunction with the colour developing medium, the single-stranded sequence of the nucleic acid signal probe can be non-with the nucleic acid promoter chain at 5 ' single-stranded ends
Protection sequence-specific combine formed can digestion nucleic acid signal probe developing body, the exonucleaseⅲ can digestion nucleic acid signal
Probe is single-stranded, obtain colour developing medium, and discharge described in can digestion nucleic acid signal probe developing body the nucleic acid promoter chain.
It should be noted that aptamer (Aptamer) is section of DNA (DNA) or RNA (ribose core
Acid) sequence, aptamer of the invention obtains and corresponding target (biomolecule) high-affinity from nucleic acid molecule libraries
With the oligonucleotide fragment of high specific.The present invention carries out specific recognition for the whole of biomolecule, and be different from order to avoid
Epidemic disease diagnostic techniques (Ag-Ab identification) and science of heredity identification technology (DGGE/TGGE/TTGE, T-RFLP, SSCP, FISH, print
Remember hybridization, quantitative PCR, genetic chip and gene sequencing etc.).And aptamer of the invention is to be based on having filtered out
Biomolecule correspondence aptamer.
It should be noted that biomolecule includes biological micromolecule (such as metal ion), large biological molecule (such as DNA,
RNA or protein etc.), virus, bacterium, fungi, the substances such as cell.
Preferably, the aptamer can pass through in-vitro screening technology-index concentration Fas lignand system evolution skill
Art screens to obtain from the molecular library of the biomolecule.The Fas lignand system evolution technology of in-vitro screening technology --- index concentration
(Systematic evolution of ligands by exponential enrichment, SELEX), from nucleic acid molecules
Oligonucleotide fragment obtained in library.Aptamer and biomolecule (including bacterium, fungi, virus) are whole in conjunction with (core
Sour aptamers from DNA molecular library, are obtained by multi-turns screen by screening process, so that aptamer and certain detail
Bacterium is specific binding, not in conjunction with bacterium target sequence), since aptamer Filtering system itself determines aptamer
It is better than the binding ability of aptamer Yu nucleic acid promoter chain with bacteria-binding capacity.
It should be noted that nucleic acid signal probe it is single-stranded 5 ' end modified by mercapto groups, the single-stranded benefit of nucleic acid signal probe
With 5 ' terminal modified mercapto groups, the surface of nano gold spherical is fixed on by way of covalent bond.
Preferably, the protection sequence is that poly thymidine repeats oligonucleotide sequence, of the thymidine
Number is 5-60.
More preferably, the protection sequence is the repetition oligonucleotide sequence of 5 thymidines, i.e., the described protection sequence is
TTTTT。
More preferably, the exonucleaseⅲ is for DNA excision enzyme III or with similar to III function of DNA excision enzyme
Enzyme.
Specifically, referring to Fig. 1, Fig. 1 is the nucleic acid double chain heterozygote of Biomolecule detection kit provided by the invention
Structure, aptamer 3 and the specific binding of nucleic acid promoter chain 4 obtain nucleic acid double chain heterozygote 2, the aptamer and
3 ' ends of the nucleic acid promoter chain are equipped with protection sequence, since the principle of exonuclease III effect double-stranded DNA limits (core
Sour exonucleaseⅢ acts on double-stranded DNA, identifies flat end or 3 ' the recessed ends single stranded DNAs of double-stranded DNA, from 3 ' to 5 ' gradually
Hydrolysing single DNA), so protection sequence is arranged in aptamer for Biomolecule detection kit of the invention
It is held with the 3 ' of nucleic acid promoter chain, so that the sequence total length that the sequence total length of nucleic acid promoter chain is more single-stranded than nucleic acid signal probe
It is long.In addition, protection sequence also can choose other nucleotide sequences, but principle selection is as follows: 1, playing nucleic acid double chain heterozygosis
Body is not hydrolyzed by exonucleaseⅢ；2, protection sequence cannot influence aptamer and biomolecule is specifically bound, and protect simultaneously
Sequence cannot influence the single-stranded specific binding of nucleic acid signal probe of nucleic acid promoter chain and the dielectric surface that develops the color.
Preferably, the colour developing medium is that can occur to assemble and the substance of colour developing.
Preferably, the colour developing medium is selected from one of nano metal ball, quantum dot or a variety of.
Preferably, the nano metal ball is selected from nano gold spherical or/and nanometer ping-pong ball.
Preferably, the nucleic acid signal probe it is single-stranded 5 ' end be also connected with colour developing medium binding sequence, the colour developing
Medium binding sequence for nucleic acid signal probe it is single-stranded with develop the color medium combination.
Preferably, the colour developing medium binding sequence is that poly thymidine repeats oligonucleotide sequence, the thymus gland
The number of pyrimidine is 5-60.
More preferably, the colour developing medium binding sequence is the repetition oligonucleotide sequence of 10 poly thymidines, i.e.,
The colour developing medium binding sequence is TTTTTTTTTT.
It should be noted that colour developing medium binding sequence effect be so that nucleic acid signal probe it is single-stranded with colour developing medium more
It is easy to combine, specifically, 5 ' ends of colour developing medium binding sequence are modified by mercapto groups, nucleic acid signal probe is single-stranded to utilize 5 ' ends
The mercapto groups of modification are fixed on the surface of nano gold spherical by way of covalent bond.
In order to further increase the sensitivity technique to biomolecule, the present invention repairs at 3 ' single-stranded ends of nucleic acid signal probe
Fluorophor is adornd, nucleic acid signal probe is single-stranded to utilize 5 ' terminal modified mercapto groups, and nanometer is fixed on by way of covalent bond
The surface of gold goal or nanometer ping-pong ball is shifted former using the excellent electric conductivity of nano gold spherical or nanometer ping-pong ball and fluorescence resonance energy
Reason completes the fluorescent quenching single-stranded to nucleic acid signal probe.Under the action of nucleic acid promoter chain and the driving of exonuclease III
Under, nucleic acid signal probe is single-stranded constantly hydrolyzed, so that fluorescein base group is de- from the surface of nano gold spherical or nanometer ping-pong ball
From solution system fluorescence restores.Enzymolysis process is repeated, the purpose to the fluorescence signal augmentation detection of biomolecule is completed.
Preferably, the single-stranded second end of the nucleic acid signal probe is also marked with fluorescein base group.Specifically, nucleic acid is believed
Number 3 ' single-stranded terminal modified fluorophors of probe.
More preferably, the fluorescein base group is selected from FAM, FAM/AMCA, FAM/TEXAS RED, AMCA, FAM/TEXAD
One of RED or TEXAD RED or a variety of.
It should be noted that nucleic acid signal probe is single-stranded when 3 ' single-stranded terminal modified fluorophor of nucleic acid signal probe
When being fixed on nano gold spherical or nanometer ping-pong ball, the electric conductivity and fluorescence resonance energy that nano gold spherical or nanometer ping-pong ball are excellent are utilized
Principle of transfer completes the fluorescent quenching of upper fluorophor single-stranded to nucleic acid signal probe.Under the action of nucleic acid promoter chain and core
Under the driving of sour exonucleaseⅢ, the nucleic acid signal probe for being fixed on nano gold spherical or nanometer ping-pong ball is single-stranded constantly hydrolyzed, thus
So that fluorophor is detached from from the surface of nano gold spherical or nanometer ping-pong ball, so that the fluorescence of reaction solution system restores, and nucleic acid is opened
Dynamic chain repeats to repeat hydrolytic nucleic acid signal probe with remaining single-stranded specific binding of nucleic acid signal probe, exonuclease III
It is single-stranded, the enzymolysis process is repeated, until whole single-stranded complete hydrolysis of nucleic acid signal probe of nano gold spherical or nano silver ball surface,
Then the aggregation for inducing nano gold spherical or nanometer ping-pong ball, realizes entire reaction solution color change, completes quick to MRSA naked eyes
The purpose of Visual retrieval, and complete the purpose to the fluorescence signal augmentation detection of biomolecule detection.
The present invention also provides a kind of biomolecule detecting methods, comprising the following steps:
Step 1, by the nucleic acid double chain heterozygote, nucleic acid signal probe developing body and exonucleaseⅲ and the biology
Molecular mixing obtains reaction solution；Wherein, the aptamer is specifically bound with the biomolecule, is discharged
To the free nucleic acid promoter chain；The core of the non-protected sequence of the nucleic acid promoter chain and the nucleic acid signal probe developing body
Acid signal probe is single-stranded to be specifically bound, and formation can digestion nucleic acid signal probe developing body；The exonucleaseⅲ enzyme
Cut it is described can digestion nucleic acid signal probe developing body, described in release can digestion nucleic acid signal probe developing body the nucleic acid promoter
Chain, obtains colour developing medium, and the colour developing medium occurs aggregation and develops the color；
Step 2 carries out color developing detection to the reaction solution, obtains testing result.
Referring to Fig. 2, Fig. 2 is the schematic illustration of Biomolecule detection kit of the invention, from fig. 1, it can be seen that nucleic acid
The aptamer of double-stranded hybrids can only be specifically bound with corresponding biomolecule, and release obtains free nucleic acid promoter
Chain；The non-protected sequence of nucleic acid promoter chain with nucleic acid signal probe is single-stranded specifically binds, formation can digestion nucleic acid signal
Probe developing body (can the nucleic acid promoter chain of digestion nucleic acid signal probe developing body protect sequence protection nucleic acid promoter chain not by core
Sour exonucleaseⅢ hydrolysis)；Exonuclease III digestion this can digestion nucleic acid signal probe developing body nucleic acid signal probe list
Chain, discharge this can digestion nucleic acid signal probe developing body nucleic acid promoter chain, obtain nano gold spherical, most nano gold sphericals are mutually sent out
Life is assembled and develops the color, so that the change of color occurs for reaction solution, realizes quick visualization detection of the naked eyes to bacterium；Meanwhile
When nucleic acid signal probe it is single-stranded 5 ' it is terminal modified have fluorescein base group, since nano gold spherical has in nucleic acid signal probe developing body
Excellent electric conductivity is completed to visit nucleic acid signal using the electric conductivity and fluorescence resonance energy principle of transfer of nano gold spherical
The fluorescent quenching of the single-stranded fluorescein base group of needle.Under the action of nucleic acid promoter chain and under the driving of exonuclease III, nucleic acid
Signal probe is single-stranded constantly hydrolyzed, so that fluorescein base group is detached from from nano gold spherical surface, leads to reaction solution system
Fluorescence restore.The nucleic acid promoter chain of release and the single-stranded combination of other nucleic acid signal probes, in the repetition of exonuclease III
Enzymolysis process is completed to detect fluorescence data by Fluorescence Spectrometer to the purpose of the fluorescence signal augmentation detection of MRSA experimental group
The present invention is by designing suitable aptamer, nucleic acid promoter chain and nucleic acid signal probe list to biomolecule
Chain, the specific binding using aptamer to biomolecule, enzyme-chain cascade reaction of triggering colour developing medium interface pass through
Amplification to the fluorescence signal of detection fluorescein base group is realized and is analyzed the fast super sensitivity detection of biomolecule.It can from experiment
Know, Biomolecule detection kit of the invention has good specificity, sensitivity and universality.The present invention provides a kind of spirit
Sensitivity height, good, quickly and accurately biomolecule the detection and analysis technology of specificity.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described.
Fig. 1 shows the structure of the nucleic acid double chain heterozygote of Biomolecule detection kit provided by the invention
Fig. 2 shows the schematic illustration of Biomolecule detection kit of the invention；
Fig. 3 shows the visible absorption spectra for the Biomolecule detection kit detection different bacterium that the embodiment of the present invention 4 provides；
Fig. 4 shows the absorbance (A for the Biomolecule detection kit detection different bacterium that the embodiment of the present invention 4 provides650/
A519) and different bacterium reaction solution visual test result；
Fig. 5 shows the fluorescence spectra for the Biomolecule detection kit detection different bacterium that the embodiment of the present invention 4 provides；
Fig. 6 shows the detection fluorescence for the Biomolecule detection kit detection various concentration MRSA that the embodiment of the present invention 5 provides
Spectrogram；
Fig. 7 shows that the Biomolecule detection kit that the embodiment of the present invention 5 provides detects various concentration MRSA in the inspection of 520nm
Survey fluorescence spectra；
Fig. 8 shows the absorbance for the Biomolecule detection kit detection various concentration MRSA that the embodiment of the present invention 5 provides
(A600/A519)；
The Biomolecule detection kit that the embodiment of the present invention 6 provides of Fig. 9 showing detects the fluorescence inspection of MRSA under different body fluid
Survey result；
Figure 10 shows the Biomolecule detection kit detection different bacterium specificity fluorescent detection that the embodiment of the present invention 7 provides
As a result；
Wherein, appended drawing reference, biomolecule 1, nucleic acid double chain heterozygote 2, aptamer 3, nucleic acid promoter chain 4, colour developing
Medium 5, nucleic acid signal probe be single-stranded 6, fluorescein base group 7, colour developing medium binding sequence 8, exonucleaseⅲ 10, protection sequence
A。
Specific embodiment
The present invention provides a kind of Biomolecule detection kit and biomolecule detecting methods, for providing a kind of spirit
Sensitivity is high, specificity is good, quickly and accurately biomolecule detection analytical technology.
The technical scheme in the embodiments of the invention will be clearly and completely described below, it is clear that described implementation
Example is only a part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, this field is common
Technical staff's every other embodiment obtained without making creative work belongs to the model that the present invention protects
It encloses.
Wherein, the present invention is with methicillin-resistant staphylococcus aureus (MRSA), P.aeruginosa, Escherichia
Coli, S.epidermidis, Listeria, S.typhimurim, L.acidophilus, Shigella, E.faecealis and
S.pneum detects specificity, sensitivity and the universality of Biomolecule detection kit of the present invention as test object.Below
The strain number of methicillin-resistant staphylococcus aureus used in embodiment (MRSA) is ATCC BAA 1766, and purchase is certainly Chinese
General Microbiological Culture preservation administrative center；The strain number of Pseudomonas aeruginosa used in following embodiment (P.aeruginosa) is
CMCC 10102 is bought from China typical culture collection center；Escherichia coli (Escherichia used in following embodiment
Coli strain number) is CMCC44103, is bought from China typical culture collection center；Used in following embodiment
The strain number of S.epidermidis is CMCC 26069, is bought from China typical culture collection center；Used in following embodiment
The strain number of Listeria be CMCC 54001, purchase is from China typical culture collection center；Used in following embodiment
The strain number of S.typhimurim is CMCC 50115, is bought from China typical culture collection center；Used in following embodiment
The strain number of L.acidophilus be CMCC99101, purchase is from China typical culture collection center；Following embodiment institute
The strain number of Shigella is CMCC 51572, is bought from China typical culture collection center；Used in following embodiment
The strain number of E.faecealis be ATCC 29212, purchase is from Shanghai Lu Wei Science and Technology Ltd.；Used in following embodiment
The strain number of S.pneum is ATCC 6305, is bought from Shanghai Lu Wei Science and Technology Ltd.；Reagent used in following embodiment and
Enzyme is commercially available；The display medium of the nucleic acid signal probe developing body of following embodiment is nano gold spherical, nucleic acid signal probe list
Chain is that DNA signal probe is single-stranded, and nucleic acid promoter chain is that DNA starts chain.
Embodiment 1
The embodiment of the present invention is to synthesize the nano gold spherical particle that partial size is 13nm, the specific steps are as follows:
The HAuCl that the trisodium citrate (3.5mL) that percent by volume is 1% is added to boiling and is quickly stirred4
In (100mL, 0.01%) aqueous solution, solution boiling 20min is kept, then cools to room temperature, obtains nano gold spherical particle solution,
And 4 DEG C save for use.
Embodiment 2
The embodiment of the present invention is that DNA signal probe is single-stranded and the combination of nano gold spherical, the specific steps are as follows:
10 μ L nano gold spherical particle solutions (100nM) of the DNA signal probe of 1 μ L single-stranded (100 μM) and embodiment 1 are mixed
It closes, rapidly joins the Citrate-HCl (500mM, pH=3) of 0.2 μ L later, hatch 10 minutes at room temperature.Then, it is centrifuged
(14000rpm, 20min), removes supernatant, be added 100 μ L buffer (buffer include 10mM tris (pH=7.5),
The MgCl of 2.5mM2With the CaCl of 0.5mM2) in, in triplicate, it is single-stranded to remove extra DNA signal probe.Acquired solution is
The single-stranded nucleic acid signal probe in conjunction with nano gold spherical of DNA signal probe develops the color liquid solution (nano gold spherical 10nM), is protected from light guarantor
It deposits stand-by.Wherein, the single-stranded sequence of DNA signal probe (5 ' -3 ') are as follows:
HS-TTTTTTTTTTAATAAAGGGATTGCT.Wherein, at 3 ' single-stranded ends of DNA signal probe, to be marked with FAM glimmering
Light element group, the single-stranded colour developing medium binding sequence of DNA signal probe are the TTTTTTTTTT (effect of colour developing medium binding sequence
It is so that DNA signal probe is single-stranded is easier with nano gold spherical to combine), and the single-stranded 5 ' ends of DNA signal probe are by mercapto groups
Modification, DNA signal probe is single-stranded to utilize 5 ' terminal modified mercapto groups, and the table of nano gold spherical is fixed on by way of covalent bond
Face.
Embodiment 3
The embodiment of the present invention is the combination that aptamer and DNA start chain, the specific steps are as follows:
DNA, which starts chain, in order to prevent influences subsequent experimental result, and by 20 μ L DNA starting chain buffer, (DNA is opened first
Dynamic chain is 3nM) mixed with the aptamer buffer (aptamer 6nM) of 20 μ L, 37 DEG C constant temperature 2 hours, obtain core
Sour double stranded heteroduplex liquid solution, wherein buffer is the MgCl of 10mM tris (pH=7.5), 2.5mM2With the CaCl of 0.5mM2.Its
In, DNA starts the sequence (5 ' -3 ') of chain are as follows:
3 ' the ends that AGCAATCCCTTTCTTTGTATTAACCGAGTCGGGGTTTTTT, DNA start chain are equipped with protection sequence,
Protection sequence is TTTTTT；
The sequence (5 ' -3 ') of aptamer are as follows:
3 ' the ends of ACCCCGACTCGGTTAATACAAATAAAGGGATTGCTTTTTT, aptamer are equipped with protection sequence
Column, protection sequence are TTTTTT, DNA start chain and aptamer protect sequence play protect nucleic acid double chain heterozygote not by
Exonuclease III hydrolysis.Since the principle of exonuclease III effect double-stranded DNA limits, (exonuclease III is acted on
Double-stranded DNA identifies flat end or 3 ' concave end end single stranded DNAs, holds from 3 ' to 5 ' and holds gradually hydrolysing single DNA), so, needle
For nucleic acid double chain heterozygote, since aptamer and DNA starting chain have specific binding sequence and protection sequence, protect
Shield sequence can not be specifically bound, and therefore, nucleic acid double chain heterozygote is cohesive terminus,cohesive termini, and 3 ' ends of nucleic acid double chain heterozygote are protruded
End, exonuclease III can not hydrolytic nucleic acid aptamers and DNA starting chains.In addition, protection sequence also can choose other cores
Nucleotide sequence, but selection principle is as follows: 1, it protects sequence to play nucleic acid double chain heterozygote and is not hydrolyzed by exonucleaseⅢ；2, it protects
Shield sequence cannot influence the specific binding of aptamer and biomolecule, while protect sequence that cannot influence DNA starting chain
With the single-stranded specific binding of DNA signal probe on nano gold spherical surface.
Embodiment 4
The embodiment of the present invention is the specificity experiments of Biomolecule detection kit, the specific steps are as follows:
Experimental group (is labeled as MRSA): by the MRSA buffer of 50 μ L, (MRSA buffer includes methicillin-resistant staphylococcus
Staphylococcus and buffer, buffer components are the MgCl of 10mM tris (pH=7.5), 2.5mM2With the CaCl of 0.5mM2,
MRSA is 108CFU/mL) it is added in the nucleic acid double chain heterozygosis liquid solution of embodiment 3,100 μ L nucleic acid signals of embodiment 2 are visited
Needle develops the color liquid solution (nano gold spherical 10nM) and exonucleaseⅢ (final concentration of 1unit/L) mixing.Reaction condition: 2 hours,
37 DEG C of constant temperature.Then reaction solution spectrum is measured with Fluorescence Spectrometer and UV-vis, as a result as in Figure 2-4.
Control group 1 (be labeled as Blank): by the buffer of 50 μ L (buffer components are 10mM tris (pH=7.5),
2.5mM MgCl2,and 0.5mM CaCl2) it is added to the nucleic acid double chain heterozygosis liquid solution of embodiment 3,100 μ L cores of embodiment 2
Acid signal probe develops the color liquid solution (nano gold spherical 10nM) and exonucleaseⅢ (final concentration of 1unit/L) mixing.React item
Part: 2 hours, 37 DEG C of constant temperature.Then reaction solution spectrum is measured with Fluorescence Spectrometer and UV-vis, as a result as in Figure 2-4.
Control group 2 (is labeled as E.coil): by the Escherichia coli buffer of 50 μ L, (Escherichia coli buffer includes large intestine bar
Bacterium and buffer, buffer components are the MgCl of 10mM tris (pH=7.5), 2.5mM2With the CaCl of 0.5mM2, E.coil is
108CFU/mL) it is added to the nucleic acid double chain heterozygosis liquid solution of embodiment 3,100 μ L nucleic acid signal probe developing bodies of embodiment 2
Solution (nano gold spherical 10nM) and exonucleaseⅢ (final concentration of 1unit/L) mixing.Reaction condition: 2 hours, 37 DEG C of constant temperature.
Then reaction solution spectrum is measured with Fluorescence Spectrometer and UV-vis, as a result as in Figure 2-4.
Control group 3 (is labeled as Shigella): by the Hayes bacillus buffer of 50 μ L, (Hayes bacillus buffer includes Hayes
Bacillus Shigella and buffer, buffer components are the MgCl of 10mM tris (pH=7.5), 2.5mM2With 0.5mM's
CaCl2, Shigella 108CFU/mL) it is added to the nucleic acid double chain heterozygosis liquid solution of embodiment 3,100 μ L nucleic acid of embodiment 2
Signal probe develops the color liquid solution (nano gold spherical 10nM) and exonucleaseⅢ (final concentration of 1unit/L) mixing.Reaction condition: 2
Hour, 37 DEG C of constant temperature.Then reaction solution spectrum is measured with Fluorescence Spectrometer and UV-vis, as a result as in Figure 2-4.
Control group 4 (is labeled as P.aeru): by the Pseudomonas aeruginosa buffer of 50 μ L, (Pseudomonas aeruginosa buffer includes green pus bar
Bacterium P.aeru and buffer, buffer components are the MgCl of 10mM tris (pH=7.5), 2.5mM2With the CaCl of 0.5mM2,
P.aeru is 108100 μ L nucleic acid signals of the nucleic acid double chain heterozygosis liquid solution, embodiment 2 that CFU/mL) are added to embodiment 3 are visited
Needle develops the color liquid solution (nano gold spherical 10nM) and exonucleaseⅢ (final concentration of 1unit/L) mixing.Reaction condition: 2 hours,
37 DEG C of constant temperature.Then reaction solution spectrum is measured with Fluorescence Spectrometer and UV-vis, as a result as in Figure 3-5.
The ultraviolet-visible absorption spectroscopy of the test solution of addition different bacterium is obtained from Fig. 3.As can be seen from Figure 4, it is adding
There is the ratio of the test solution of MRSA to absorb light intensity (A650/A519) it is significantly higher than the test solution added with other bacteriums, and energy
MRSA is enough differentiated by macroscopicization.The aptamer of 3 nucleic acid double chain heterozygote of embodiment can only occur special with MRSA
Property combine, release obtains free DNA starting chain；The non-protected sequence of DNA starting chain and the single-stranded generation of DNA signal probe are special
Property combine, formation can digestion nucleic acid signal probe developing body (can digestion nucleic acid signal probe developing body DNA starting chain protection
Sequence protection DNA starting chain is not hydrolyzed by exonuclease III)；Exonuclease III digestion this can digestion nucleic acid signal probe
The DNA signal probe of developing body is single-stranded, discharge this can digestion nucleic acid signal probe developing body DNA starting chain, obtain nanogold
Ball, most nano gold sphericals mutually occur to assemble and develop the color, so that the change of color occurs for reaction solution, from the red of control group 1-4
Discoloration realizes that naked eyes detect the quick visualization of bacterium at the purple of experimental group；Meanwhile it being received in nucleic acid signal probe developing body
Meter Jin Qiu has excellent electric conductivity, utilizes the electric conductivity and fluorescence resonance energy principle of transfer of nano gold spherical, completion pair
The fluorescent quenching of the single-stranded FAM fluorescein base group of DNA signal probe.Under the action of DNA starts chain and exonuclease III
Under driving, DNA signal probe is single-stranded constantly hydrolyzed, so that FAM fluorescein base group is detached from from nano gold spherical surface, causes
The fluorescence of reaction solution system restores.The DNA starting chain of release and the single-stranded combination of others DNA signal probe, in Exonucleolytic
The repetition enzymolysis process of enzyme III is completed to be detected the purpose of the fluorescence signal augmentation detection of MRSA experimental group by Fluorescence Spectrometer
Fluorescence data out.
Embodiment 5
The embodiment of the present invention is the sensitivity experiment of Biomolecule detection kit, the specific steps are as follows:
MRSA is added in the nucleic acid double chain heterozygosis liquid solution of the embodiment 3 of 50 μ L, so that nucleic acid double chain heterozygote is molten
The concentration of MRSA is 0,1,10,10 in liquid2、103、104、105、106、107With 108CFU/ml, the 100 μ L nucleic acid letter of embodiment 2
Number probe colour developing liquid solution (nano gold spherical 10nM) and exonucleaseⅢ (final concentration of 1unit/L) mix.Reaction condition: 2 is small
When, 37 DEG C of constant temperature.Then reaction solution spectrum is measured with Fluorescence Spectrometer and UV-vis, as a result as illustrated in figs. 5-7, from Fig. 6-8
In obtain the detection range and detection sensitivity of MRSA, the detection limit of fluorescent spectrometry and UV-vis absorption spectrometry to MRSA
It is attained by single bacteria detection level, realizes highly sensitive detection.
Embodiment 6
The embodiment of the present invention is that Biomolecule detection kit detects MRSA measurement experiment in different body fluid, specific steps
It is as follows:
By the buffer (MgCl of 10mM tris (pH=7.5), 2.5mM of experimental group, control group 1-4 in embodiment 42
With the CaCl of 0.5mM2) change into respectively cerebrospinal fluid (labeled as CSF), urine (labeled as Urine), saliva (labeled as Spit) and
Serum solution (is labeled as Serum), then measures reaction solution spectrum with Fluorescence Spectrometer, as a result as shown in figure 9, can from Fig. 9
Know, detection kit of the invention is suitable for a variety of detection liquid, and buffer, cerebrospinal fluid, urine, saliva and serum solution are suitable
With detection kit of the invention has the universality of detection environment.
Embodiment 7
The embodiment of the present invention is that the universality of Biomolecule detection kit is tested, the specific steps are as follows:
For different types of bacterium, not homotactic aptamer, DNA starting chain and DNA signal probe chain are designed
It is single-stranded.Its sequence is as shown in the table:
Its experimental procedure such as embodiment 4 is similar, the spectrum of the reaction solution of different bacterium is measured with Fluorescence Spectrometer, as a result
As shown in Figure 10, as can be seen from Figure 10, Biomolecule detection kit of the invention is suitable for the detection of a variety of different bacteriums, this hair
Bright detection kit has the universality of test object.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (10)
1. a kind of Biomolecule detection kit characterized by comprising
Nucleic acid double chain heterozygote, nucleic acid signal probe developing body and exonucleaseⅲ；
Wherein, nucleic acid double chain heterozygote includes aptamer and nucleic acid promoter chain, and the aptamer and the nucleic acid open
The same end of dynamic chain is additionally provided with protection sequence, the non-guarantor of the non-protected sequence of the aptamer and the nucleic acid promoter chain
Sequence-specific is protected to combine；The aptamer specific recognition and in conjunction with the biomolecule, and the aptamer
It is higher than the associativity of the aptamer and the nucleic acid promoter chain with the associativity of the biomolecule；
The nucleic acid signal probe developing body includes that colour developing medium and nucleic acid signal probe are single-stranded, and the nucleic acid signal probe is single-stranded
5 ' end in conjunction with the colour developing medium, the single-stranded sequence of the nucleic acid signal probe can be non-protected with the nucleic acid promoter chain
Sequence-specific combine, the exonucleaseⅲ can digestion nucleic acid signal probe it is single-stranded.
2. Biomolecule detection kit according to claim 1, which is characterized in that the aptamer passes through external
Screening technique-index concentration Fas lignand system evolution technology screens to obtain from the molecular library of the biomolecule.
3. Biomolecule detection kit according to claim 1, which is characterized in that the protection sequence is poly thymus gland
Pyrimidine repeats oligonucleotide sequence, and the number of the thymidine is 5-60.
4. Biomolecule detection kit according to claim 1, which is characterized in that the colour developing medium is that can gather
The substance for collecting and developing the color.
5. Biomolecule detection kit according to claim 4, which is characterized in that the colour developing medium is selected from nanogold
Belong to one of ball, quantum dot or a variety of.
6. Biomolecule detection kit according to claim 5, which is characterized in that the nano metal ball is selected from nanometer
Gold goal or/and nanometer ping-pong ball.
7. Biomolecule detection kit according to claim 6, which is characterized in that the nucleic acid signal probe is single-stranded
5 ' ends are also connected with colour developing medium binding sequence, and the colour developing medium binding sequence is single-stranded for nucleic acid signal probe and colour developing is situated between
The combination of matter.
8. Biomolecule detection kit according to claim 5, which is characterized in that the colour developing medium binding sequence is
Poly thymidine repeats oligonucleotide sequence, and the number of the thymidine is 5-60.
9. Biomolecule detection kit according to claim 5, which is characterized in that the nucleic acid signal probe is single-stranded
3 ' ends are also marked with fluorescein base group.
10. a kind of biomolecule detecting method, which comprises the following steps:
Step 1, by the nucleic acid double chain heterozygote, nucleic acid signal probe developing body and exonucleaseⅲ and the biomolecule
Mixing, obtains reaction solution；
Step 2 carries out color developing detection to the reaction solution, obtains testing result.
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Cited By (4)
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| CN114807313A (en) * | 2022-01-28 | 2022-07-29 | 东北农业大学 | Method for detecting escherichia coli by using exonuclease III and enhanced test strip |
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