CN102876807A - Liquid chip non-diagnostic method for joint detection of three rash and fever viruses and preparation method of liquid chip - Google Patents
Liquid chip non-diagnostic method for joint detection of three rash and fever viruses and preparation method of liquid chip Download PDFInfo
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Abstract
The invention discloses a liquid chip non-diagnostic method for joint detection of three rash and fever viruses and a preparation method of a liquid chip. The liquid chip non-diagnostic method includes: performing PCR (polymerase chain reaction) amplification for the three rash and fever viruses by primers with biotin labelings, and designing specificity capture probes according to nucleotide sequences in a PCR amplification area; enabling the capture probes to be coupled with corresponding coding microspheres respectively, and enabling the capture probes to be combined with PCR products on a certain condition specifically; and adding streptavidin-phycoerythrin to be combined with biotins on the PCR products captured on the microspheres, using a liquid chip Luminex system for detection, and performing qualitative and quantitative analysis for a to-be-detected object by stimulating red classified fluorescence on a microsphere matrix and phycoerythrin combined on the microsphere surface by specificity reaction. The liquid chip non-diagnostic method is high in detection sensitivity and good in specificity.
Description
Technical field
The present invention relates to a kind ofly can detect simultaneously Enterovirus 71 (Enterovirus 71), CA group 16(Coxsackievirus A 16) and the non-diagnostic methods of liquid-phase chip of Measles virus (Measles virus) and to the preparation method and application of its liquid-phase chip.
Background technology
Heating companion's eruption syndrome be generate heat, eruption is cardinal symptom, simultaneously with one group of disease of other clinical symptom.Common pathogenic agent comprise Enterovirus 71 (Enterovirus 71, EV71), CA group 16(Coxsackievirus A 16, CA16), Measles virus (Measles virus, MV), rubella virus, dengue virus etc.Wherein, EV71 and CA16 are respectively the main pathogens that causes severe and light disease hand foot mouth disease, and Measles virus is the main pathogens of measles.This class disease just has infectivity when latent period.Therefore, disease carry out in early days examination to this syndrome cause of disease, to subsequently isolation, take corresponding prevention and control measure etc. to have directive significance.
PCR is called for short in polymerase chain reaction (polymerase chain reaction), is a kind of amplification in vitro method that n DNA copies of simulating.The appearance of round pcr brings a revolution to molecular diagnosis.Round pcr has become the basis of molecular diagnosis.The detection method of PCR product is that agarose gel electrophoresis detects at present, and sepharose is only about can distinguish the dna fragmentation that differs 100bp.The PCR product that fragment length is more or less the same or equates, agarose gel electrophoresis is unable with incapability.In addition, agarose gel electrophoresis produces fluorescence by the ultraviolet excitation fluorescence dye and judges whether to exist the PCR product, and detection sensitivity is relatively low.And agarose gel electrophoresis is judged product by the size of fragment, and poor specificity can not be distinguished the big or small similar fragment of non-specific amplification.And dyestuff commonly used such as ethidium bromide etc. have carinogenicity, often operate healthy unfavorable.
Liquid-phase chip (liquid chip) also claims suspending chip (suspension array), is a kind of very flexibly multifunctional examining survey technology platform, can carry out the researchs such as the biomacromolecule detections such as albumen, nucleic acid, acceptor and part discriminance analysis.Liquid-phase chip mainly by fluorescence-encoded micro-beads and the two bundle laser detection of coupling probe, to the analyte qualitative and quantitative analysis, can be finished 100 kinds of different biologicallies in the reacting hole.Can realize the Multiple detection to nucleic acid.
Summary of the invention
Problem for the prior art existence, the object of the present invention is to provide a kind of detection sensitivity high, specificity is good, is particularly useful for the similar length fragment of multiple PCR products and is not easily distinguishable, and can distinguish 100 kinds of different PCR products in theory, realize the Multiple detection of 100 kinds of different factors.
Existing cause of disease detection technique is subject to the predicament of Multiple detection ability, and many suspicious samples need to carry out the examination of many cause of diseases, the objective of the invention is non-diagnostic method of liquid-phase chip by three kinds of viruses of a kind of joint-detection are provided and its preparation method and application, accompany the rapid detection of eruption cause of diseases for three kinds of heatings a kind of sensitivity, special, quick, reliable method are provided.
The technical solution used in the present invention is: select the specific gene fragment of three kinds of viruses, design is for the Auele Specific Primer of three kinds of viruses; Then design is positioned at the specific probe of three kinds of viruses of amplification region, the coding microball of the different numberings of probe coupling, the liquid-phase chip method that can detect simultaneously above three kinds of viruses is set up out in preparation, when using, at first use above-mentioned primer that three kinds of viruses are carried out the multiplex PCR amplified production, there is the coding microball of probe to be combined with coupling, react with streptavidin-phycoerythrin (SA-PE) again, pass through afterwards the suspending chip detection system (such as Luminex 100, Luminex 200, Bio-plex, Liquichip etc.), realize three kinds of heatings are accompanied out the disposable detection of exanthema virus.
For achieving the above object, the invention provides a kind of non-diagnostic methods of liquid-phase chip of accompanying out exanthema virus for three kinds of heatings of joint-detection, comprising:
1) uses with biotin labeled Auele Specific Primer and carry out three kinds of heating companion's eruption virus PCR increasings;
2) coupling capture probe arranged coding microball under certain conditions capture probe special be combined with biotin labeled PCR product;
3) adding the vitamin H of SA-PE on the PCR product of catching on the microballoon is combined, utilize suspending chip Luminex system to detect, by excite redness classification fluorescence on the microballoon matrix and microsphere surface through specific reaction in conjunction with upper phycoerythrin, thing to be detected is carried out qualitative and quantitative analysis.
Further, described heating accompanies out exanthema virus to comprise Enterovirus 71 (EV71), CA group 16(CA16) and Measles virus (MV).
Further, the primer sequence of described EV 71, CA16 and MV is:
Reverse 5 ' end adds the Biotin mark in the further above-mentioned sequence.
Further, described RT-PCR reaction system is: 10 * PCR buffer, 2.5 μ L, dNTPs 2.5 μ L, MgCl
2(25mM) 5 μ L, RNase Inhibitor(40U/ μ L) 0.5 μ L, AMV RTase XL(5U/ μ L) 0.5 μ L, AMV-optionized Taq (5U/ μ L) 0.5 μ L, each 1 μ L of upstream and downstream primer (10uM), RNA3 μ L, RNase-free ddH
2O supplies 30 μ L.Reaction conditions: 50 ℃ of 30min; 94 ℃ of 2min; 94 ℃ of 30s, 55 ℃ of 30s, 72 ℃ of 45s, 30 circulations; 72 ℃ of 10min.
Further, described step 2) and the method in the step 3), may further comprise the steps:
1) each 3500 of every kind of coding microballs getting the good detection probes of coupling are in hybridization solution, and making its total amount is 33 μ L, calculates corresponding add-on according to the microballoon count results;
2) add 5～17 μ L with biotin labeled multiplex PCR amplified production in each pipe, making its final volume is 50 μ L, the piping and druming mixing.
3) 92 ℃ ~ 95 ℃ sex change 10min～15min.
4) hybridize certain hour under the hybridization temperature.
5) be transferred to the filter plate suction filtration and remove unconjugated complementary probe chain.
6) add 1 * TMAC liquid of 75 μ L 4ng/ μ L SA-PE to each hole, the room temperature lucifuge is hatched 10min again, and suction filtration removes unconjugated SA-PE.
7) add 75 μ L, 1 * TMAC solution to each hole, vibration makes microballoon resuspended again.
8) after reaction finished, liquid-phase chip Luminex system detected, and by exciting the redness classification fluorescence on the microballoon matrix, the numbering of microballoon was identified; By exciting the phycoerythrin of microsphere surface, read fluorescence intensity MFI numerical value and analytical data.
The invention provides a kind of preparation method of above-mentioned liquid-phase chip, the method comprises:
1) according to PCR product to be detected, select design to be positioned at the special capture probe of amplification region;
2) capture probe and complementary probe are synthetic;
3) corresponding capture probe coupling coding microball;
4) checking of capture probe coupling microballoon.
Further, the capture probe of described EV 71, CA16 and MV and complementary sequence sequence thereof are:
Each probe 5 ' end adds 12 C or 20 T add-NH again in the further above-mentioned sequence
2Modify; RC-probe 5 ' end adds the Biotin mark.
Further, the method in the described step 3) when capture probe and microballoon coupling may further comprise the steps:
A) choose respectively different coding microballs with COOH of numbering, with vortex oscillation device vibration microballoon suspension, microballoon is mixed;
B) get respectively about 1.25 ⅹ 10 of above-mentioned microballoon
6Individual, be transferred to respectively centrifuge tube, the centrifugal 3～5min of 14000g, careful sucking-off supernatant;
C) 2-(n-morpholino) the ethyl sulfonic acid solution of adding 50 μ L 0.1mol/L, vibration 20～30S, ultrasonic 20～30s makes microballoon resuspended;
D) with distilled water synthetic oligonucleotide probe is diluted to 0.1mmol/L;
E) probe with 1 μ L dilution is added in the microsphere suspension liquid, the vibration mixing;
F) the EDC solution that adds the freshly prepared 10mg/mL of 2.5 μ L is to microballoon and the mixed liquid of probe, and mixing vibrates;
G) with aluminium foil parcel centrifuge tube lucifuge, 400～600rpm vibration on the vortex oscillation device, incubated at room 30min;
H) again add freshly prepared 10mg/mL EDC;
I) again 400～600rpm vibration on the vortex oscillation device, the room temperature lucifuge is hatched 30min;
J) with 0.02%PBST 1ml washing 1 time, centrifugal 14000g 3～5min;
K) move and abandon supernatant, microballoon is resuspended among the 1ml 0.1%SDS, and washing is centrifugal;
L) move and abandon supernatant, microballoon is resuspended among the 100 μ L pH8.0TE, and mixing has been hanged in vibration, namely obtains the good detection microballoon of coupling.
Further, the verification method of coupling microballoon in the described step 4) may further comprise the steps:
A) get each 3500 of every kind of good detection probes of coupling in hybridization solution, making its total amount is 33 μ L, calculates corresponding add-on according to the microballoon count results;
B) add 5～17 μ L with biotin labeled complementary probe chain (RC-probe) in each pipe, making its final volume is 50 μ L, the piping and druming mixing.
C) 92 ℃ ~ 95 ℃ sex change 10min.
D) 45 ℃ ~ 55 ℃ lower hybridization certain hours of hybridization temperature.
E) be transferred to the filter plate suction filtration and remove unconjugated complementary probe chain.
F) add 1 * TMAC liquid of 75 μ L 4ng/ μ L SA-PE to each hole, the room temperature lucifuge is hatched 10min again, and suction filtration removes unconjugated SA-PE.
G) add 75 μ L, 1 * TMAC solution to each hole, vibration makes microballoon resuspended again.
H) after reaction finished, liquid-phase chip Luminex system detected, and by exciting the redness classification fluorescence on the microballoon matrix, the numbering of microballoon was identified; By exciting the phycoerythrin of microsphere surface, read fluorescence intensity MFI numerical value and analytical data.
I) result judges: blank MFI is lower than 100, and coated microballoon MFI is greater than 2000 use; If coated microballoon MFI is less than 2000, then explanation is coated unsuccessful, should again be coated with.
The present invention relates to utilize the application of the above-mentioned three kinds of heating companion eruption Detecting methods of above-mentioned liquid-phase chip compound detection, comprise: 1. the extraction of sample nucleic acid to be detected and reverse transcription, 2. carry out multi-PRC reaction, wherein prepare certain concentration of component, certain volume is the PCR reaction system of 30 μ l for example, 3. capture probe is caught the PCR product: coupling is had the microballoon of probe mix with the multiple PCR products of biotinylation mark, 95 ℃ of sex change 5～10 minutes, hybridized 10～30 minutes for 45～60 ℃, probe can specificity be caught corresponding PCR product, and then adds streptavidin-phycoerythrin.
The present invention compares with traditional PCR detection method, and advantage is:
1. by the signal amplifying system of instrument and the amplification of vitamin H avidin, the present invention is higher than agarose gel electrophoresis to the detection sensitivity of PCR product.
2. catch the PCR product by specific probe, detection specificity is much better than with fragment length the PCR product to be judged.
3. to the similar or the same fragment of fragment length size, can carry out Division identification equally, thereby make the design of multiple PCR primer be tending towards flexible.
Description of drawings
Fig. 1 is 3 kinds of virus multiple pcr amplification electrophoresis result;
Fig. 2 is the fluorescence intensity dose response curve of the DNA concentration-coding microball surface of EV71;
Fig. 3 is the fluorescence intensity dose response curve of the DNA concentration-coding microball surface of CA16;
Fig. 4 is the fluorescence intensity dose response curve of the DNA concentration-coding microball surface of Measles virus (MV).
Embodiment
The present invention relates to three kinds of heatings of joint-detection and accompany out method for detecting suspension chip of exanthema virus and its preparation method and application, the present invention will be further described below by embodiment, but the present invention is subjected to the restriction of the following example never in any form.
Embodiment 1: respectively the cDNA of three kinds of viruses is carried out the optimization of multi-PRC reaction condition with the primer of vitamin H
Determine that at first heating companion eruption cause of disease comprises the diagnostic fragment of Enterovirus 71, CA group 16 and Measles virus, GenBank by NCBI obtains candidate gene sequence, utilize the software design primer, and synthetic correlated series, downstream primer 5 ' end biotin labeling, see the following form, and these primers are diluted to 10 μ mol/L.
Detect the primer of 3 kinds of heating companion eruption cause of diseases
1. by following system preparation multi-PRC reaction system, totally be 30 μ l:
10×PCR buffer： 3μl
Taq E： 0.4μl
dNTPs： 0.6μl
EV71 primer (10 μ M): each 1 μ l
CA16 primer (10 μ M): each 1 μ l
MV primer (10 μ M): each 1 μ l
Template cDNA:2 μ l
DdH
2O: add to 30 μ l
2. carry out the PCR reaction by following reaction conditions
Denaturation:
94℃ 2min
30 circulations:
94℃ 30s
55℃ 30s
72℃ 45s
Extend:
72℃ 10min
After reaction finishes with 1.2% agarose gel electrophoresis inspection amplification situation, PCR negative control: except not adding template and with ddH
2Outside O replaced, all the other systems were identical.The running gel imaging results is seen Fig. 1, three kinds of viruses of presentation of results all can specific amplification out.
Embodiment 2: the preparation of liquid-phase chip
(1) capture probe and microballoon coupling
1. choose respectively the coding microball of different numberings, with vortex oscillation device vibration microballoon suspension, microballoon is mixed.
2. get respectively about 1.25 ⅹ 10 of above-mentioned microballoon
6Individual, be transferred to respectively centrifuge tube, centrifugal 3～5 minutes of 14000g, careful sucking-off supernatant.
3. add 2-(n-morpholino) the ethyl sulfonic acid solution of 50 μ l 0.1mol/L, vibrated 20～30 seconds, ultrasonic 20～30 seconds, make microballoon resuspended.
4. with distilled water synthetic oligonucleotide probe is diluted to 0.1mmol/L.
5. the probe with 1～5 μ l dilution is added in the microsphere suspension liquid, the vibration mixing.
6. add the EDC solution of the freshly prepared 10mg/mL of 2.5 μ l to microballoon and the mixed liquid of probe, the vibration mixing.
7. with aluminium foil parcel centrifuge tube lucifuge, 400～600rpm vibration on the vortex oscillation device, incubated at room 30 minutes.
8. again add freshly prepared 10mg/mL EDC.
9. again 400～600rpm vibration on the vortex oscillation device, the room temperature lucifuge was hatched 30 minutes.
10. with 0.02%PBST 1ml washing 1 time, the centrifugal 3-5 of 14000g minute.
Abandon supernatant 11. move, microballoon is resuspended among the 1ml 0.1%SDS, and washing is centrifugal.
Abandon supernatant 12. move, microballoon is resuspended among the 100 μ l pH8.0TE, and mixing has been hanged in vibration, namely obtains the good detection microballoon of coupling.
13. with the quantity of cell counter counting microballoon, converse the unit concentration of every kind of microballoon.
Keep in Dark Place 14. the good detection microballoon of coupling is placed on 4 ℃, the microballoon of general every kind of probe coupling is preserved separately, during use, and the microballoon kind that selection will mix according to test item.
(2) checking of capture probe coupling coding microball
1. get each 3500 mixing of above-mentioned various coupling microballoon, be sub-packed in the PCR pipe and (calculate corresponding add-on according to the microballoon count results), making its total amount is 33 μ L
2. the complementary probe that adds respectively three kinds of viruses of 5～17 μ l in each pipe adds water and supplies that to make its final volume be 50 μ l, the piping and druming mixing.
3.92～95 ℃ of sex change 10 minutes.
4.45～60 ℃ were reacted 10～30 minutes.
5. be transferred to 96 hole filter plate suction filtrations and remove unconjugated PCR product.
6. add 75 μ l 4ng/ μ l SA-PE to each hole, the room temperature lucifuge was hatched 10 minutes again, and suction filtration removes unconjugated SA-PE.
7. add 75 μ l suspension to each hole, vibration makes microballoon resuspended again.
8. reaction finishes to detect by the suspending chip detection system, detects gained fluorescent value MFI and all is higher than 2000.
Embodiment 3: sensitivity detects
The cDNA masterplate dilution different concns gradient of exanthema virus is accompanied out in three kinds of heatings, carries out respectively the amplification of multiplex PCR and the multiplex PCR amplification of hybrid template, amplified production is carried out the liquid-phase chip method detect:
A) get each 3500 of every kind of good detection probes of coupling in hybridization solution, making its total amount is 33 μ L, calculates corresponding add-on according to the microballoon count results;
B) add 5～17 μ L multiplex PCR amplified productions in each pipe, adding water, to make its final volume be 50 μ L, the piping and druming mixing.
C) 92 ℃ ~ 95 ℃ sex change 10min.
D) 45 ℃ ~ 55 ℃ lower hybridization certain hours of hybridization temperature.
E) be transferred to the filter plate suction filtration and remove unconjugated complementary probe chain.
F) add 1 * TMAC liquid of 75 μ L 4ng/ μ L SA-PE to each hole, the room temperature lucifuge is hatched 10min again, and suction filtration removes unconjugated SA-PE.
G) add 75 μ L, 1 * TMAC solution to each hole, vibration makes microballoon resuspended again.
H) after reaction finished, suspending chip Luminex system detected, and by exciting the redness classification fluorescence on the microballoon matrix, the numbering of microballoon was identified; By exciting the phycoerythrin of microsphere surface, read fluorescence intensity MFI numerical value and analytical data.
Concrete detected result sees the following form:
The liquid-phase chip method detects the increase result of different gradient concentration EV71cDNA templates of multiplex PCR
The typical curve equation of the detection EV71 that obtains: FI=-1.24657+ (465.047+1.24657)/((1+ (Conc/226.075)
-0.820515))
1.80342, drawing as calculated minimum detected value is 17.2pg/tes, obtains canonical plotting and sees accompanying drawing 2.
The liquid-phase chip method detects the increase result of different gradient concentration CA16 templates of multiplex PCR
Obtain detecting the typical curve equation of CA16: FI=-6.19033+ (303.4+6.19033)/((1+ (Conc/928.593)
-1.50301))
0.464424, the minimum detected value that calculates is 28.9pg/tes, the canonical plotting that obtains is seen accompanying drawing 3.
The liquid-phase chip method detects the increase result of different gradient concentration MV templates of multiplex PCR:
Obtain detecting the typical curve equation of MV virus: FI=0.24393+ (7630.07-0.24393)/((1+ (Conc/741.585)
-1.40692))
0.886855, drawing as calculated lowest detectable limit: 2.14pg/ml, the canonical plotting that obtains is seen accompanying drawing 4.
The liquid-phase chip method detects the result of three kinds of different gradient concentration viral template of multiplex PCR amplification:
Embodiment 4, pattern detection
1. the extraction of sample amplifying nucleic acid and reverse transcription: use RNA viruses nucleic acid extraction kit and reverse transcription test kit to carry out.
2. carry out reverse transcription reaction (AMV reversed transcriptive enzyme method) by following reaction conditions
DEPC water 4.5ul
Random primer 1ul
Template RN 1ul
Slightly centrifugal, 100 ℃ of boiling water baths 1 minute add
dNTP 0.5ul
5×buffer 2ul
AMV reversed transcriptive enzyme 1ul
Slightly centrifugal, sealed membrane sealing, 42 ℃ of water-baths 90 minutes.3 minutes deactivation AMV of 100 ℃ of boiling water baths.
3. carry out immediately the detection of multiplex PCR amplification and liquid-phase chip method.
The result judges:
When the MFI of sample to be detected value is judged to the positive more than three times the time for this detection background strength of signal.The PCR product of the positive that detection is obtained is sent to order-checking, and sequencing result is compared, and the result shows it is the corresponding virus of liquid-phase chip positive findings.
Can draw from the above results, liquid-phase chip of the present invention and detection method have following superiority:
1. sensitive: compare to common multi-PRC reaction, sensitivity of the present invention can improve from the embodiment of following two aspects: there is the amplification system of signal in (1) detecting instrument; (2) avidin that connects of the vitamin H on the PCR product band and the fluorescence dye phycoerythrin amplification of being combined.
2. special: the employing Nucleic Acid Probe Technique is carried out specific identification to the PCR product of vitamin H on the mark, and unconventional electrophoresis method is identified by PCR product clip size.
3. high-throughput: detect when one time PCR reaction one-time detection can realize plurality of target virus.
4. precise and high efficiency: integrate nucleic acid amplification in vitro, making nucleic acid molecular hybridization, coding microball, biotin labeling, fluoroscopic examination and Flow Cytometry in one, realize simultaneously detection, the evaluation of nucleic acid samples, make the result accurate, efficient work.
5, chip manufacturing is convenient: only with the primer that designs target viral to be checked, and the optimize PCR condition, probe corresponding to mark namely can be assembled into detection system in coding microball.
6, the Multiple detection target is adjustable flexibly: can detect target according to difference, select corresponding primer and microballoon, form the detection system of different target combination.
SEQUENCE LISTING
＜110〉China Inst. of Quarantine Inspection Sciences
＜120〉be used for three kinds of heatings of joint-detection and accompany out non-diagnostic methods of liquid-phase chip of exanthema virus and preparation method thereof
＜130〉invention
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 23
<212> DNA
<213> EV71-Forward
<400> 1
ATA ATA GCA YTR GCG GCA GCC CA 23
<210> 2
<211> 20
<212> DNA
<213> EV71-Reverse
<400> 2
AGA GGG AGR TCT ATC TCY CC 20
<210> 3
<211> 23
<212> DNA
<213> CA16-Forward
<400> 3
TTG CAG ACA TGA TTG ACC AG 20
<210> 4
<211> 20
<212> DNA
<213> CA16-Reverse
<400> 4
TGT TCT GTG TAC CCC TGG TG 20
<210> 5
<211> 23
<212> DNA
<213> MV-Forward
<400> 5
ACG GCT TGT TCT TGG GTG AG 20
<210> 6
<211> 20
<212> DNA
<213> MV-Reverse
<400> 6
AAC TTC GGA GGG ATA GGG TG 20
Claims (10)
1. one kind is used for the non-diagnostic methods of liquid-phase chip that exanthema virus is accompanied out in three kinds of heatings of joint-detection, it is characterized in that this non-diagnostic methods comprises:
1) uses with biotin labeled primer companion's eruption virus multiple pcr amplification that generates heat;
2) coupling capture probe arranged coding microball under certain conditions, capture probe special be combined with biotin labeled PCR product;
3) adding the vitamin H of SA-PE on the PCR product of catching on the microballoon is combined, utilize suspending chip Luminex system to detect, by excite redness classification fluorescence on the microballoon matrix and microsphere surface through specific reaction in conjunction with upper phycoerythrin, thing to be detected is carried out qualitative and quantitative analysis.
2. non-diagnostic methods as claimed in claim 1 is characterized in that, described heating accompanies out exanthema virus to comprise Enterovirus 71, CA group 16 and Measles virus.
3. non-diagnostic methods as claimed in claim 2 is characterized in that, the primer sequence of described Enterovirus 71, CA group 16 and Measles virus is:
4. non-diagnostic methods as claimed in claim 3 is characterized in that, Reverse 5 ' end adds the Biotin mark in the described primer sequence.
5. non-diagnostic methods as claimed in claim 1 is characterized in that, the reaction system of described pcr amplification is: cDNA template 2ul, and PCR premixed liquid 15ul, each viral upstream and downstream primer (10umol/L) 1ul, deionized water is supplied 30ul.Reaction conditions: 50 ℃ of 30min; 94 ℃ of 2min; 94 ℃ of 30s, 55 ℃ of 30s, 72 ℃ of 45s, 30 circulations; 72 ℃ of 10min.
6. preparation method who is used for non-its liquid-phase chip of diagnostic methods of liquid-phase chip that three kinds of heatings of joint-detection accompany out exanthema virus, the method comprises:
1) according to PCR product to be detected, select design to be positioned at the special capture probe of amplification region;
2) capture probe and complementary probe are synthetic;
3) corresponding capture probe coupling coding microball;
4) checking of capture probe coupling microballoon.
7. preparation method as claimed in claim 6 is characterized in that, capture probe and the complementary sequence sequence thereof of described EV 71, CA16 and MV are:
8. as claimed in claim 7, it is characterized in that, in the described sequence each probe 5 ' end add 12 C or 20 T add again-NH2 modifies; RC-probe 5 ' end adds the Biotin mark.
9. liquid-phase chip preparation method as claimed in claim 6 is characterized in that, described step 2) method when middle probe and microballoon coupling, may further comprise the steps:
A) choose respectively different coding microballs of numbering, with vortex oscillation device vibration microballoon suspension, microballoon is mixed;
B) get respectively about 1.25 ⅹ 10 of above-mentioned microballoon
6Individual, be transferred to respectively centrifuge tube, the centrifugal 3～5min of 14000g, careful sucking-off supernatant;
C) 2-(n-morpholino) the ethyl sulfonic acid solution of adding 50 μ L0.1mol/L, vibration 20～30S, ultrasonic 20～30s makes microballoon resuspended;
D) with distilled water synthetic oligonucleotide probe is diluted to 0.1mmol/L;
E) probe with 1～5 μ L dilution is added in the microsphere suspension liquid, the vibration mixing;
F) the EDC solution that adds the freshly prepared 10mg/mL of 2.5 μ L is to microballoon and the mixed liquid of probe, and mixing vibrates;
G) with aluminium foil parcel centrifuge tube lucifuge, 400～600rpm vibration on the vortex oscillation device, incubated at room 30min;
H) again add freshly prepared 10mg/mL EDC;
I) again 400～600rpm vibration on the vortex oscillation device, the room temperature lucifuge is hatched 30min;
J) with 0.02%PBST 1ml washing 1 time, centrifugal 14000g 3～5min;
K) move and abandon supernatant, microballoon is resuspended among the 1ml 0.1%SDS, and washing is centrifugal;
L) move and abandon supernatant, microballoon is resuspended among the 100 μ L pH8.0TE, and mixing has been hanged in vibration, namely obtains the good detection microballoon of coupling.
10. preparation method as claimed in claim 6 is characterized in that, the verification method of coupling microballoon in the described step 4) may further comprise the steps:
1) get each 3500 of every kind of good detection probes of coupling in hybridization solution, making its total amount is 33 μ L, calculates corresponding add-on according to the microballoon count results;
2) add 5～17 μ L with biotin labeled complementary probe chain in each pipe, making its final volume is 50 μ L, the piping and druming mixing;
3) 92 ℃ ~ 95 ℃ sex change 10min;
4) hybridize certain hour under the hybridization temperature;
5) be transferred to the filter plate suction filtration and remove unconjugated complementary probe chain;
6) add 1 * TMAC liquid of 75 μ L 4ng/ μ LSA-PE to each hole, the room temperature lucifuge is hatched 10min again, and suction filtration removes unconjugated SA-PE;
7) add 75 μ L1 * TMAC solution to each hole, vibration makes microballoon resuspended again;
8) after reaction finished, Luminex system detected, and by exciting the redness classification fluorescence on the microballoon matrix, the numbering of microballoon was identified; By exciting the phycoerythrin of microsphere surface, read fluorescence intensity MFI numerical value and analytical data;
9) result judges: blank MFI is lower than 100, and coated microballoon MFI is greater than 2000 use; If coated microballoon MFI is less than 2000, then explanation is coated unsuccessful.
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| CN105907888A (en) * | 2016-04-14 | 2016-08-31 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | Making method and use of gene chip for detecting nine rash and fever pathogens |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105907888A (en) * | 2016-04-14 | 2016-08-31 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | Making method and use of gene chip for detecting nine rash and fever pathogens |
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