CN108531550A

CN108531550A - Marking and identifying method of particle carrier and application thereof

Info

Publication number: CN108531550A
Application number: CN201810102290.9A
Authority: CN
Inventors: 杜权; 王华全
Original assignee: Wuhan Sunma Biotechnology Co ltd
Current assignee: Du Quan
Priority date: 2018-02-01
Filing date: 2018-02-01
Publication date: 2018-09-14

Abstract

The invention discloses a marking and identifying method of a particle carrier and application thereof. The labeling molecule specifically labels the particulate support by contacting and binding an amount of at least one detectable labeling molecule to an amount of the particulate support under certain coupling conditions. Identifying the type of the labeled particulate carrier by qualitatively and quantitatively detecting the labeled molecule coupled to the particulate carrier. The invention realizes the accurate quantitative identification of the particle carrier by utilizing the artificially synthesized labeled molecules with a certain amount of detectable groups, and obviously improves the stability and the distinguishing degree of the same type of particle carrier labels. Based on the marking and identifying method of the particle carrier, the invention also provides a detection method of nucleic acid molecules and a detection method of microorganisms, which can carry out rapid, convenient, qualitative and quantitative detection on dozens of known nucleic acid molecules and dozens of known microorganisms and have the advantages of high flux, high precision and good stability.

Description

The label and discrimination method of a kind of particulate carrier and its application

Technical field

The present invention relates to nucleic acid detection technique fields, and in particular to the label and discrimination method of a kind of particulate carrier and its answers With.

Background technology

As the carrier of gene and expressing gene, detection of nucleic acids is an important side in biomedical research and application Face.Currently, the nucleic acid detection technique of mainstream has Northern blot, RT-PCR, real-time fluorescence quantitative PCR, genetic chip and two For sequencing technologies.

Northern blot are used to detect the RNA of specificity, are most classical gene expression detection methods, can it is qualitative or The expression of gene is quantitatively detected, but detection flux is relatively low and complex for operation step, technology is more demanding.

RT-PCR technology is broadly divided into qualitative RT-PCR and two kinds quantitative of real-time fluorescence quantitative RT-PCR, is commonly used in The detection of mRNA gene expression abundances can determine purpose RNA by the signal strength between quantitative comparison internal standard RNA and target RNA The relative amount of molecule realizes the quantitative detection to target nucleic acid molecules.

According to the type of probe, real-time fluorescence quantitative RT-PCR uses the PCR instrument coupled with excitation optical detection device, Fluorescence signal is detected during PCR in real time.With this technology, relative quantification, absolute quantitation can be carried out to DNA, RNA sample And qualitative analysis.The technological merit of real-time fluorescence quantitative RT-PCR includes：1) it is detected the range of linearity of sample rna initial concentration It is wide；2) fluorescence monitoring reaction sensitivity is high, and the high 3) sample of accuracy need not carry out amplification post analysis, it is only necessary to sample be known in advance This rough abundance range.Real-time fluorescence quantitative RT-PCR is widely used, and is had made great progress.In the SARS epidemic phase Between, it was once used to detect sars coronavirus.The detection sensitivity ratio Northern blot highers of RT-PCR, the RNA samples needed Amount is also less.But since it is related to two continuous enzymatic reaction systems, technical sophistication degree higher, this is also to the control of detection process Make the higher requirement proposed.

Northernblot, RT-PCR, the flux of three detection schemes of real-time fluorescence quantitative PCR are relatively low, are only suitable for minority It is detected while several nucleic acid molecules.What it is in view of biosystem is a complicated regulated and control network, and the variation of any point is all It can cause being adaptively adjusted for whole network.Include gene in recent years to realize the Multiple detection to gene or gene expression High-throughput nucleic acid detection scheme including chip and two generation sequencing technologies has obtained swift and violent development.High-throughput nucleic acid is detected in skill Breakthrough in art also brings bright foreground for its clinical application.

Skill was sequenced with the sequence of a parallel determination hundreds of thousands to millions of DNA moleculars in two generations, and sequencing reading length is generally More than 40 to 100 a bases.High-throughput advantage can carry out the genome or transcript profile of species comprehensive and careful Analysis.Experiment flow generallys include four sample of nucleic acid preparation, sequencing library structure, sequencing reaction and data analysis steps.Root According to the uniqueness of technical solution, two current generations sequencing mainly has extensive parallel signature sequencing, polonies, 454 burnt phosphorus Sour sequencing, Illumina (Solexa) sequencing, ABI SOLiD sequencing, ionic semiconductor be sequenced, DNA nanometers Ball sequencing etc..

The technical principle of genetic chip is to use oligonucleotides fabricated in situ or micro- printing means, by a large amount of probe fragments Cure in an orderly manner in the surface of support, high-density DNA microarray is made, whether has sequence complementary therewith in sample for detecting Row.By carrying out laser confocal scanning to slide, the fluorescence intensity of each point on microarray is measured, is calculated various in sample to be tested The expression of gene.Currently, genetic chip has been employed successfully in detection, gene diagnosis and drug screening of gene expression amount etc. Life science.The great advantage of biochip technology is analysis throughput height, moreover it is possible to detect gene expression amount in two groups of samples Difference.But low, quantitative poor specificity that there is sensitivity needs expensive instrument and signal analysis software and is prepared in probe There is comparable technical difficulty with hybridization link.

Invention content

In order to overcome the above-mentioned deficiency of the prior art, it is an object of the invention to provide a kind of label of particulate carrier and discriminating sides Method and its application, the present invention utilize mark molecule artificial synthesized, with a certain amount of detectable group, realize and are carried to microballoon The accurate quantitative mark of body is significantly improved to the stability of the microsphere supported label of same class and can discrimination.And by the particle The label and discrimination method of carrier are applied to the detection of nucleic acid and microorganism.

To achieve the goals above, the present invention is achieved by the following technical solutions：

A kind of label and discrimination method of particulate carrier, include the following steps：

1) a kind of particulate carrier is obtained, the particulate carrier surface has at least one group being coupled；

2) obtain at least one detectable mark molecule, each mark molecule contain it is at least one can independent detection mark Note group and it is at least one can the group that is coupled of coupling group with particulate carrier surface；

3) under certain coupling condition, make a certain amount of detectable mark molecule of at least one and a certain amount of particle Carrier is contacted and is coupled, and mark molecule is made specifically to mark the particulate carrier；

4) by detecting the mark molecule with particulate carrier coupling, differentiate the type of the labeled particulate carrier, institute The detection mode for stating mark molecule is at least one of qualitative detection or quantitative detection.

Further, the particulate carrier is selected from Ago-Gel microballon, sephadex microballon, cellulose bead, gathers Acrylamide gel, polyacrylic acid ester liposome composite particles, chitosan particle, gold-magnetic particles, bead.

Further, a diameter of 10 nanometers~1000 microns of the particulate carrier.

Further, it is described can coupling group be selected from antigen molecule, antibody molecule, streptavidin, biotin.

Further, the detectable mark molecule is selected from oligonucleotide molecules, peptide molecule, polysaccharide molecule, tree-like Macromolecule, artificial synthesized macromolecule.

A kind of detection method of nucleic acid molecules, includes the following steps：

2) obtain at least one detectable label molecule, each mark molecule contain it is at least one can independent detection label Group and it is at least one can with particulate carrier surface can the group that is coupled of coupling group；

3) a kind of capture probe molecule is obtained, which visits molecule needle and contain one section of specific nucleic acid sequence and at least one The group that kind can be coupled, the nucleic acid sequence can form a degree of complementary pairing with detected target nucleic acid molecules, The group being coupled can with particulate carrier surface can coupling group occur coupled action；

4) make detectable label molecule, capture probe molecule and particulate carrier surface can coupling group be coupled, obtain one Kind can specifically bind object to be measured nucleic acid molecules and the specific detection carrier with detectable label；

5) detectability label is carried out to target nucleic acid molecules to be measured；

6) under nucleic acid hybridization conditions, the labeled object to be measured nucleic acid molecules that step 5) obtains is made to be obtained with step 4) Specific detection carrier contact, make object to be measured nucleic acid molecules and be coupled to detection carrier surface capture probe progress it is miscellaneous It hands over, obtains the combination of object to be measured nucleic acid molecules and specific detection carrier；

7) combination for obtaining the object to be measured nucleic acid molecules and specific detection carrier in step 6), detects the detection The type and intensity of mark molecule on carrier and the type and intensity of the detectable label on object to be measured nucleic acid determine The presence of object to be measured nucleic acid molecules and content.

Further, the capture probe is selected from oligonucleotides molecule, DNA oligo molecule contains simultaneously There are ribonucleic acid and DNA nucleic acid oligomer molecule.

Further, the synthetic method of specific detection carrier is in step 4)：Under certain coupling condition, make first A certain amount of at least one detectable label molecule is contacted and is combined with a certain amount of particulate carrier, keeps mark molecule special Mark the particulate carrier to property；Then a certain amount of capture probe molecule and a certain amount of labeled particulate carrier are carried out It contacts and combines, obtain the specific detection carrier that can specifically bind target nucleic acid molecules.

Further, the synthetic method of specific detection carrier is in step 4)：Under certain coupling condition, make certain At least one detectable label molecule, a kind of capture probe molecule of amount are contacted with a certain amount of particulate carrier, make label Molecule, capture probe molecule are combined with particulate carrier surface, obtain can specifically bind target nucleic acid molecules and with can Detect the specific detection carrier of label.

The present invention also provides the application of the detection method of above-mentioned nucleic acid molecules, the detection method application of the nucleic acid molecules In gene expression detection.

Based on the detection method of above-mentioned nucleic acid molecules, the present invention also provides a kind of detection reagent of nucleic acid molecules, the inspections Test agent includes particulate carrier, detectable label molecule, capture probe molecule and corresponding coupling buffer, Hybridization Buffer Liquid and label detection reagent, the particulate carrier surface have at least one can coupling group, the detectable label molecule contains Have it is at least one can independent detection labelling groups and it is at least one can with particulate carrier can coupling group coupling group Or molecule, the capture probe molecule can be coupled with particulate carrier and can be with object to be measured making nucleic acid molecular hybridizations.

The present invention also provides a kind of detection methods of microorganism, and detection method includes the following steps for this：

2) obtain at least one detectable mark molecule, each mark molecule contain it is at least one can independent detection mark Remember group and at least one group that can be coupled with the group of particulate carrier being coupled；

3) a kind of capture probe molecule is obtained, which visits molecule needle and contain one section of specific nucleic acid sequence and at least one The group that kind can be coupled, the target nucleic acid molecules that the nucleic acid sequence can contain with detected Institute of Micro-biology form a degree of Complementary pairing, the group being coupled can with particulate carrier surface can coupling group occur coupled action；

4) detectable label molecule, capture probe molecule is made to be combined with particulate carrier surface, acquisition can be specifically bound The target nucleic acid molecules that tested Institute of Micro-biology contains and the specific detection carrier with detectable label；

5) it obtains and is detected the target nucleic acid molecules that Institute of Micro-biology is contained, carry out detectability label；

6) under nucleic acid hybridization conditions, make the specificity of the labeled nucleic acid molecules that step 5) obtains and step 4) acquisition Carrier contact is detected, target nucleic acid molecules is made to be hybridized with the capture probe for detecting carrier surface, obtains target nucleic acid molecules With the combination of specific detection carrier；

7) combination for obtaining the object to be measured nucleic acid molecules and specific detection carrier in step 6), detects the mark of carrier The type and intensity for the sub type and the detectable label in intensity and target nucleic acid molecules of scoring determine tested microorganism In contained nucleic acid molecules presence and its content, so that it is determined that be detected microorganism presence and its quantity.

Further, the synthetic method of specific detection carrier is in step 4)：Under certain coupling condition, make first A certain amount of detectable mark molecule of at least one is contacted and is combined with a certain amount of particulate carrier, keeps mark molecule special The particulate carrier is marked anisotropicly；Then by a certain amount of capture probe molecule and a certain amount of labeled particulate carrier into Row is contacted and is combined, and acquisition can specifically bind specific detection carrier of the tested Institute of Micro-biology containing target nucleic acid molecules.

Further, the synthetic method of specific detection carrier is in step 4)：Under certain coupling condition, make certain The detectable mark molecule of at least one of amount, a kind of capture probe molecule are contacted with a certain amount of particulate carrier, make mark Sub, capture probe molecule of scoring is combined with particulate carrier surface, and acquisition can specifically bind tested Institute of Micro-biology's core containing target Acid molecule and the specific detection carrier with detectable label.

Finally, the present invention provides the application of the detection method of the microorganism, the microorganism detection method is answered Detection for clinical pathogenic microorganism, food microorganisms, agricultural product microorganism, livestock and poultry sex pheromone.

Compared with prior art, the beneficial effects of the present invention are：

(1) present invention utilizes mark molecule artificial synthesized, with a certain amount of detectable group, realizes and is carried to particle The accurate quantitative mark of body, significantly improves the stability marked to same class particulate carrier and can discrimination.

(2) label and identification method of the particulate carrier based on the present invention, present invention provides a kind of nucleic acid molecules Detection method and a kind of detection method of microorganism, can be to microorganism known to nucleic acid molecules known to tens of kinds and tens of kinds Quick, easily qualitative and quantitative detection is carried out, has the advantages that high throughput, high-precision, stability are good.

Description of the drawings

Fig. 1 is the fluorescent image of detection of particles carrier in embodiment 1.

Quantitative analysis results of the Fig. 2 by the sample of nucleic acid that different labeled microparticles capture in embodiment 1.

Fig. 3 is the optimization analysis result to particulate carrier quantity in embodiment 2.

Fig. 4 is the optimization analysis result to hybridization temperature in embodiment 2.

Fig. 5 is the optimum results to hybridization time in embodiment 2.

Fig. 6 is the optimum results to hybridizing volume conditions in embodiment 2.

Fig. 7 is a kind of capture probe in embodiment 3 to the detection specificity result of study of single nucleic acid sample.

Fig. 8 is a kind of capture probe in embodiment 3 to the detection specificity result of study of mixing sample of nucleic acid.

Fig. 9 is the linear standard curve of different microRNA analogs concentration and fluorescence intensity in embodiment 3.

Figure 10 is salmonella, Shigella, staphylococcus aureus, enterocolitis Yale Salmonella, list in embodiment 4 Increase the standard curve of Listeria reference molecules.

Figure 11 be embodiment 4 in campylobacter jejuni, enterorrhagia escherichia coli, vibrio parahemolyticus, comma bacillus, The standard curve of Vibrio vulnificus reference molecules.

Figure 12 is the standard curve of hepatitis B, hepatitis C virus reference molecules in embodiment 5.

Specific implementation mode

With reference to specific embodiment and attached drawing, the present invention is further explained.It should be appreciated that these embodiments are only used for It is bright the present invention and cannot be used for limiting the scope of the invention.It can be from material, method and reaction condition etc. to institute of the present invention Disclosure is improved, and all these improvement should all be within the scope of the invention.No special explanation, the present invention are real It is commercial goods to apply reagent used by example, and database used in the embodiment of the present invention is disclosed online database.Under Test method without specific conditions in row embodiment, usually according to normal condition, such as Sambrook et al. writes《Point Son clone：Laboratory manual》(NewYork:Cold Spring Harbor Laboratory Press, 1989) described in Condition, or according to the normal condition proposed by manufacturer.

Wherein, the particulate carrier is selected from Ago-Gel microballon, sephadex microballon, cellulose bead, polypropylene Acrylamide gel, polyacrylic acid ester liposome composite particles, chitosan particle, gold-magnetic particles, bead, preferably Ago-Gel Microballon；A diameter of 10 nanometers~1000 microns of the particulate carrier, preferably 100 nanometers~200 microns, more preferably 10 is micro- Rice~100 microns；It is described can coupling group be selected from antigen molecule, antibody molecule, streptavidin, biotin, preferably chain is affine Element；The detectable mark molecule is selected from oligonucleotide molecules, peptide molecule, polysaccharide molecule, tree form modification, artificial synthesized Macromolecule, preferably oligonucleotide molecules, peptide molecule, tree form modification, more preferably oligonucleotide molecules；It is described can be only The labelling groups of vertical detection are selected from fluorophor, isotope, phosphatase, antigen, preferably fluorophor.

Label based on above-mentioned particulate carrier and discrimination method, the present invention propose a kind of detection method of nucleic acid molecules, Include the following steps：

Wherein, the capture probe is selected from oligonucleotides molecule, DNA oligo molecule contains simultaneously There are ribonucleic acid and DNA nucleic acid oligomer molecule, preferably DNA oligo molecule；It is special in step 4) Property detection carrier synthetic method be：Under certain coupling condition, make a certain amount of at least one detectable label point first It is sub to be contacted and combined with a certain amount of particulate carrier, so that mark molecule is specifically marked the particulate carrier；Then will A certain amount of capture probe molecule is contacted and is combined with a certain amount of labeled particulate carrier, and acquisition specific can be tied Close the specific detection carrier of target nucleic acid molecules；Or the synthetic method of specific detection carrier is in step 4)：Certain Coupling condition under, make a certain amount of at least one detectable label molecule, a kind of capture probe molecule and a certain amount of particle Carrier is contacted, and so that mark molecule, capture probe molecule is combined with particulate carrier surface, acquisition can specifically bind target Nucleic acid molecules and with detectable label specific detection carrier.

The detection method of above-mentioned nucleic acid molecules is applied to gene expression detection, is such as applied to prepare the detection examination of nucleic acid molecules Agent, which includes particulate carrier, detectable label molecule, capture probe molecule and corresponding coupling buffer, miscellaneous Hand over buffer solution and label detection reagent, the particulate carrier surface have at least one can coupling group, the detectable label Molecule contain it is at least one can independent detection labelling groups and it is at least one can with particulate carrier can coupling group be coupled Group or molecule, the capture probe molecule can with particulate carrier be coupled and can be with object to be measured making nucleic acid molecular hybridization.

The detection method of above-mentioned nucleic acid molecules can also be applied to detection microorganism, a kind of detection method of microorganism, packet Include following steps：

Capture probe is selected from oligonucleotides molecule, DNA oligo point in mentioned microorganism detection method Son contains ribonucleic acid and DNA nucleic acid oligomer molecule simultaneously；The synthesis side of specific detection carrier in step 4) Method is：Under certain coupling condition, make a certain amount of detectable mark molecule of at least one and a certain amount of particle first Carrier is contacted and is combined, and mark molecule is made specifically to mark the particulate carrier；Then by a certain amount of capture probe Molecule is contacted and is combined with a certain amount of labeled particulate carrier, and acquisition can specifically bind tested Institute of Micro-biology and contain The specific detection carrier of target nucleic acid molecules；The synthetic method of specific detection carrier may be in step 4)：Certain Coupling condition under, make the detectable mark molecule of a certain amount of at least one, a kind of capture probe molecule with it is a certain amount of micro- Grain carrier is contacted, and so that mark molecule, capture probe molecule is combined with particulate carrier surface, acquisition can specifically bind quilt Target nucleic acid molecules contained by micrometer biology and the specific detection carrier with detectable label.

The detection method of above-mentioned microorganism can be applied to clinical pathogenic microorganism, food microorganisms, agricultural product microorganism, The detection of livestock and poultry sex pheromone.

Embodiment 1：The experiment flow of detection of nucleic acids scheme

Detection of nucleic acids scheme provided by the invention includes six operating procedures：Fluorescent marker, the capture probe of particulate carrier It contains, the analysis of sample of nucleic acid extraction, the fluorescent marker of sample of nucleic acid, solution hybridization and hybridization signal.Now in 3T3-L1 cells MicroRNA-146b-5p (5 '-ugagaacugaauuccauaggcu, nucleotide sequence such as SEQ ID NO：Shown in 1) expression For level detection, illustrate technical solution provided by the invention.

1, the fluorescent marker of particulate carrier：The particulate carrier that the present embodiment uses is purchased from GE Healthcare Bio- Agarose microbeads (the Streptavidin of Sciences companiesHigh Performance, 17-5113- 01), microsphere surface coupling has the streptavidin molecule that can be combined with biotin.For fluorescent marker (fluorescence-encoded) agarose The mark molecule of microballoon is artificial synthesized few DNA molecule, sequence such as SEQ ID NO：Shown in 2, the ends 3' are equal Containing biotin group, can be coupled with the streptavidin molecular radical on agarose microbeads surface.In addition, the two molecules The ends 5'- all have detectable fluorophor, the fluorescent marker of mark molecule 1 is：5’-Alexa488-aaaaaaaaaa- The fluorescent marker of biotin, mark molecule 2 is：5’-Texas Red-aaaaaaaaaa-biotin.

Label reaction：In the overall reaction system of 75 μ L, be added a certain amount of agarose microbeads (the present embodiment reaction in, It is added 5 × 10³A agarose microbeads).A certain amount of mark molecule 1 and a certain amount of mark molecule 2, the total concentration of the two is added For 0.2pmol/ μ L (in the present embodiment reaction, the mark molecule 1 and 0.1pmol/ μ L mark molecules 2 of 0.1pmol/ μ L is added). According to the relative amount that mark molecule 1 and mark molecule 2 is added, identifiable differentiation can be carried out to different agarose microbeads Coding.

2, capture probe contains：According to the nucleic acid sequence of microRNA-146b-5p, the capture of design and the complementation of its sequence Probe, sequence capture probe such as SEQ ID NO：Shown in 3.Synthetic capture probe, in its ends 5' couple biotin group (capture-146b-5p:5 '-biotin-aaaaaaaaaaagcctatggaattcagttctca), it can be with agarose The streptavidin group of microsphere surface is coupled.

Containing for capture probe reacts as follows：In 100 μ L reaction systems, it is added 5 × 10³A agarose microbeads, end are dense Spend the internal reference trapping nucleic acids probe of 10pM, internal reference trapping nucleic acids probe sequence such as SEQ ID NO：(capture- shown in 4 reference：5 '-biotin-aaaaaaaaaaagtcagtcagtcagtcagtcagtc), the capture- of final concentration 40pM 146b-5p capture probes.

3, sample of nucleic acid extracts：Using the DMEM medium culture 3T3-L1 cells of GIBCO companies, contain in culture solution 10% fetal calf serum, the penicillin of 100units/mL and 100 μ g/mL streptomysins (Life Technologies) cultivate item Part is 37 DEG C, 5%CO₂.Utilize the Trizol reagents of Invitrogen, the total serum IgE of extraction culture cell.

In order to detect the quality of total serum IgE sample, a certain amount of RNA samples are taken, are loaded on 8% denaturation PAGE gels, It is separated by electrophoresis.18S, the integrality of 28S rRNA bands and the relationship of content are observed, determines extracted total serum IgE sample Whether quality is qualified.For the qualified total serum IgE sample of detection, 2000 ultramicrospectrophotometers of NanoDrop are utilized (NanoDrop Technologies) is quantified, and is stored in spare in -80 DEG C of refrigerators.

4, the fluorescent marker of sample of nucleic acid：Using the Label IT Cy5 kits of Mirus companies, to the total serum IgE of acquisition And the internal reference nucleic acid participated in, internal reference nucleic acid sequence such as SEQ ID NO：(reference DNA shown in 5：5'- Gactgactgactgactgactgact fluorescent marker) is carried out.It is operated according to the specification of kit, step is specifically such as Under：1) 5 μ g total serum IgE samples are taken, DNase/RNase-free deionized waters are added to 40 μ L, 10 × Labeling is added 5 μ L, Label IT Reagent of BufferA, 5 μ L, total reaction volume are 50 μ L；2) reaction system is placed under the conditions of 37 DEG C It is incubated 1 hour；3) sample after G50Microspin purification column purification tags is used, is reacted for subsequent solution hybridization.

5, solution hybridization：In the solution hybridization system of 40 μ L, a certain amount of labeled agarose microbeads are added, are added The total serum IgE sample of a certain amount of label.Make reaction system under certain hybridization temperature under the conditions of be incubated the regular hour, complete miscellaneous Hand over reaction.The agarose microbeads amount that the present embodiment hybridization reaction is added is 1 × 10³, the total serum IgE sample size of addition is 400ng, miscellaneous It is 40 minutes to hand over 25 DEG C of temperature, hybridization time.

6, hybridization signal is analyzed：Use the Operetta High Content Screening of Perkin Elmer companies AndAnalysis systems obtain microsphere supported hybridization fluorescent image.Using the system Columbus image datas storage and Analysis system (Columbus Image Data Storage andAnalysis system), analyzes the fluorescent image of acquisition, Qualitative and quantitative analysis is carried out to target nucleic acid.Analytic process is specific as follows：

1) removal of background fluorescence：Include a certain amount of reference without any coated original form in each reaction Microballoon, for removing reasons for its use fluorescence signal in detection process.Using these with reference to microballoon, each fluorescence channel is determined Background value.In the case where removing fluorescence background, subsequent analysis is completed.

2) agarose microbeads is fluorescence-encoded：By calculating each microballoon A488 fluorescent values and Texas Red fluorescent values Ratio determines the fluorescence-encoded ratio (A488/Texas Red) of the microballoon.Using the theoretical value of certain microsphere fluorescence coding as base Standard determines the microballoon positioned at a reference value attachment, the candidate population as the fluorescence-encoded micro-beads.It obtains in each candidate population Between 80% microballoon individual, be used for subsequent analysis.

3) fluorescent quantitation of target nucleic acid：The Cy5 fluorescent values for detecting each coding microball, subtract background fluorescence activity and Due to error fluorescent value caused by fluorescence interference, the fluorescent measurement of the microballoon target nucleic acid is obtained.

4) the opposite and absolute quantitation of target nucleic acid：Based on the content of the internal reference nucleic acid molecules participated in, pass through calculating The ratio of target nucleic acid molecules Cy5 fluorescent values and internal reference nucleic acid molecules fluorescent value, calculates the relative expression quantity of target nucleic acid molecules. The absolute content of target nucleic acid molecules can also be calculated by establishing the calibration curve of target nucleic acid molecules and reference molecules.

When being separately added into the mark molecule 1 and mark molecule 2 of different relative amounts, microsphere supported detection image such as Fig. 1 It is shown, (Alexa indicated in Fig. 1 is that the Texas indicated in fluorescent dye Alexa488, Fig. 1 is fluorescent dye Texas Red, Alexa:Texas=4:1 indicates that the ratio between the concentration of Alexa 488 and Texas Red concentration are 4:1), target nucleic acid molecules are quantitative (A indicates Alexa to testing result in figure as shown in Figure 2:Texas=4:1, B indicates Alexa:Texas=2:1, C indicates Alexa: Texas=1:2, D indicate Alexa:Texas=1:4).The result shows that according to the opposite of mark molecule 1 and mark molecule 2 is added Content can carry out identifiable differentiation coding to different agarose microbeads, can not only be distinguished with not by fluoroscopic examination With fluorescence-encoded agarose microbeads, moreover it is possible to realize the qualitative and quantitative analysis of target nucleic acid molecules.

Embodiment 2：The technical optimization of detection of nucleic acids scheme

In order to establish stable solution hybridization scheme, the present invention has carried out system optimization to hybridization conditions.Experiment material and Experimentation is specific as follows.

A, microsphere supported：The Streptavidin of GE Healthcare Bio-Sciences companies High Performance agarose microbeads carriers.

B, microballoon mark molecule：

Mark molecule 1：5’-Alexa 488-AAAAAAAAAA-biotin

Mark molecule 2：5’-Texas Red-AAAAAAAAAA-biotin

C, sample of nucleic acid is detected：With four kinds of microRNA analogs of chemical synthesis, as detection sample, RNA sequence is such as SEQ ID NO：6~SEQ ID NO：Shown in 9.

mmu-miR-146b-5p：5’-ugagaacugaauuccauaggcu

mmu-miR-222-3p：5’-agcuacaucuggcuacugggu

mmu-miR-302a-3p：5’-uaagugcuuccauguuuugguga

mmu-miR-let-7d-5p：5’-agagguaguagguugcauaguu

D, capture probe：Based on the sequence for being detected microRNA, the capture probe of specific effect, sequence are designed Row such as SEQ ID NO：10~SEQ ID NO：Shown in 13.

Cap-miRNA146b：5’-biotin-aaaaaaaaaaagcctatggaattcagttctca

Cap-miRNA222：5’-biotin-aaaaaaaaaaacccagtagccagatgtagct

Cap-miRNA302a：5’-biotin-aaaaaaaaaatcaccaaaacatggaagcactta

Cap-miRNAlet7d：5’-biotin-aaaaaaaaaaaactatgcaacctactacctct

E, sample labeling reagent：The Label IT Cy5 kits of Mirus companies.

F, Image Acquisition and data analysis：Perkin companies Operetta High Content Screening and Analysis systems and Columbus image data storage and analysis systems.

1, the optimization of microsphere supported usage amount

According to the scheme that embodiment 1 describes, with the mixture for four kinds of mark molecules that total concentration is 0.2pmol/ μ L, fluorescence Encode four kinds of agarose microbeads.Wherein, for contain Cap-miRNA146b capture probes agarose microbeads it is fluorescence-encoded For：Concentration=4 of concentration/mark molecule 2 of mark molecule 1:1, the agarose for containing Cap-miRNA222 capture probes The fluorescence-encoded of microballoon be：Concentration=2 of concentration/mark molecule 2 of mark molecule 1:1, it is caught for containing Cap-miRNA302a Obtaining the fluorescence-encoded of the agarose microbeads of probe is：Concentration=1 of concentration/mark molecule 2 of mark molecule 1:2, for containing The fluorescence-encoded of agarose microbeads of Cap-miRNA let7d capture probes be：Concentration/mark molecule 2 of mark molecule 1 it is dense Degree=1:4.

According to the scheme that embodiment 1 describes, four kinds of capture probes are contained respectively in agarose microbeads surface.Each agar Sugared microballoon only contains a kind of capture probe, respectively Cap-miRNA146b, Cap-miRNA222, Cap-miRNA302a and Cap- miRNAlet7d。

According to the scheme that embodiment 1 describes, the Label IT Cy5 kits provided using Mirus companies close chemistry At four kinds of microRNA analogs molecules (microRNA-146b, microRNA-222, microRNA-302a and MicroRNA-let7d Cy5 fluorescent markers) are carried out.

According to the scheme that embodiment 1 describes, in the hybridization system of 40 μ L, the different captures that contain that four kinds of equivalent are added are visited The agarose microbeads of needle.As shown in figure 3, the addition of each agarose microbeads is respectively 1 × 10 in each reaction³、2×10³、3 ×10³、4×10³、5×10³、6×10³With 7 × 10³.The microRNA of the fluorescent marker of four kinds of equivalent is added into each reaction Analog, each 12.5ng.It is incubated 40 minutes under the conditions of 25 DEG C, completes hybridization reaction.

According to the scheme that embodiment 1 describes, the Operetta High Content of Perkin Elmer companies are utilized Screening andAnalysis systems and Columbus image data storage and analysis systems, obtain and analyze hybridization after it is micro- The fluorescent image of balloon borne body, the results are shown in Figure 3 for acquisition.It was found that with the increase of microsphere supported usage amount, each microballoon carries The hybridization signal of target nucleic acid of body capture gradually reduces, this is consistent with being contemplated to be.According to this experimental result, determine rear In continuous experiment, the usage amount of each hybridization reaction agarose microbeads is 2 × 10³。

2, the optimization of hybridization temperature scheme

According to the scheme that embodiment 1 describes, with the mixture for four kinds of mark molecules that total concentration is 0.2pmol/uL, fluorescence Encode four kinds of agarose microbeads.Wherein, for contain Cap-miRNA146b capture probes agarose microbeads it is fluorescence-encoded For：Concentration=4 of concentration/mark molecule 2 of mark molecule 1:1, the agarose for containing Cap-miRNA222 capture probes The fluorescence-encoded of microballoon be：Concentration=2 of concentration/mark molecule 2 of mark molecule 1:1, it is caught for containing Cap-miRNA302a Obtaining the fluorescence-encoded of the agarose microbeads of probe is：Concentration=1 of concentration/mark molecule 2 of mark molecule 1:2, for containing The fluorescence-encoded of agarose microbeads of Cap-miRNA let7d capture probes be：Concentration/mark molecule 2 of mark molecule 1 it is dense Degree=1:4.

According to the scheme that embodiment 1 describes, in the hybridization system of 40 μ L, the different captures that contain that four kinds of equivalent are added are visited The quantity of the agarose microbeads of needle, each microballoon is 2 × 10³.The fluorescent marker of four kinds of equivalent is added into each reaction MicroRNA analogs, each 12.5ng.Hybridization reaction is carried out using three kinds of different hybridization temperature schemes.Wherein a schemes： Hybridize 10 minutes under the conditions of 65 DEG C, hybridization temperature is then reduced to room temperature rapidly, continues hybridization 50 minutes.B schemes：Room temperature is miscellaneous It hands over 60 minutes.C schemes：Hybridize 10 minutes under the conditions of 65 DEG C；Hybridization temperature is reduced to 55 DEG C, is hybridized 10 minutes；It will hybridization Temperature continues to be reduced to 45 DEG C, hybridizes 10 minutes；Hybridization temperature is reduced to 35 DEG C, is hybridized 10 minutes；Hybridization temperature is reduced To 25 DEG C, hybridize 10 minutes；Terminate hybrid process.

According to the scheme that embodiment 1 describes, the Operetta High Content of Perkin Elmer companies are utilized Screening andAnalysis systems and Columbus image data storage and analysis systems, obtain and analyze hybridization after it is micro- The fluorescent image of balloon borne body is as shown in Figure 4.It was found that influence of the different crossing schemes to experimental result is little.According to this experiment As a result, determining using (25 DEG C) incubation schemes of room temperature.

3, the optimization of hybridization time scheme

According to the scheme that embodiment 1 describes, in the hybridization system of 40 μ L, the different captures that contain that four kinds of equivalent are added are visited The usage amount of the agarose microbeads of needle, each microballoon is 2 × 10³.The fluorescent marker of four kinds of equivalent is added into each reaction MicroRNA analogs, each 12.5ng.Three reactions are incubated 10 minutes, 20 minutes, 30 minutes respectively under the conditions of 25 DEG C With 40 minutes.

According to the scheme that embodiment 1 describes, the Operetta High Content of Perkin Elmer companies are utilized Screening andAnalysis systems and Columbus image data storage and analysis systems, obtain and analyze hybridization after it is micro- The fluorescent image of balloon borne body is as shown in Figure 5.The results show that with the extension of incubation time, hybridization signal is stepped up, this with it is pre- Phase is consistent.According to this experimental result, the crossing scheme for being subsequently incubated 40 minutes using 25 DEG C is determined.

4, hybridize the optimization of volume approach

According to embodiment 1 describe scheme, 40 μ L, 60 μ L, 80 μ L, 100 μ L hybridization system in, be added four kinds of equivalent The agarose microbeads for containing different capture probes, the usage amount of each microballoon is 2 × 10³.Four kinds are added into each reaction The microRNA analogs of the fluorescent marker of equivalent, each 12.5ng.It is incubated 40 minutes under the conditions of 25 DEG C, it is anti-to complete hybridization It answers.

According to the scheme that embodiment 1 describes, the Operetta High Content of Perkin Elmer companies are utilized Screening andAnalysis systems and Columbus image data storage and analysis systems, obtain and analyze hybridization after it is micro- The fluorescent image of balloon borne body is as shown in Figure 6.The results show that influence of the hybridization volume to signal detection is little, follow-up study is determined Hybridization volume be 40 μ L.

Embodiment 3：The technical identification of detection of nucleic acids scheme

In order to verify the sensitivity and specificity of technical solution of the present invention, the present invention has carried out the technology of detection of nucleic acids scheme Checking research.Experiment material and experimentation are specific as follows.

B, microballoon mark molecule：

Mark molecule 1：5’-Alexa 488-AAAAAAAAAA-biotin

Mark molecule 2：5’-Texas Red-AAAAAAAAAA-biotin

mmu-miR-146b-5p：5’-UGAGAACUGAAUUCCAUAGGCU

mmu-miR-222-3p：5’-AGCUACAUCUGGCUACUGGGU

mmu-miR-302a-3p：5’-UAAGUGCUUCCAUGUUUUGGUGA

mmu-miR-let-7d-5p：5’-AGAGGUAGUAGGUUGCAUAGUU

D, capture probe：Based on the sequence for being detected microRNA, the special capture probe influenced is designed, sequence is such as SEQ ID NO：10~SEQ ID NO：Shown in 13.

Cap-miRNA146b：5’-biotin-aaaaaaaaaaAGCCTATGGAATTCAGTTCTCA

Cap-miRNA222：5’-biotin-aaaaaaaaaaACCCAGTAGCCAGATGTAGCT

Cap-miRNA302a：5’-biotin-aaaaaaaaaaTCACCAAAACATGGAAGCACTTA

Cap-miRNAlet7d：5’-biotin-aaaaaaaaaaAACTATGCAACCTACTACCTCT

E, sample labeling reagent：The Label IT Cy5 kits of Mirus companies.

1, the single pattern detection of special Journal of Sex Research-of detection scheme

According to the scheme that embodiment 1 describes, in the hybridization system of four 40 μ L, the difference that contains that four kinds of equivalent are added is caught The agarose microbeads of probe are obtained, the usage amount of each microballoon is 2 × 10³.A kind of fluorescence mark is respectively added into each hybridization system The microRNA analogs 12.5ng of note.It is incubated 40 minutes under the conditions of 25 DEG C, completes hybridization reaction.

According to the scheme that embodiment 1 describes, the Operetta High Content of Perkin Elmer companies are utilized Screening andAnalysis systems and Columbus image data storage and analysis systems, obtain and analyze hybridization after it is micro- The fluorescent image of balloon borne body is as shown in Figure 7.The experimental results showed that when single pattern detection, a kind of capture probe be only capable of capture with The microRNA analog molecules of its sequence complementation have preferable specificity.

2, the special Journal of Sex Research of detection scheme-mixing sample detection

According to the scheme that embodiment 1 describes, in the hybridization system of four 40 μ L, the difference that contains that four kinds of equivalent are added is caught The agarose microbeads of probe are obtained, the usage amount of each microballoon is 2 × 10³.The fluorescence mark of four kinds of equivalent is added into each reaction The microRNA analogs of note, each 12.5ng.It is incubated 40 minutes under the conditions of 25 DEG C, completes hybridization reaction.

According to the scheme that embodiment 1 describes, the Operetta High Content of Perkin Elmer companies are utilized Screening andAnalysis systems and Columbus image data storage and analysis systems, obtain and analyze hybridization after it is micro- The fluorescent image of balloon borne body.Obtain the testing result comparative analysis figure of single sample as shown in Figure 8, mixing sample, experiment knot Fruit, which shows mixing sample not, influences detectability of the technical program to target molecule, when mixing sample detects, a kind of capture Probe is only capable of the microRNA analog molecules of capture and the complementation of its sequence, has preferable specificity.

3, the sensitivity study of detection scheme

According to the scheme that embodiment 1 describes, in 40 μ L hybridization systems, four kinds of equivalent of addition contain different capture probes Agarose microbeads, the usage amount of each microballoon is 2 × 10³.A kind of fluorescent marker of various concentration is added into each reaction MicroRNA analogs, be incubated 40 minutes under the conditions of 25 DEG C, complete hybridization reaction.

According to the scheme that embodiment 1 describes, the Operetta High Content of Perkin Elmer companies are utilized Screening andAnalysis systems and Columbus image data storage and analysis systems, obtain and analyze hybridization after it is micro- The fluorescent image of balloon borne body, is obtained that the results are shown in Figure 9 by fluorescent image.According to the calibration curve established (calibration curve), discovery have reached pM grades to the detection sensitivity of different microRNA analogs, and one There is good linear dependence in a larger range.

Embodiment 4：The detection of food related microorganisms

In order to realize that the present embodiment research has been carried out in the quantitative detection of food related microorganisms, the present invention.Experiment material and Experimentation is specific as follows.

B, microballoon mark molecule：

Mark molecule 1：5’-Alexa 488-aaaaaaaaaa-biotin

Mark molecule 2：5’-Texas Red-aaaaaaaaaa-biotin

C, microorganism specificity capture probe, sequence such as SEQ ID NO：14~SEQ ID NO：Shown in 33：

Salmonella Cap-invA-1：5'-aaaaaaaaaagtgaaattatcgccacgttcgggcaa

Salmonella Cap-invA-2：5'-aaaaaaaaaatcatcgcaccgtcaaaggaacc

Shigella Cap-ipaH-1：5'-aaaaaaaaaagttccttgaccgcctttccgataccgtc

Shigella Cap-ipaH-2：5'-aaaaaaaaaagccggtcagccaccctctgagagtac

Staphylococcus aureus Cap-femA-1：5'-aaaaaaaaaaaaaaaagcacataacaagcg

Staphylococcus aureus Cap-femA-2：5'-aaaaaaaaaagataaagaagaaaccagcag

Enterocolitis Yale Salmonella Cap-16s-1：5'-aaaaaaaaaaaataccgcataacgtcttcg

Enterocolitis Yale Salmonella Cap-16s-2：5'-aaaaaaaaaacttcttctgcgagtaacgtc

Listeria monocytogenes Cap-prfA-1：5'-aaaaaaaaaagatacagaaacatcggttggc

Listeria monocytogenes Cap-prfA-2：5'-aaaaaaaaaagtgtaatcttgatgccatcag

Campylobacter jejuni Cap-VS1-1：5'-aaaaaaaaaagatatgtatgattttatcttgc

Campylobacter jejuni Cap-VS1-2：5'-aaaaaaaaaagaatgaaattttagaatgggg

Enterorrhagia escherichia coli Cap-efbE-1：5'-aaaaaaaaaaattgcgctgaagcctttg

Enterorrhagia escherichia coli Cap-efbE-2：5'-aaaaaaaaaacgagtacattggcatcgtg

Vibrio parahemolyticus Cap-tlH-1：5'-aaaaaaaaaaaaagcggattatgcagaagcactg

Vibrio parahemolyticus Cap-tlH-2：5'-aaaaaaaaaagctactttctagcattttctctgc

Comma bacillus Cap-ompW-1：5'-aaaaaaaaaacaccaagaaggtgactttattgtg

Comma bacillus Cap-ompW-2：5'-aaaaaaaaaagaacttataacccgcg

Vibrio vulnificus Cap-vvhA-1：5'-aaaaaaaaaaccgcggtacaggttggcgca

Vibrio vulnificus Cap-vvhA-2：5'-aaaaaaaaaacgccacccactttcgggcc

D, sample labeling reagent：The Label IT Cy5 kits of Mirus companies.

E, Image Acquisition and data analysis：Perkin companies Operetta High Content Screening and Analysis systems and Columbus image data storage and analysis systems.

In order to keep detection Stability and veracity, devise 2 capture probe molecules for each microorganism, will be every 2 microorganisms (4 capture probes of meter) are detected as one group, and grouping situation is as follows.

First group：Salmonella Cap-invA-1, salmonella Cap-invA-2, Shigella Cap-ipaH-1, will are congratulated Salmonella Cap-ipaH-2；Second group：Staphylococcus aureus Cap-femA-1, staphylococcus aureus Cap-femA-2, small intestine Colitis Yale Salmonella Cap-16s-1, enterocolitis Yale Salmonella Cap-16s-2；Third group：Listeria monocytogenes Cap- PrfA-1, Listeria monocytogenes Cap-prfA-2, campylobacter jejuni Cap-VS1-1, campylobacter jejuni Cap-VS1-2；The Four groups：Enterorrhagia escherichia coli Cap-efbE-1, enterorrhagia escherichia coli Cap-efbE-2, vibrio parahemolyticus Cap-tlH-1, vibrio parahemolyticus Cap-tlH-2；5th group：Comma bacillus Cap-ompW-1, comma bacillus Cap-ompW-2, Vibrio vulnificus Cap-vvhA-1, Vibrio vulnificus Cap-vvhA-2.

According to the scheme that embodiment 1 describes, with the mixture for four kinds of mark molecules that total concentration is 0.2pmol/uL, fluorescence Encode four kinds of agarose microbeads.Wherein, the fluorescence of the agarose microbeads for containing salmonella Cap-invA-1 capture probes It is encoded to：Concentration=4 of concentration/mark molecule 2 of mark molecule 1:1, it is visited for containing salmonella Cap-invA-2 captures The fluorescence-encoded of the agarose microbeads of needle be：Concentration=2 of concentration/mark molecule 2 of mark molecule 1:1, for wrapping shiga The fluorescence-encoded of agarose microbeads of bacterium Cap-ipaH-1 capture probes be：The concentration of concentration/mark molecule 2 of mark molecule 1 =1:2, the fluorescence-encoded of agarose microbeads for containing Shigella Cap-ipaH-2 capture probes be：Mark molecule 1 Concentration=1 of concentration/mark molecule 2:4.

The fluorescence-encoded of agarose microbeads for containing staphylococcus aureus Cap-femA-1 capture probes be：Label Concentration=4 of concentration/mark molecule 2 of molecule 1:1, the fine jade for containing staphylococcus aureus Cap-femA-2 capture probes The fluorescence-encoded of lipolysaccharide microballoon be：Concentration=2 of concentration/mark molecule 2 of mark molecule 1:1, it is used for packet enterocolitis You are at the fluorescence-encoded of agarose microbeads of Salmonella Cap-16s-1 capture probes：Concentration/mark molecule 2 of mark molecule 1 it is dense Degree=1:2, the fluorescence-encoded of agarose microbeads for containing enterocolitis Yale Salmonella Cap-16s-2 capture probes be： Concentration=1 of concentration/mark molecule 2 of mark molecule 1:4.

The fluorescence-encoded of agarose microbeads for containing Listeria monocytogenes Cap-prfA-1 capture probes be：Label point Concentration=4 of concentration/mark molecule 2 of son 1:1, the agarose for containing Listeria monocytogenes Cap-prfA-2 capture probes The fluorescence-encoded of microballoon be：Concentration=2 of concentration/mark molecule 2 of mark molecule 1:1, it is used for packet campylobacter jejuni Cap- The fluorescence-encoded of the agarose microbeads of VS1-1 capture probes be：Concentration=1 of concentration/mark molecule 2 of mark molecule 1:2, it uses It is in the fluorescence-encoded of agarose microbeads for containing campylobacter jejuni Cap-VS1-2 capture probes：The concentration of mark molecule 1/ Concentration=1 of mark molecule 2:4.

The fluorescence-encoded of agarose microbeads for containing enterorrhagia escherichia coli Cap-efbE-1 capture probes be： Concentration=4 of concentration/mark molecule 2 of mark molecule 1:1, for containing the Cap-efbE-2 captures of enterorrhagia escherichia coli The fluorescence-encoded of the agarose microbeads of probe be：Concentration=2 of concentration/mark molecule 2 of mark molecule 1:1, it is secondary molten for wrapping The fluorescence-encoded of agarose microbeads of courageous and upright vibrios Cap-tlH-1 capture probes be：Concentration/mark molecule 2 of mark molecule 1 Concentration=1:2, the fluorescence-encoded of agarose microbeads for containing vibrio parahemolyticus Cap-tlH-2 capture probes be：Label Concentration=1 of concentration/mark molecule 2 of molecule 1:4.

The fluorescence-encoded of agarose microbeads for containing comma bacillus Cap-ompW-1 capture probes be：Mark molecule 1 Concentration/mark molecule 2 concentration=4:1, the agarose microbeads for containing comma bacillus Cap-ompW-2 capture probes It is fluorescence-encoded to be：Concentration=2 of concentration/mark molecule 2 of mark molecule 1:1, it is caught for containing Vibrio vulnificus Cap-vvhA-1 Obtaining the fluorescence-encoded of the agarose microbeads of probe is：Concentration=1 of concentration/mark molecule 2 of mark molecule 1:2, for containing The fluorescence-encoded of agarose microbeads of Vibrio vulnificus Cap-vvhA-2 capture probes be：Concentration/mark molecule 2 of mark molecule 1 Concentration=1:4.

According to the scheme that embodiment 1 describes, the capture probe grouping of internal reference nucleic acid, microorganism specificity is contained in agar Sugared microsphere surface, each agarose microbeads only contain a kind of capture probe.

According to national standards《Various pathogens rapid detection method PCR methods in SNT 1869-2007 food》Described in Technical solution, the microorganism in food is carried out increasing bacterium and extracts nucleic acid compositions therein, is studied for the present embodiment.

According to the scheme that embodiment 1 describes, the Label IT Cy5 kits provided using Mirus companies, to acquisition Microbial nucleic acids sample and internal reference nucleic acid (the internal reference nucleic acid sequence such as SEQ ID NO participated in：Shown in 6, reference DNA：5'-gactgactgactgactgactgact) sample carries out Cy5 fluorescent markers.

According to the scheme that embodiment one describes, in a 40 μ L hybridization systems, be added one group of four kinds of equivalent, contain not With the agarose microbeads of capture probe, the usage amount of each microballoon is 2 × 10³.Above-mentioned acquisition, phase are added into reaction system The sample of nucleic acid 400ng for the 2 kinds of microorganism fluorescent markers answered is incubated 40 minutes under the conditions of 25 DEG C, completes hybridization reaction.

According to the scheme that embodiment 1 describes, the Operetta High Content of Perkin Elmer companies are utilized Screening andAnalysis systems and Columbus image data storage and analysis systems, obtain and analyze hybridization after it is micro- The fluorescent image of balloon borne body.Using the standard calibration curve (as shown in Figure 10 and Figure 11) of target nucleic acid molecules and reference molecules, Calculate the content of target nucleic acid molecules.Homogenization processing is carried out to the testing result of two capture probes, is obtained in detection sample The nucleic acid content of food related microorganisms is as follows：

Salmonella nucleic acid content：586pM

Shigella nucleic acid content：264pM

Staphylococcus aureus nucleic acid content：79pM

Enterocolitis Yale Salmonella nucleic acid content：430pM

Listeria monocytogenes nucleic acid content：114pM

Campylobacter jejuni nucleic acid content nucleic acid content：34pM

Enterorrhagia escherichia coli nucleic acid content：210pM

Vibrio parahemolyticus nucleic acid content：46pM

Comma bacillus nucleic acid content：25pM

Wound vibrio content：137pM

Embodiment 5：The detection of clinical pathogenic microorganism

In order to realize that the present embodiment research has been carried out in the quantitative detection of pathogenic microorganism, the present invention.Experiment material and experiment Process is specific as follows.

B, microballoon mark molecule：

Mark molecule 1：5’-Alexa 488-aaaaaaaaaa-biotin

Mark molecule 2：5’-Texas Red-aaaaaaaaaa-biotin

C, pathogenic microorganism specificity capture probe, sequence such as SEQ ID NO：34~SEQ ID NO：Shown in 37：

Hepatitis B Cap-HBV-1：5'-aaaaaaaaaaccggaaagcttgactttttcacc

Hepatitis B Cap-HBV-2：5'-aaaaaaaaaaccggaaagcttgtcaaaaagtt

Hepatitis C virus Cap-HCV-1：5'-aaaaaaaaaaacccaacactactcggctag

Hepatitis C virus Cap-HCV-2：5'-aaaaaaaaaaatcactcccctgtgaggaac

D, sample labeling reagent：The Label IT Cy5 kits of Mirus companies.

In order to keep detection Stability and veracity, devise 2 capture probe molecules for each microorganism, according to The scheme that embodiment 1 describes, with the mixture for four kinds of mark molecules that total concentration is 0.2pmol/uL, fluorescence-encoded four kinds of agar Sugared microballoon.Wherein, the fluorescence-encoded of agarose microbeads for containing hepatitis B Cap-HBV-1 capture probes is：Label point Concentration=4 of concentration/mark molecule 2 of son 1:1, the agarose microbeads for containing hepatitis B Cap-HBV-2 capture probes Fluorescence-encoded be：Concentration=2 of concentration/mark molecule 2 of mark molecule 1:1, it is caught for containing hepatitis C virus Cap-HCV-1 Obtaining the fluorescence-encoded of the agarose microbeads of probe is：Concentration=1 of concentration/mark molecule 2 of mark molecule 1:2, for containing The fluorescence-encoded of agarose microbeads of hepatitis C virus Cap-HCV-2 capture probes be：Concentration/mark molecule 2 of mark molecule 1 Concentration=1:4.

According to the scheme that embodiment 1 describes, the capture probe of internal reference nucleic acid, pathogenic microorganism specificity is grouped contain in Agarose microbeads surface, each agarose microbeads only contain a kind of capture probe.

The tissue samples from hepatitis B patient, hepatitis C patients source are obtained, according to conventional technique scheme, Extract nucleic acid compositions therein.

According to the scheme that embodiment 1 describes, the Label IT Cy5 kits provided using Mirus companies, to acquisition Sample of nucleic acid and internal reference nucleic acid (the internal reference nucleic acid sequence such as SEQ ID NO participated in：Shown in 6, reference DNA：5'- Gactgactgactgactgactgact) sample carries out Cy5 fluorescent markers.

According to the scheme that embodiment 1 describes, in a 40 μ L hybridization systems, four kinds of equivalent of addition contain different captures The usage amount of the agarose microbeads of probe, each microballoon is 2 × 10³.The fluorescent marker of above-mentioned acquisition is added into reaction system Sample of nucleic acid 400ng, be incubated 40 minutes under the conditions of 25 DEG C, complete hybridization reaction.

According to the scheme that embodiment 1 describes, the Operetta High Content of Perkin Elmer companies are utilized Screening andAnalysis systems and Columbus image data storage and analysis systems, obtain and analyze hybridization after it is micro- The fluorescent image of balloon borne body.Using the standard calibration curve (as shown in figure 12) of target nucleic acid molecules and reference molecules, mesh is calculated Mark the content of nucleic acid molecules.Homogenization processing is carried out to the testing result of two capture probes, obtains the core that cause of disease closes microorganism Acid content is as follows：

Hepatitis A Virus nucleic acid content：125pM

Hepatitis C virus nucleic acid content：178pM

The foregoing is only a preferred embodiment of the present invention, the present invention application range include and be not limited to Lower aspect.

1, the detection of clinical pathogenic microorganism, including bacterium, virus, mycoplasma, Chlamydia, fungi, Richettsia, spiral Body；It further include the inspection of the detection of drug resistant gene, the detection of cell factor, the detection and human gene express spectra of tumor markers It surveys.

2, the detection of food related microorganisms, including：1) detection of total plate count；2) detection of coliform；3) it causes a disease The detection of bacterium, such as salmonella, Shigella, staphylococcus aureus, hemolytic streptococcus, escherichia coli, secondary haemolysis Property vibrios, bacillus cereus, comma bacillus, vibrio parahemolyticus, Listeria Monocytogenes, Vibrio vulnificus, meat The detection of malicious clostridium, lactic acid bacteria etc.；4) detection of mould and saccharomycete；5) viral detection, as hepatitis virus, swine fever virus, Newcastle disease virus, horse root of Dahurian angelica kirschner virus, foot and mouth disease virus, hydrophobin, the detection of swine vesicular disease virus.

3, the detection of agricultural product related microorganisms includes：1) detection of total plate count；2) detection of coliform；3) it causes a disease The detection of bacterium, such as salmonella, Shigella, staphylococcus aureus, hemolytic streptococcus, escherichia coli, secondary haemolysis Property vibrios, bacillus cereus, comma bacillus, vibrio parahemolyticus, Listeria Monocytogenes, Vibrio vulnificus, meat The detection of malicious clostridium, lactic acid bacteria etc.；4) detection of mould and saccharomycete；5) viral detection, as hepatitis virus, swine fever virus, Newcastle disease virus, horse root of Dahurian angelica kirschner virus, foot and mouth disease virus, hydrophobin, the detection of swine vesicular disease virus.

4, the detection of livestock and poultry sex pheromone specifically includes following microorganism type and type：

One kind is the detection of animal pathogenic microorganism, including foot and mouth disease virus, highly pathogenic avian influenza virus, swine pox Virus, African swine fever virus, African horse sickness virus, rinderpest virus, PPR virus, contagious bovine pleuropneumonia filiform branch Substance, bovine spongiform encephalopathy cause of disease, itch cause of disease.

Two classes are the detection of animal pathogenic microorganism, including swine fever virus, newcastle disease virus, hydrophobin, sheep Acne/goat capripoxvirus, blue tongue virus, rabbit hemorrhagic disease virus, Bacillus anthracis, Brucella.

Three classes are the detections of animal pathogenic microorganism, including：

1) many animals are total to illness pathogenic microorganism：Low pathogenicity influenza virus, pseudorabies virus, clostridium tetani, Clostridium chauvei, johne's bacillus, enteropathogenic E. Coli, salmonella, Pasteurella, is caused a disease at Mycobacterium tuberculosis Property streptococcus, Li bacillus, C.perfringens, Aeromonas hydrophila, clostridium botulinum, clostridium septicum and other cause Characteristic of disease clostridium, chlamydia psittaci, actinomyces, Leptospira.

2) detection of cattle disease pathogenic microorganism, the pathogenic microorganism include malignant catarrh virus, bovine leucosis disease Poison, bovine epizootic fever virus, infectious bovine rhinotrachetis virus, bovine viral diarrhea/bovine diarrhoea virus, ox genitals campylobacter, Schistosoma japonicum.

3) detection of sheep and goat disease pathogenic microorganism, the pathogenic microorganism include caprine arthritis/encephalomyelitis Virus, Mei Di/Wei Sina diseases virus, infectious labial dermatitis virus.

4) detection of swine disease pathogenic microorganism, the pathogenic microorganism include that japanese encephalitis virus, pig breeding and breathing are comprehensive Close syndrome virus, pig parvoviral, pig circular ring virus, Porcine epidemic diarrhea virus, transmissible gastro-enteritis virus, brickpox bar The close spiral of bacterium, B.bronchisepticai, actinobacillus pleuropneumoniae, haemophilus parasuis, mycoplasma hyopneumoniae, pig Body.

5) detection of horse disease pathogenic microorganism, the pathogenic microorganism include equine infectious anemia virus, the scorching disease of equine arteritis Poison, horse disease toxicity abortion virus, horse Coryzavirus, pseudomonas mallei, Pseudomonas Pseudomallei, imitation leather subcutaneous ulcer histoplasma capsulatum, Ulcerative lymphangitis Corynebacterium pseudotuberculosis.

6) detection of poultry diease pathogenic microorganism, the pathogenic microorganism includes duck plague virus, virulent duck enteritis virus, small Goose plague virus, chicken infectivity bursa of Fabricius virus, chicken Marek's disease virus, avian leukosis/sarcoma virus, fowl reticular endothelium group The sick virus of proliferation, Chicken Infectious Anemia Virus, avian infectious laryngotracheitis virus, avian infectious bronchitis virus, chicken is knitted to subtract Egg syndrome virus, fowlpox virus, avian viral arthritis virus, avian infectious encephalomyelitis virus, haemophilus paragallinarum, chicken Virus mycoplasma, Eimeria species.

7) detection of rabbit pathogenic microorganism, the pathogenic microorganism include infectious myxomatosis virus, hare hometown pull rod Bacterium, Rabbit Bordetella bronchiseptic stain, rabbit coccidia.

8) detection of aquatic animal disease pathogenic microorganism, the pathogenic microorganism include popular Hematopoietic Necrosis's disease Poison, infectious hematopoietic necrosis's poison, cherry salmon virus, viral haemorrhagic septicaemia virus, fancy carp blister sore Poison, channel catfish virus, viral encephalopathy and retina virus, infectious pancreas necrosis virus, red-sea bream iridovirus, paddlefish The polygonal baculovirus of irido virus, midgut gland necrosis baculovirus, infectious hypodermal and hematopoietic necrosis virus, core, shrimp Oviposition death syndrome virus, soft-shelled turtle parotitis disease virus, Taura syndrome virus, White Spot Syndrome Virus, yellow head disease disease Poison, grass carp hemorrhage virus, huichun viremia virus, Bao spherical virus, salmon infectious anemia virus.

9) detection of honeybee disease pathogenic microorganism, the pathogenic microorganism include American foul brood larva bacillus, Europe Continent foulbrood Bee venoms, chalk disease Ascosphaera apis, Nosema apis worm, instep gland mite, Ya Shi varoa mites.

10) detection of other zoosis pathogenic microorganisms, the pathogenic microorganism include canine distemper virus, canine parvovirus Poison, hepatitis infectiosa canis virus, canine coronavirus, canine parainfluenza virus, cat whiting Leukopenia syndrome virus, Aleutian disease disease Poison, mink enteritis virus.

The foregoing is only a preferred embodiment of the present invention, but scope of protection of the present invention is not limited thereto, Any one skilled in the art in the technical scope disclosed by the present invention, according to the technique and scheme of the present invention and its Inventive concept is subject to equivalent substitution or change, should be covered by the protection scope of the present invention.

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<400> 25

aaaaaaaaaa gaatgaaatt ttagaatggg g 31

<210> 26

<211> 28

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 26

aaaaaaaaaa attgcgctga agcctttg 28

<210> 27

<211> 29

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 27

aaaaaaaaaa cgagtacatt ggcatcgtg 29

<210> 28

<211> 34

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 28

aaaaaaaaaa aaagcggatt atgcagaagc actg 34

<210> 29

<211> 34

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 29

aaaaaaaaaa gctactttct agcattttct ctgc 34

<210> 30

<211> 34

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 30

aaaaaaaaaa caccaagaag gtgactttat tgtg 34

<210> 31

<211> 26

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 31

aaaaaaaaaa gaacttataa cccgcg 26

<210> 32

<211> 30

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 32

aaaaaaaaaa ccgcggtaca ggttggcgca 30

<210> 33

<211> 29

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 33

aaaaaaaaaa cgccacccac tttcgggcc 29

<210> 34

<211> 33

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 34

aaaaaaaaaa ccggaaagct tgactttttc acc 33

<210> 35

<211> 32

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 35

aaaaaaaaaa ccggaaagct tgtcaaaaag tt 32

<210> 36

<211> 30

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 36

aaaaaaaaaa acccaacact actcggctag 30

<210> 37

<211> 30

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 37

aaaaaaaaaa atcactcccc tgtgaggaac 30

Claims

1. the label and discrimination method of a kind of particulate carrier, which is characterized in that include the following steps：

2) obtain at least one detectable mark molecule, each mark molecule contain it is at least one can independent detection label base Group and it is at least one can the group that is coupled of coupling group with particulate carrier surface；

3) under certain coupling condition, make a certain amount of detectable mark molecule of at least one and a certain amount of particulate carrier It is contacted and is coupled, mark molecule is made specifically to mark the particulate carrier；

4) by detecting the mark molecule with particulate carrier coupling, differentiate the type of the labeled particulate carrier, the mark Sub detection mode of scoring is at least one of qualitative detection or quantitative detection.

2. the label and discrimination method of a kind of particulate carrier according to claim 1, which is characterized in that the particulate carrier Selected from Ago-Gel microballon, sephadex microballon, cellulose bead, polyacrylamide gel, polyacrylic acid ester liposome Composite particles, chitosan particle, gold-magnetic particles, bead.

3. the label and discrimination method of a kind of particulate carrier according to claim 1, which is characterized in that the particulate carrier A diameter of 10 nanometers~1000 microns.

4. the label and discrimination method of a kind of particulate carrier according to claim 1, which is characterized in that it is described can coupled base Group is selected from antigen molecule, antibody molecule, streptavidin, biotin.

5. a kind of particulate carrier according to claim 1 is in label and discrimination method, which is characterized in that described detectable Mark molecule is selected from oligonucleotide molecules, peptide molecule, polysaccharide molecule, tree form modification, artificial synthesized macromolecule.

6. a kind of detection method of nucleic acid molecules, which is characterized in that include the following steps：

2) obtain at least one detectable label molecule, each mark molecule contain it is at least one can independent detection label base Group and it is at least one can with particulate carrier surface can the group that is coupled of coupling group；

3) a kind of capture probe molecule is obtained, capture spy molecule needle contains one section of specific nucleic acid sequence and at least one can The group of coupling, the nucleic acid sequence can form a degree of complementary pairing with detected target nucleic acid molecules, described The group that can be coupled can with particulate carrier surface can coupling group occur coupled action；

4) make detectable label molecule, capture probe molecule and particulate carrier surface can coupling group be coupled, obtain a kind of energy Enough specific binding object to be measured nucleic acid molecules and with detectable label specific detection carrier；

6) under nucleic acid hybridization conditions, make the spy of the labeled object to be measured nucleic acid molecules that step 5) obtains and step 4) acquisition Opposite sex detection carrier contact, makes object to be measured nucleic acid molecules be hybridized with the capture probe for being coupled to detection carrier surface, obtains Obtain the combination of object to be measured nucleic acid molecules and specific detection carrier；

7) combination for obtaining the object to be measured nucleic acid molecules and specific detection carrier in step 6), detects the detection carrier On mark molecule type and intensity and the detectable label on object to be measured nucleic acid type and intensity, determine to be measured The presence of target nucleic acid molecules and content.

7. a kind of detection method of nucleic acid molecules according to claim 6, which is characterized in that the capture probe is selected from widow Poly- ribonucleic acid molecule, contains ribonucleic acid and DNA nucleic acid oligomer point at DNA oligo molecule simultaneously Son.

8. a kind of detection method of nucleic acid molecules according to claim 6, which is characterized in that specific detection in step 4) The synthetic method of carrier is：Under certain coupling condition, make a certain amount of at least one detectable label molecule and one first Quantitative particulate carrier is contacted and is combined, and mark molecule is made specifically to mark the particulate carrier；It then will be a certain amount of Capture probe molecule contacted and combined with a certain amount of labeled particulate carrier, acquisition can specifically bind target The specific detection carrier of nucleic acid molecules.

9. a kind of detection method of nucleic acid molecules according to claim 6, which is characterized in that specific detection in step 4) The synthetic method of carrier is：Under certain coupling condition, make a certain amount of at least one detectable label molecule, a kind of capture Probe molecule is contacted with a certain amount of particulate carrier, and mark molecule, capture probe molecule is made to be combined with particulate carrier surface, Obtain can specifically bind target nucleic acid molecules and with detectable label specific detection carrier.

10. the application of the detection method of the nucleic acid molecules as described in any one of claim 6-9, which is characterized in that described The detection method of nucleic acid molecules is applied to gene expression detection.

11. a kind of detection reagent of nucleic acid molecules, which is characterized in that including particulate carrier, detectable label molecule, capture probe Molecule and corresponding coupling buffer, hybridization buffer and label detection reagent, the particulate carrier surface have at least one Kind can coupling group, the detectable label molecule contain it is at least one can independent detection labelling groups and at least one Can with particulate carrier can coupling group coupling group or molecule, the capture probe molecule can with particulate carrier be coupled and energy With object to be measured making nucleic acid molecular hybridization.

12. a kind of detection method of microorganism, which is characterized in that include the following steps：

2) obtain at least one detectable mark molecule, each mark molecule contain it is at least one can independent detection label base Group and at least one group that can be coupled with the group of particulate carrier being coupled；

3) a kind of capture probe molecule is obtained, capture spy molecule needle contains one section of specific nucleic acid sequence and at least one can The group of coupling, the target nucleic acid molecules that the nucleic acid sequence can contain with detected Institute of Micro-biology form a degree of complementation Pairing, the group being coupled can with particulate carrier surface can coupling group occur coupled action；

4) detectable label molecule, capture probe molecule is made to be combined with particulate carrier surface, acquisition can specifically bind tested The target nucleic acid molecules that Institute of Micro-biology contains and the specific detection carrier with detectable label；

6) under nucleic acid hybridization conditions, make the specific detection of the labeled nucleic acid molecules that step 5) obtains and step 4) acquisition Carrier contacts, and target nucleic acid molecules is made to be hybridized with the capture probe for detecting carrier surface, obtains target nucleic acid molecules and spy The combination of opposite sex detection carrier；

7) combination for obtaining the object to be measured nucleic acid molecules and specific detection carrier in step 6) detects the label point of carrier The type and intensity of the type and the detectable label in intensity and target nucleic acid molecules of son determine institute in tested microorganism Presence containing nucleic acid molecules and its content, so that it is determined that being detected presence and its quantity of microorganism.

13. a kind of detection method of microorganism according to claim 12, which is characterized in that specific detection in step 4) The synthetic method of carrier is：Under certain coupling condition, make first a certain amount of detectable mark molecule of at least one with A certain amount of particulate carrier is contacted and is combined, and mark molecule is made specifically to mark the particulate carrier；It then will be certain The capture probe molecule of amount is contacted and is combined with a certain amount of labeled particulate carrier, and acquisition can specifically bind quilt The specific detection carrier of target nucleic acid molecules contained by micrometer biology.

14. a kind of detection method of microorganism according to claim 12, which is characterized in that specific detection in step 4) The synthetic method of carrier is：Under certain coupling condition, the detectable mark molecule of a certain amount of at least one, one kind is made to catch It obtains probe molecule to be contacted with a certain amount of particulate carrier, mark molecule, capture probe molecule and particulate carrier surface is made to tie It closes, obtains the specific detection that can specifically bind tested Institute of Micro-biology containing target nucleic acid molecules and with detectable label Carrier.

15. the application of the detection method of the microorganism as described in any one of claim 12-14, which is characterized in that described Microorganism detection method is applied to the inspection of clinical pathogenic microorganism, food microorganisms, agricultural product microorganism, livestock and poultry sex pheromone It surveys.