CN103255231A - Nucleic acid parallel testing liquid-phase chip as well as preparation method and application thereof - Google Patents
Nucleic acid parallel testing liquid-phase chip as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of molecular biology and clinical test, and particularly discloses a nucleic acid parallel testing liquid-phase chip for respiratory tract infection pathogen diagnosis as well as a preparation method and application thereof. The nucleic acid parallel testing liquid-phase chip mainly comprises microspheres, probes, specific primers, biotinylated universal primers and streptavidin-phycoerythrin corresponding to respiratory tract infection relevant pathogens, and is characterized in that the microspheres are respectively coupled with the specific primers for 11 respiratory tract infection relevant pathogens to form microspheres No.11, No.15, No.22, No.28, No.36, No.42, No.49, No.55, No.56, No.71 and No.75; and the probes are aminated specific probes for 11 pathogen target genes. According to the nucleic acid parallel testing liquid-phase chip, the preparation method and the application, various respiratory tract infection relevant pathogens are parallelly detected, and 11 pathogens can be detected at the same time, so that great convenience is provided for laboratories and clinical tests of different diseases.
Description
(1) technical field
The invention belongs to Protocols in Molecular Biology and clinical detection technique field, particularly a kind of nucleic acid liquid phase chip for parallel detection for the respiratory tract infection pathogen diagnosis and preparation method thereof and application.
(2) background technology
Respiratory tract infection is common a kind of transmissible disease.Common disease is because of pathogenic agent, and minority is caused by bacterium.The patient is of all ages, sex, occupation and regional, has stronger infectivity, and if untimely treatment, inflammation can involve other organs corresponding symptom takes place, and constitutional symptom also can increase the weight of.Common complication has sinusitis paranasal sinusitis, otitis media, eye binding film inflammation etc.; Young and weak infant, upper respiratory tract infection be development downwards easily, causes bronchitis and pneumonia; Minority also also can cause complication such as septicemia, meningitis and the ephritis at whole body and other positions to debility child when infectation of bacteria is arranged.
In the therapeutic process of respiratory tract infection, a lot of clinicians always select microbiotic for use " empirical ", ignore Study on etiologic agents.But microbiotic is to antibacterial, and pathogenic infection is a bit acted on not to be had yet.All being caused by pathogenic agent more than 90% and have in whole respiratory tract infection, is not bacterium.Therefore if each respiratory tract infection patient uses microbiotic, then having all is unnecessary more than 90%.So not only cause the waste of drug resource, also cause antibiotic abuse, thereby cause bacterial drug resistance; Different infectation of bacteria in addition, selected microbiotic kind, dose, fate are also different, and therefore the pathogenic agent of clear and definite respiratory tract infection is a basic norm that should become clinical application.
At present, respiratory tract infection laboratory diagnosis common method is the pathogen culture method, ELISA (enzyme-linked immunosorbent assay), molecular biology reverse transcription polymerase chain reaction (RT-PCR) or real-time RT-PCR(rRT-PCR) etc., these methods once can only detect a kind of pathogenic agent, in actual application, detect not only loaded down with trivial details but also need a large amount of relatively reagent and clinical samples, the testing cost height, if therefore can use new technology that these pathogenic agent are detected simultaneously, will provide great convenience to the diagnosis and treatment of this class disease.The high-throughout characteristics of at present increasingly mature biochip technology can realize the disposable joint-detection of multiple pathogenic agent.Liquid-phase chip technology is a kind of of biochip, it is except having the high-throughout characteristics of common biochip (being solid phase chip), also have advantages such as high stability that solid phase chip do not have, hypersensitivity, so the development of respiratory tract infection related diseases substance liquid phase chip for parallel detection and the exploitation needs that catered to present laboratory and clinical diagnosis.
(3) summary of the invention
The present invention provides a kind of detect rapidly accurate, time saving and energy saving nucleic acid liquid phase chip for parallel detection and preparation method thereof and application in order to remedy the deficiencies in the prior art.
The present invention is achieved through the following technical solutions:
A kind of nucleic acid liquid phase chip for parallel detection, main microballoon by corresponding respiratory tract infection related diseases substance, probe, Auele Specific Primer, biotinylated universal primer and Streptavidin-phycoerythrin is formed, it is characterized in that: described respiratory tract infection related diseases substance comprises respiratory syncytial virus A, respiratory syncytial virus B, influenza virus A, influenza virus B, parainfluenza virus 1, parainfluenza virus 2, parainfluenza virus 3, mumps virus, the human corona virus, hsv-1 and hsv-2 be pathogenic agent in totally 11, described microballoon be respectively with form at the specific probe coupling of above-mentioned 11 kinds of respiratory tract infection related diseases substances 11,15,22,28,36,42,49,55,56,71 and No. 75 microballoons, described probe are the amidized specific probes at above-mentioned 11 kinds of pathogenic agent target genes; Described Auele Specific Primer can carry out specific amplification to above-mentioned 11 kinds of pathogenic agent; Described universal primer is the nucleotide sequence irrelevant with above-mentioned 11 kinds of pathogenic agent target genes, and wherein oppositely universal primer 5 ' end has biotin labeling.
The present invention is according to causing the diversified characteristics of respiratory tract infection pathogenic agent, methods such as ELISA, the RT-PCR that adopts at present detection and real-time RT-PCR once can only detect a kind of inconvenience of pathogenic agent technology, the needs of loaded down with trivial details time-consuming and laboratory and clinical diagnosis, selection is at the specific probe of respiratory tract infection related diseases substance target gene, respectively with not homochromy number microballoon coupling, mix according to detecting needs, tentatively prepare the liquid-phase chip that can carry out disposable parallel detection to above-mentioned respiratory tract infection related diseases substance.
The pathogenic agent index that the present invention detects is as follows:
The present invention utilizes bioinformation to gain knowledge and relevant information biology software, all above-mentioned respiratory tract infection pathogen nucleic acid sequences that can retrieve among the Genbank are carried out homology analysis, find out conservative region, design primer, probe at corresponding gene fragment.
Described each bar probe 5 ' end has an amino, and its sequence is:
RSVA：5’——CCC?TCC?ACT?CAA?CCT?CCT?CC；
RSVB：5’——CAA?CAA?GCC?AGA?TCA?AGA?AC；
INFA：5’——GCA?GTT?AAA?CTG?TAT?AGA?AA；
INFB：5’——TCT?ACT?TCA?GCC?CTA?TAA?AA；
PIV1：5’——TAG?ATC?TTC?AGG?TTG?GCA?CT；
PIV2：5’——TGG?TGG?TCT?CAT?AAG?TGG?GA；
PIV3：5’——TTC?TCG?GGT?ATG?GAG?GTC?TT；
Mumps?Virus：5’——CCA?ATG?AAA?TTG?CTG?CCT?AT；
HCOV：5’——ATA?TGA?AAG?GCA?AGA?TGT?TT；
HSV-1：5’——TCC?AGG?ACG?CCG?CGA?CGC?CT；
HSV-2：5’——GAA?CGC?AGC?CCC?GCT?GGA?GC。
Described Auele Specific Primer 5 ' end has the universal primer label, and oppositely universal primer 5 ' end has a vitamin H sign, and its sequence is:
Specific primer sequence:
RSVA：5’——GAA?CTA?CAC?AGC?TCG?CCT?CC，
5’——GTG?AAG?GGC?TTG?GAT?TGC?CT；
RSVB：5’——TAG?CCT?CGG?CAA?ACC?ACA?AA，
5’——TTC?CAA?GCT?GGG?GAT?TCT?GG；
INFA：5’——ACG?CTT?TGT?CCA?AAA?TGC?CC，
5’——TAT?GAG?GCC?CAT?GCA?ACT?GG；
INFB：5’——AGC?CGG?AAA?ATG?TCG?ATC?ACC，
5’——GGT?TCC?GTT?CAC?CAA?CAC?TC；
PIV1：5’——CTT?AGG?TGC?CGA?AGG?TAG?GC，
5’——TCC?TGG?TCG?AGA?CAG?GAC?TT；
PIV2：5’——GCA?GTT?GGA?AGC?GGG?ATC?TA，
5’——CAG?CCT?GTT?CGC?TGG?AGT?TA；
PIV3：5’——GCG?GCA?TTA?TAC?CCA?TCT?GTT?G，
5’——ACC?CAG?TTG?TGT?TGC?AGA?TTG；
Mumps?Virus：5’——ACG?CTG?TGA?GAT?TGA?CGG?TT，
5’——GTT?GGA?GGG?AGG?TCA?TCT?GC；
HCoV：5’——ACT?GGC?AGT?TGG?TGG?AGT?TTT，
5’——GTG?CCA?AGT?TTG?ACA?CCC?TG；
HSV-1：5’——CGC?AAA?TCC?CAC?CAA?ACT?GG，
5’——TTT?GGG?GCT?TTT?TGA?GTG?CG；
HSV-2：5’——CGA?GTG?CCC?CTA?CAA?CAA?GT，
5’——GTG?TGA?TCT?CCG?TCC?AGT?CG；
The universal primer sequence:
5’——CAG?GCC?ACG?TTT?TGT?CAT?GC，
5’——TTC?TTT?GCG?TTA?TGT?CTC?TG。
The preparation method of nucleic acid liquid phase chip for parallel detection of the present invention mainly comprises the steps:
(1) microballoon and amidized probe coupling form couplet, choose the carboxyl microballoon of 11 kinds of different fluorescence color respectively, washing, activated carboxyl microballoon; Add specificity amination probe at respiratory syncytial virus A, respiratory syncytial virus B, influenza virus A, influenza virus B, parainfluenza virus 1, parainfluenza virus 2, parainfluenza virus 3, mumps virus, human corona virus, hsv-1, hsv-2 respectively accordingly with the order of 11,15,22,28,36,42,49,55,56,71, No. 75 microballoons; Add the EDC mixing, under the room temperature, be placed on the rotating speed of 200～250 rpm and hatch 120 minutes on the shaking table; Repeat 1～2 time; With PBS-TBN washing 2～3 times; Obtain the couplet that 11 kinds of different specific probes and microballoon form respectively; The number of every kind of microballoon couplet of tally's volume is respectively at keeping in Dark Place under 4 ℃ of conditions; During use, select to mix according to test item;
(2) couplet, Auele Specific Primer, the biotin labeled universal primer of activation, the Streptavidin-phycoerythrin with specific probe and microballoon coupling disposes in proportion, obtains respiratory tract infection related diseases substance liquid phase chip for parallel detection.
The application of nucleic acid liquid phase chip for parallel detection of the present invention in preparation respiratory tract infection clinical diagnosis and detection reagent, main application method is:
The microballoon couplet of probe will be marked with, Auele Specific Primer, biotin labeled universal primer as the activation of reporter molecules, Streptavidin-phycoerythrin disposes in proportion, probe and the combination of purpose nucleic acid specificity accordingly, the reporter molecules that has green report fluorescence also with the specific combination of Streptavidin-phycoerythrin, with sample on the mixture to Luminex 200, adopt liquid technology that the microballoon couplet is divided into individual cells stream, utilize red, green two bundle laser detection obtain optical signal, processing data just can finish to biological respinse in real time, quantitative analysis.
The present invention is when using, add pathogen nucleic acid RT-PCR product to be checked, purpose PCR product is caught by the specific probe on the microballoon, add Streptavidin-phycoerythrin (SA-PE) again, vitamin H on the pathogen nucleic acid RT-PCR product and Streptavidin-phycoerythrin (SA-PE) combination, by the liquid-phase chip detection system, realize the disposable qualitative and quantitative detection to above-mentioned respiratory tract infection related diseases substance at last.
Utilize respiratory tract infection pathogenic agent liquid phase chip for parallel detection of the present invention, can realize the various respiratory road is infected the parallel detection of related diseases substance, can detect 11 kinds of pathogenic agent simultaneously, also can optionally detect wherein several pathogenic agent according to laboratory and clinical needs, laboratory and the clinical detection of convenient different disease need.
Method of the present invention is owing to carry out the detection of various respiratory road pathogen infection in a reaction system, different probes can carry out combination with different molecules of interest, the reaction back can be distinguished different detection reaction by laser for detecting color numbers of sphere matrix, both had the qualitative or quantitative characteristics that RT-PCR or real-time RT-PCR detect, has high-throughout advantage again, thereby can be more time saving and energy saving, saved the detection cost simultaneously.
(4) embodiment
Embodiment 1: the coupling of the microballoon of probe and known numbering
(1) choose 11 kinds of different fluorescence color (11,15,22,28,36,42,49,55,56,71, No. 75 microballoons) carboxylated microballoons (Luminex company) respectively, with whirlpool oscillator vibration microballoon suspension, time 20s mixes microballoon.
(2) get above-mentioned each number carboxyl microballoon about 2 * 10 respectively
3Individual, transfer in the centrifuge tube the centrifugal 2min of 〉=8000 * g, precipitation carboxyl microballoon respectively.
(3) remove supernatant, add 100 μ ldH
2O, with the resuspended microballoon of whirlpool oscillator vibration 20s, the centrifugal 2min of 〉=8000 * g, precipitation carboxyl microballoon.
(4) remove supernatant, add the biphosphate sodium salt solution of 80 μ l, 100 mM, pH=6.2, with the vibrate carboxyl microballoon of the resuspended washing of 20 s of whirlpool oscillator.
(5) the Sulfo-NHS(dH of adding 10 μ l, 50mg/ml
2The O dilution), with the vibration gently of whirlpool oscillator.
(6) the EDC(dH of adding 10 μ l, 50mg/ml
2The O dilution), with the vibration gently of whirlpool oscillator.Incubated at room 20min gently shakes once with the whirlpool oscillator every 10min.The centrifugal 2min of 〉=8000 * g, the carboxyl microballoon of precipitation activation.
(7) remove supernatant, add the MES of 250 μ l, 50mM, pH=5.0, whirlpool oscillator vibration 20s, the carboxyl microballoon of resuspended activation.The centrifugal 2min of 〉=8000 * g, the carboxyl microballoon after the washing of precipitate.
(8) repeating step is 7 twice, with the MES washing of 50mM, pH=5.0 2 times.
(9) MES of adding 100 μ l, 50mM, pH=5.0 is with whirlpool oscillator vibration 20s.In the microballoon of mixing, add the 1nmol specific probe respectively, be settled to 500 μ l with the MES of 50mM, pH=5.0, with whirlpool oscillator mixing.
(10) at room temperature be placed on the shaking table (200rpm) and hatch 2h.The centrifugal 2min of 〉=8000 * g, the good microballoon of precipitation coupling.
(11) remove supernatant, add 300 μ lPBS-TBN, whirlpool oscillator vibration 30s.At room temperature be placed on the shaking table (200rpm) and hatch 30min.The centrifugal 2min of 〉=8000 * g, the good microballoon of precipitation coupling.
(12) remove supernatant, add 1ml PBS-TBN, whirlpool oscillator vibration 30s.The centrifugal 2min of 〉=8000 * g, the good microballoon of precipitation coupling.
(13) repeating step 12 once washs 2 times with PBS-TBN.
(14) add 500 μ lPBS-TBN, the microballoon that resuspended coupling is good and washing is good.
(15) with the quantity of cell counter counting microballoon, converse the concentration of every kind of microballoon.
(16) the good microballoon of coupling is placed on 4 ℃ and keeps in Dark Place, the microballoon of general every kind of probe coupling is preserved separately, during use, selects to mix according to test item.
Embodiment 2: the application of respiratory tract infection pathogenic agent liquid phase chip for parallel detection of the present invention in clinical detection
Utilize respiratory tract infection pathogenic agent liquid phase chip for parallel detection of the present invention to detect the techniqueflow of respiratory tract infection pathogenic agent:
1. the extraction of pathogen nucleic acid: can use business-like test kit to extract RNA, from clinical samples or pathogen isolation culture, extract pathogenic agent RNA as using QIGEN Viral RNA mini Extraction Kit (CAT:52904); Extract 40 parts of sample nucleic acid altogether, wherein 30 parts is the known positive sample of hypotype, 5 parts of negative contrasts, and 5 parts is uncorrelated pathogenic agent sample.According to requirement of experiment, other establishes a blank.Sample information such as following table:
(1) adds the AVL damping fluid of 560 μ l in the 1.5 ml centrifuge tubes;
(2) in this centrifuge tube, add 140 μ l clinical samples or pathogen isolation culture again;
(3) place 10 min under the room temperature, instantaneous centrifugal then;
(4) add 560 μ l dehydrated alcohols, abundant mixing is 15s at least, and is instantaneous centrifugal;
(5) mixing solutions that carefully adds about 630 μ l is to the QIAamp post, and on collection tube, centrifugal 5 s of 8000rpm make liquid pass through filter membrane then with the QIAamp column sleeve;
(6) discard liquid in the collection tube, repeat the 6th step;
(7) discard the AW1 damping fluid of the careful 750 μ l of adding of liquid in the collection tube, the centrifugal 5s of 8000rpm makes liquid pass through filter membrane;
(8) discard the AW2 damping fluid of the careful 750 μ l of adding of liquid in the collection tube, centrifugal 5 s of 8000rpm make liquid pass through filter membrane;
(9) discard liquid in the collection tube, 13000rpm recentrifuge 1min;
(10) the QIAamp post is put into the centrifuge tube of a clean 1.5ml;
(11) add the EB damping fluid of 30 μ l on the film, leave standstill 2 ~ 5 min then under the room temperature;
(12) in whizzer with the centrifugal 1min of the speed of 8000rpm, the centrifugal liquid that gets off is required nucleic acid solution.
2. RT-PCR amplification
The reagent that first round pcr amplification adopts is Qiagen single stage method RT-PCR Kit, Cat No. 210212, and reaction system disposes as following table:
Response procedures is as follows:
Second reagent of taking turns the pcr amplification employing is Qiagen multiplex PCR Kit, Cat No. 206143, and reaction system disposes as following table:
Response procedures is as follows:
Gained RT-PCR product places 4 ℃ of preservations, and is to be checked.
3. liquid-phase chip detects
(1) take out respectively above-mentioned preparation at each 500 of the coupling microballoons of every kind of pathogenic agent, mix by equal proportion, be divided in 96 orifice plates, contain each 500 of the coupling microballoons of various specific probes in each hole, add RT-PCR product 2 μ l to be checked;
(2) 52 ℃ of hybridization 30min;
(3) add Streptavidin-phycoerythrin (SA-PE) 100 μ l, mixing is hatched 30min for 52 ℃;
(4) add the resuspended microballoon of 100 μ lPBS-TBN, be used for liquid-phase chip and detect;
(5) during liquid-phase chip detected, red fluorescence defined the kind of microballoon, determined the pathogen type of testing sample, and is namely qualitative; Green fluorescence is measured the average fluorescent strength of the SA-PE of microballoon combination, according to fluorescent value, determines pathogenic agent content, and is namely quantitative.
Detected result is as shown in the table:
4. interpretation of result
(1) this reagent can carry out accurate somatotype to 30 parts of pathogen nucleic acid samples, and specificity is up to 100%.
(2) good reproducibility, 90% detection batch within variance coefficient (CV)＜10%.
(3) detect 5 parts of known negative sample, all there is not false positive in result's demonstration.
(4) detect 5 parts of other pathogenic agent samples, the result shows all negative, does not have the situation of false retrieval.
Multiple respiratory pathogen liquid-phase chip molecule differential diagnosis kit can be carried out somatotype detection accurately to 26 kinds of pathogenic agent hypotypes that cause respiratory tract infection, and specificity and repeatability are all good.Further after the clinical verification, can be used as the examination of respiratory tract infection pathogenic agent.
Claims (6)
1. nucleic acid liquid phase chip for parallel detection, main microballoon by corresponding respiratory tract infection related diseases substance, probe, Auele Specific Primer, biotinylated universal primer and Streptavidin-phycoerythrin is formed, it is characterized in that: described respiratory tract infection related diseases substance comprises respiratory syncytial virus A, respiratory syncytial virus B, influenza virus A, influenza virus B, parainfluenza virus 1, parainfluenza virus 2, parainfluenza virus 3, mumps virus, the human corona virus, hsv-1 and hsv-2 be pathogenic agent in totally 11, described microballoon be respectively with form at the specific probe coupling of above-mentioned 11 kinds of respiratory tract infection related diseases substances 11,15,22,28,36,42,49,55,56,71 and No. 75 microballoons, described probe are the amidized specific probes at above-mentioned 11 kinds of pathogenic agent target genes; Described Auele Specific Primer can carry out specific amplification to above-mentioned 11 kinds of pathogenic agent; Described universal primer is the nucleotide sequence irrelevant with above-mentioned 11 kinds of pathogenic agent target genes, and wherein oppositely universal primer 5 ' end has biotin labeling.
2. nucleic acid liquid phase chip for parallel detection according to claim 1 is characterized by, and described each bar probe 5 ' end has an amino, and its sequence is:
RSVA：5’——CCC?TCC?ACT?CAA?CCT?CCT?CC；
RSVB：5’——CAA?CAA?GCC?AGA?TCA?AGA?AC；
INFA：5’——GCA?GTT?AAA?CTG?TAT?AGA?AA；
INFB：5’——TCT?ACT?TCA?GCC?CTA?TAA?AA；
PIV1：5’——TAG?ATC?TTC?AGG?TTG?GCA?CT；
PIV2：5’——TGG?TGG?TCT?CAT?AAG?TGG?GA；
PIV3：5’——TTC?TCG?GGT?ATG?GAG?GTC?TT；
Mumps?Virus：5’——CCA?ATG?AAA?TTG?CTG?CCT?AT；
HCOV：5’——ATA?TGA?AAG?GCA?AGA?TGT?TT；
HSV-1：5’——TCC?AGG?ACG?CCG?CGA?CGC?CT；
HSV-2：5’——GAA?CGC?AGC?CCC?GCT?GGA?GC。
3. nucleic acid liquid phase chip for parallel detection according to claim 1 is characterized by, and described Auele Specific Primer 5 ' end has the universal primer label, and oppositely universal primer 5 ' end has a vitamin H sign, and its sequence is,
Specific primer sequence:
RSVA：5’——GAA?CTA?CAC?AGC?TCG?CCT?CC，
5’——GTG?AAG?GGC?TTG?GAT?TGC?CT；
RSVB：5’——TAG?CCT?CGG?CAA?ACC?ACA?AA，
5’——TTC?CAA?GCT?GGG?GAT?TCT?GG；
INFA：5’——ACG?CTT?TGT?CCA?AAA?TGC?CC，
5’——TAT?GAG?GCC?CAT?GCA?ACT?GG；
INFB：5’——AGC?CGG?AAA?ATG?TCG?ATC?ACC，
5’——GGT?TCC?GTT?CAC?CAA?CAC?TC；
PIV1：5’——CTT?AGG?TGC?CGA?AGG?TAG?GC，
5’——TCC?TGG?TCG?AGA?CAG?GAC?TT；
PIV2：5’——GCA?GTT?GGA?AGC?GGG?ATC?TA，
5’——CAG?CCT?GTT?CGC?TGG?AGT?TA；
PIV3：5’——GCG?GCA?TTA?TAC?CCA?TCT?GTT?G，
5’——ACC?CAG?TTG?TGT?TGC?AGA?TTG；
Mumps?Virus：5’——ACG?CTG?TGA?GAT?TGA?CGG?TT，
5’——GTT?GGA?GGG?AGG?TCA?TCT?GC；
HCoV：5’——ACT?GGC?AGT?TGG?TGG?AGT?TTT，
5’——GTG?CCA?AGT?TTG?ACA?CCC?TG；
HSV-1：5’——CGC?AAA?TCC?CAC?CAA?ACT?GG，
5’——TTT?GGG?GCT?TTT?TGA?GTG?CG；
HSV-2：5’——CGA?GTG?CCC?CTA?CAA?CAA?GT，
5’——GTG?TGA?TCT?CCG?TCC?AGT?CG；
The universal primer sequence:
5’——CAG?GCC?ACG?TTT?TGT?CAT?GC，
5’——TTC?TTT?GCG?TTA?TGT?CTC?TG。
4. the preparation method of nucleic acid liquid phase chip for parallel detection according to claim 1 is characterized by, and mainly comprises the steps:
(1) microballoon and amidized probe coupling form couplet, choose the carboxyl microballoon of 11 kinds of different fluorescence color respectively, washing, activated carboxyl microballoon; Add specificity amination probe at respiratory syncytial virus A, respiratory syncytial virus B, influenza virus A, influenza virus B, parainfluenza virus 1, parainfluenza virus 2, parainfluenza virus 3, mumps virus, human corona virus, hsv-1, hsv-2 respectively accordingly with the order of 11,15,22,28,36,42,49,55,56,71, No. 75 microballoons; Add the EDC mixing, under the room temperature, be placed on the rotating speed of 200～250 rpm and hatch 120 minutes on the shaking table; Repeat 1～2 time; With PBS-TBN washing 2～3 times; Obtain the couplet that 11 kinds of different specific probes and microballoon form respectively; The number of every kind of microballoon couplet of tally's volume is respectively at keeping in Dark Place under 4 ℃ of conditions; During use, select to mix according to test item;
(2) couplet, Auele Specific Primer, the biotin labeled universal primer of activation, the Streptavidin-phycoerythrin with specific probe and microballoon coupling disposes in proportion, obtains respiratory tract infection related diseases substance liquid phase chip for parallel detection.
5. the application of nucleic acid liquid phase chip for parallel detection according to claim 1 in preparation respiratory tract infection clinical diagnosis and detection reagent.
6. the application of nucleic acid liquid phase chip for parallel detection according to claim 5, it is characterized in that: the microballoon couplet that will be marked with probe, Auele Specific Primer, biotin labeled universal primer as the activation of reporter molecules, Streptavidin-phycoerythrin disposes in proportion, probe and the combination of purpose nucleic acid specificity accordingly, the reporter molecules that has green report fluorescence also with the specific combination of Streptavidin-phycoerythrin, with sample on the mixture to Luminex 200, adopt liquid technology that the microballoon couplet is divided into individual cells stream, utilize red, green two bundle laser detection obtain optical signal, processing data just can finish to biological respinse in real time, quantitative analysis.
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| 石莹等: "液相芯片技术在传染病快速诊断中的应用", 《中国国境卫生检疫杂志》, vol. 34, no. 5, 31 October 2011 (2011-10-31) * |
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