CN101629214A - Hand-foot-and-mouth disease (HFMD) related virus parallel detection liquid-phase chip and preparation method and application thereof - Google Patents
Hand-foot-and-mouth disease (HFMD) related virus parallel detection liquid-phase chip and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a hand-foot-and-mouth disease (HFMD) related virus parallel detection liquid-phase chip and a preparation method and an application thereof. The HFMD related virus parallel detection liquid-phase chip in the invention mainly comprises microballoons, specific probes, biotinylated universal primer and streptavidin-phycoerythrin, which is characterized in that microballoons in the chip are Number 26, Number 36 and Number 46 microballoons; the specific probes are aminated specific probes aiming at virus VP1 area of EV71, CoxA16 and Echo9; the universal primer is enterovirus universal primer marked by activated biotin. In the invention, different probes can combine with different target molecules, different detection reactions can be classified by using laser for detecting color numbers of sphere matrix after reaction, thus having the quantitative or qualitative characteristics and high throughput advantage, thereby saving time and power and detection cost greatly.
Description
(1) technical field
The present invention relates to a kind of liquid-phase chip and preparation method thereof and application, relate in particular to a kind of hand foot mouth disease correlated virus liquid phase chip for parallel detection and preparation method thereof and application, belong to Protocols in Molecular Biology and clinical detection technique field.
(2) background technology
(hand-foot-mouth disease HFMD) has another name called the dermexanthesis stomatitis to hand foot mouth disease, is caused by enterovirus infection, and clinical manifestation fash occurs with positions such as heating and hand, foot, oral cavities or bleb is a kind of acute common transmittable disease of principal character.Children's pilosity, infant below 3 years old and 3 years old is easier to fall ill.The HFMD infectivity is strong, and the route of transmission complexity is propagated soon, can cause at short notice and be very popular.
Cause that the pathogenic agent of HFMD is mainly 2,4,5,7,9,10,16 types of Coxsackie virus (Coxsackie virus) the A group of pico+ribonucleic acid+virus section (Picornaviridae) enteron aisle genus, 1,2,3,4,5 types of Coxsackie B virus group; Part Echo virus (ECHO-viruses) and enterovirns type 71.The most common is CA group 16 types (CA16) and enterovirns type 71 (EV71).The HFMD state of an illness that different type enterovirus infections cause also has nothing in common with each other.Compare with enteroviruses such as ECHO with CA16, the large percentage of the HFMD severe infection that the EV71 type causes can cause multiple diseases such as BBE, aseptic meningitis, poliomyelitis sample paralysis, herpangina, neurogenic pulmonary edema, pulmonary apoplexy, viral myocarditis.Severe infant case fatality rate can reach 10% to 25%.
HFMD laboratory diagnosis common method is in the serology and experiment at present, the etiology cell inoculation, 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 virus, not only loaded down with trivial details but also need a large amount of relatively reagent and clinical samples for the detection of virus spectrum in actual application, the testing cost height, if therefore can use new technology that these viruses 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 virus.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 needs of present laboratory and clinical diagnosis have been catered in the development of hand foot mouth disease correlated virus liquid phase chip for parallel detection and exploitation.
(3) summary of the invention
According to causing the diversified characteristics of hand foot mouth disease correlated virus, detect the ELISA that adopts at present hand foot mouth disease, methods such as RT-PCR and real-time RT-PCR once can only detect a kind of inconvenience of virus technology, the needs of loaded down with trivial details time-consuming and laboratory and clinical diagnosis, the problem to be solved in the present invention provides a kind of hand foot mouth disease correlated virus liquid phase chip for parallel detection and preparation method thereof and application, not only can detect the multiple virus that causes hand foot mouth disease simultaneously, and detect rapidly, accurately, time saving and energy saving, help realizing finding the morning of hand foot mouth disease, early diagnose and early treatment.
The present invention is achieved through the following technical solutions:
Hand foot mouth disease correlated virus liquid phase chip for parallel detection of the present invention, mainly by microballoon, specific probe, biotinylated universal primer, Streptavidin-phycoerythrin is formed, and it is characterized in that described microballoon is 26,36 and No. 46 microballoons; Described specific probe is the amidized specific probe at EV71, CoxA16 and Echo9 virus VP 1 zone, and universal primer is activated biotin labeled enterovirus universal primer.
Wherein: described EV71 specific probe and No. 26 microballoons form couplet, and CoxA16 specific probe and No. 36 microballoons form couplet, and Echo9 specific probe and No. 46 microballoons form couplet; Described couplet is a kind of sphere matrix.
Excite redness classification fluorescence on the sphere matrix with red laser, determine types according to the color of sphere matrix is different; The biotin labeling of described universal primer combines with Streptavidin-phycoerythrin, excite phycoerythrin with green laser, measure the quantity of bonded report fluorescence molecule on the sphere matrix, be used for determining indirectly bonded hand foot mouth disease correlated virus nucleic acid on the sphere matrix content.
The present invention has determined 3 kinds of specific probes at the VP1 zone that are used for the hand foot mouth disease diagnosis altogether, and utilizes described probe to detect its corresponding viral nucleic acid respectively, is respectively: EV71 viral nucleic acid, CoxA16 viral nucleic acid and Echo9 viral nucleic acid.3 kinds of specific probes at hand foot mouth disease correlated virus VP1 zone involved in the present invention are on the basis of a large amount of clinical applications, confirm that the substantial connection that has of true and hand foot mouth disease is selected.
Preferably, described biotinylated universal primer is: through the biotin labeled universal primer at enterovirus of overactivation.
Preferably, described 26,36 and No. 46 microballoons are through washing, activatory carboxyl microballoon.
The biotinylated universal primer hand foot mouth disease correlated virus nucleic acid that is used to increase, simultaneously with vitamin H on the RT-PCR product mark of amplification, biotin labeling can with liquid-phase chip microsphere bonded RT-PCR product with detect the fluorescence coupling, concentration with bonded RT-PCR product combines with the detection intensity of fluorescence indirectly, thereby realizes the concentration of every kind of virus is carried out quantitative assay.
Mentality of designing of the present invention is: select the specific probe at hand foot mouth disease correlated virus VP1 zone, 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 hand foot mouth disease correlated virus.When using, add viral 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 viral nucleic acid RT-PCR product combines with Streptavidin-phycoerythrin (SA-PE), by the liquid-phase chip detection system, realize disposable qualitative and quantitative detection at last to above-mentioned hand foot mouth disease correlated virus.
The preparation method of hand foot mouth disease correlated virus liquid phase chip for parallel detection of the present invention the steps include:
(1) specific probe and microballoon coupling form couplet
Choose the carboxyl microballoon respectively 26,36 and No. 46, washing; The activated carboxyl microballoon; Add specific probe respectively accordingly with said sequence at EV71, CoxA16 and Echo9 virus VP 1 zone; Add the EDC mixing; Under the room temperature, be placed on the rotating speed of 200～250rpm and hatch 120 minutes on the shaking table; Repeat 1～2 time; With PBS-TBN washing 2～3 times; Get EV71 specific probe and No. 26 microballoons formation couplets, CoxA16 specific probe and No. 36 microballoons form couplets, and Echo9 specific probe and No. 46 microballoons form couplets; 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) preparation of hand foot mouth disease liquid-phase chip
With the sphere matrix that is marked with different probe is that specific probe mixes with microballoon link coupled couplet, tentatively obtains hand foot mouth disease correlated virus liquid phase chip for parallel detection of the present invention.
(3) selection of biotinylation universal primer
Preferred biotinylated universal primer is to be used to increase hand foot mouth disease correlated virus nucleic acid.
The biotin labeling of universal primer is used for Streptavidin-phycoerythrin is attached to microballoon.
The application of brothers' mouth correlated virus liquid phase chip for parallel detection of the present invention in preparation experiment chamber and clinical detection reagent.
Utilize hand foot mouth disease correlated virus liquid phase chip for parallel detection of the present invention, can realize parallel detection to multiple hand foot mouth disease correlated virus, can detect 3 kinds of enteroviruses simultaneously, also can optionally measure wherein several viruses according to laboratory and clinical needs, the laboratory and the clinical detection of convenient different disease need.
Method of the present invention is owing to carry out the detection of multiple hand foot mouth disease correlated virus 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 26,36 and No. 46 carboxyl 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 l dH
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, 100mM, pH=6.2, with the carboxyl microballoon of the resuspended washing of whirlpool oscillator vibration 20s.
5. the Sulfo-NHS that adds 10 μ l, 50mg/ml (uses dH
2The O dilution), with the vibration gently of whirlpool oscillator.
6. the EDC that adds 10 μ l, 50mg/ml (uses dH
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, precipitation activatory carboxyl microballoon.
7. remove supernatant, add the MES of 250 μ l, 50mM, pH=5.0, whirlpool oscillator vibration 20s, resuspended activatory carboxyl microballoon.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. the MES that adds 100 μ l, 50mM, pH=5.0 is with whirlpool oscillator vibration 20s.In the microballoon of mixing, add the specific probe of 1nmol respectively, be settled to 500 μ l, with whirlpool oscillator mixing with the MES of 50mM, pH=5.0 at EV71, CoxA16 and Echo9 virus VP 1 zone.
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 l PBS-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, with PBS-TBN washing 2 times.
14. add 500 μ l PBS-TBN, the microballoon that resuspended coupling is good and washing is good, promptly get EV71 specific probe and No. 26 microballoons formation couplets, CoxA16 specific probe and No. 36 microballoons form couplets, and Echo9 specific probe and No. 46 microballoons form couplets.
15., converse the concentration of every kind of microballoon with the quantity of cell counter counting microballoon.
Keep in Dark Place 16. the good microballoon of coupling is placed on 4 ℃, general every kind of probe link coupled microballoon is preserved separately, during use, selects to mix according to test item.
Embodiment 2: the application of hand foot mouth disease correlated virus liquid phase chip for parallel detection of the present invention in clinical detection
Utilize hand foot mouth disease correlated virus liquid phase chip for parallel detection of the present invention to detect the techniqueflow of hand foot mouth disease correlated virus:
1. the extraction of viral nucleic acid: can use business-like test kit to extract RNA, from clinical samples or viral separation and Culture thing, extract viral RNA as using QIGEN ViralRNA mini Extraction Kit (CAT:52904);
(1) the AVL damping fluid of adding 560 μ l in a 1.5ml centrifuge tube;
(2) in this centrifuge tube, add 140 μ l clinical samples or viral separation and Culture thing again;
(3) place 10min under the room temperature, instantaneous then centrifugal;
(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, the centrifugal 5s of 8000rpm makes 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, the centrifugal 5s of 8000rpm makes 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) the EB damping fluid of adding 30 μ l on film leaves standstill 2-5min under the room temperature then;
(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
(1) on the ice face, melts viral RNA sample and various PCR reagent;
(2) join following reagent master solution:
2×RT-PCR?Buffer………………………………………25.0μl
dNTPs(2.5mM?each)……………………………………2.0μl
Universal primer (justice, antisense primer) ... each 1.0 μ l
The RNA enzyme inhibitors ... 0.5 μ l
TaqDNA polysaccharase (5u/ μ l) ... 0.5 μ l
AMV reversed transcriptive enzyme (10u/ μ l) ... 1.0 μ l
Template ribonucleic acid ... 3.0 μ l
RNase?Free?d?H
2O………………………………………16.0μl
50.0μl
(3) carry out the RT-PCR reaction on the PCR instrument, reactions steps is as follows:
42℃………………………45min
94℃………………………2min
1 circulation
94℃…………………………15s
58℃…………………………30s
72℃…………………………1min
35 circulations
72℃…………………………5min
1 circulation
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 virus, 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 l PBS-TBN, be used for liquid-phase chip and detect;
(5) during liquid-phase chip detected, red fluorescence defined the kind of microballoon, determined the Virus Type of testing sample, and is promptly qualitative; Green fluorescence is measured the average fluorescent strength of microballoon bonded SA-PE, according to fluorescent value, determines viral level, and is promptly quantitative.
Claims (6)
1. hand foot mouth disease correlated virus liquid phase chip for parallel detection, mainly by microballoon, probe, biotinylated random primer, Streptavidin-phycoerythrin is formed, and it is characterized in that: described microballoon is 26,36 and No. 46 microballoons; Described probe is the amidized specific probe at EV71, CoxA16 and Echo9 virus VP 1 zone; Described biotinylated random primer is activated biotin labeled universal primer at enterovirus; The combination of the VP1 regiospecificity of described probe energy EV71, CoxA16 and Echo9 viral nucleic acid, described biotinylated random primer can carry out specific amplification to enterovirus;
Wherein: the amidized specific probe of described EV71 and No. 26 microballoons form couplet, and the amidized specific probe of CoxA16 and No. 36 microballoons form couplet, and the amidized specific probe of Echo9 and No. 46 microballoons form couplet; Described couplet is a kind of sphere matrix.
2. hand foot mouth disease correlated virus liquid phase chip for parallel detection as claimed in claim 1 is characterized in that: described biotinylated random primer is the biotinylation random primer at enterovirus.
3. hand foot mouth disease correlated virus liquid phase chip for parallel detection as claimed in claim 1 is characterized in that: described 26,36 and No. 46 microballoons are through washing, activatory carboxyl microballoon.
4. the preparation method of the described hand foot mouth disease correlated virus of claim 1 liquid phase chip for parallel detection, it is characterized in that: step is as follows:
(1) microballoon and amidized probe coupling form couplet
Choose the carboxyl microballoon respectively 26,36 and No. 46, washing; The activated carboxyl microballoon; Add specific probe respectively accordingly with said sequence at EV71, CoxA16 and Echo9 virus VP 1 zone; Add the EDC mixing; Under the room temperature, be placed on the rotating speed of 200～250rpm and hatch 120 minutes on the shaking table; Repeat 1～2 time; With PBS-TBN washing 2～3 times; Get EV71 specific probe and No. 26 microballoons formation couplets, CoxA16 specific probe and No. 36 microballoons form couplets, and Echo9 specific probe and No. 46 microballoons form couplets; 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) specific probe and microballoon link coupled couplet, the biotin labeled random primer of activatory, Streptavidin-phycoerythrin are prepared in proportion, obtained hand foot mouth disease correlated virus liquid phase chip for parallel detection of the present invention.
5. the application of the described brothers' mouth of claim 1 correlated virus liquid phase chip for parallel detection in preparation hand foot mouth disease clinical diagnosis and detection reagent.
6. as the application of brothers' mouth correlated virus liquid phase chip for parallel detection as described in the claim 5, it is characterized in that: the microballoon couplet that will be marked with probe, as the biotin labeled random primer of the activatory of reporter molecules, Streptavidin-phycoerythrin disposes in proportion, probe combines with the corresponding target nucleic acid specificity, the reporter molecules that has green report fluorescence also combines with Streptavidin-phycoerythrin is specific, 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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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102312018A (en) * | 2011-06-09 | 2012-01-11 | 深圳国际旅行卫生保健中心 | Real-time polymerase chain reaction (RT-PCR) detecting kit and non-diagnostic detection method for throat swab specimen hand-foot-and-mouth disease Cox A16 virus |
| CN102353780A (en) * | 2010-06-04 | 2012-02-15 | 北京庄笛浩禾生物医学科技有限公司 | Coxsackie virus A16 (CA16) detection testing strip |
| CN102586476A (en) * | 2012-01-18 | 2012-07-18 | 深圳市普瑞康生物技术有限公司 | Preparation and application of gene chip for detecting important enteric causative viruses |
| CN103255231A (en) * | 2013-04-24 | 2013-08-21 | 山东艾克韦生物技术有限公司 | Nucleic acid parallel testing liquid-phase chip as well as preparation method and application thereof |
| CN104073569A (en) * | 2014-07-21 | 2014-10-01 | 广州市妇女儿童医疗中心 | Molecular marker used for diagnosing extremely severe case of hand-foot-and-mouth disease and testing method as well as kit |
| CN109609628A (en) * | 2019-01-31 | 2019-04-12 | 中国科学院武汉病毒研究所 | It is a kind of for detecting the primer sets and kit of thalassaemia mutations body |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102353780A (en) * | 2010-06-04 | 2012-02-15 | 北京庄笛浩禾生物医学科技有限公司 | Coxsackie virus A16 (CA16) detection testing strip |
| CN102312018A (en) * | 2011-06-09 | 2012-01-11 | 深圳国际旅行卫生保健中心 | Real-time polymerase chain reaction (RT-PCR) detecting kit and non-diagnostic detection method for throat swab specimen hand-foot-and-mouth disease Cox A16 virus |
| CN102312018B (en) * | 2011-06-09 | 2013-04-17 | 深圳国际旅行卫生保健中心 | Real-time polymerase chain reaction (RT-PCR) detecting kit and non-diagnostic detection method for throat swab specimen hand-foot-and-mouth disease Cox A16 virus |
| CN102586476A (en) * | 2012-01-18 | 2012-07-18 | 深圳市普瑞康生物技术有限公司 | Preparation and application of gene chip for detecting important enteric causative viruses |
| CN103255231A (en) * | 2013-04-24 | 2013-08-21 | 山东艾克韦生物技术有限公司 | Nucleic acid parallel testing liquid-phase chip as well as preparation method and application thereof |
| CN103255231B (en) * | 2013-04-24 | 2015-01-07 | 山东艾克韦生物技术有限公司 | Nucleic acid parallel testing liquid-phase chip as well as preparation method and application thereof |
| CN104073569A (en) * | 2014-07-21 | 2014-10-01 | 广州市妇女儿童医疗中心 | Molecular marker used for diagnosing extremely severe case of hand-foot-and-mouth disease and testing method as well as kit |
| CN104073569B (en) * | 2014-07-21 | 2016-06-22 | 广州市妇女儿童医疗中心 | For diagnosing the molecular marked compound of hand-foot-mouth disease pole serious symptom and detection method thereof and test kit |
| CN109609628A (en) * | 2019-01-31 | 2019-04-12 | 中国科学院武汉病毒研究所 | It is a kind of for detecting the primer sets and kit of thalassaemia mutations body |
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Application publication date: 20100120 |