CN103543275A - Method for rapidly detecting PP65 based on magnetic bead - Google Patents

Method for rapidly detecting PP65 based on magnetic bead Download PDF

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CN103543275A
CN103543275A CN201310549511.4A CN201310549511A CN103543275A CN 103543275 A CN103543275 A CN 103543275A CN 201310549511 A CN201310549511 A CN 201310549511A CN 103543275 A CN103543275 A CN 103543275A
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magnetic bead
antigen
aptamer
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张波
夏宇
王永虎
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First Affiliated Hospital of TMMU
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Abstract

The invention discloses a method for rapidly detecting PP65 based on a magnetic bead, belonging to the field of molecular biology. According to the method, the immunofluorescence technology is adopted to detect HCMV (human cytomegalovirus) PP65 antigen, an aptamer with specificity to PP65 is utilized for constructing a magnetic bead-aptamer-antigen-monoclonal antibody complex, a related reagent system is optimized and the corresponding operational process is established, and the PP65 detecting efficiency is improved. Aiming at the problems that the traditional detection method has long detection time and high detection expense and is limited by clinical application, the method adopts the magnetic bead to rapidly detect PP65, and the HCMV disease can be early predicted; the method has the advantages of few flow processes, simplicity in operation, short detection time, convenience in popularization and the like and can provide theoretical and practice foundation for automated inspection.

Description

A kind of method based on magnetic bead fast detecting PP65
Technical field
The present invention relates to the detection method of human cytomegalovirus, relate in particular to a kind of method based on magnetic bead fast detecting PP65, belong to biology field.
Background technology
Human cytomegalovirus (h μ Man cytomegalovirus, HCMV) belongs to herpetoviridae second group bleb subfamily, is a kind of duplex DNA virus.HCMV is worldwide distribution, wide-scale distribution in crowd, and virus is hidden for a long time in host cell, is subclinical infection.Work as gestation, easily cause fetal in utero to infect, cause miscarriage, premature labor, stillborn foetus or fetus congenital malformation; Work as bone-marrow transplantation, during the Organ Transplantation Patients immune function depressions such as liver, kidney, heart, latent virus is activated, rises in value and severe infections occurs, and will cause patient's organ transplant failure even dead.In recent years, there are some researches show that cytomegalovirus infection and some diseases associated with inflammation and proliferative diseases have close relationship, comprise some angiocardiopathy and cancer.Now, cytomegalovirus infection causes great threat to human health, therefore the early diagnosis that HCMV is infected seems particularly important.Research shows, once there is the clinical symptoms that HCMV infects, antiviral drugs can not be controlled this sick pathology process, this means clinical symptoms that antiviral drugs must infect at HCMV occur before just use, this just depends on the analysis in laboratory.
In HCMV experiment detection technique, viral separation is the goldstandard detecting, but length consuming time, result is affected greatly by the sample holding time.Antibody test need just can detect after patient infection's certain hour, affected by patient's autoimmune state larger, and can not distinguish latent infection and Active infection.Antigenemia detection technique is quick, a responsive diagnostic techniques, but traditional detection method is virus antigen detection carrier mainly with leucocyte, has limited its clinical practice.And its sensitivity and specificity of diagnostic nucleic acid is all good, but testing cost is high and method for extracting nucleic acid need to improve.
PP65 is the early protein of HCMV virus replication, and it comes across the early stage of virus infections, within 3-4 hour after cell infection, start to occur, and be the abundantest albumen of HCMV viral level.Its function is that viral DNA is anchored on the nuclear membrane of infected cell, synthetic in the golgiosome of endochylema, then transports back in core.Along with going deep into of research, HCMV-PP65 antigen is considered to HCMV and infects the early antigen of expressing, and PP65 antigenic analysis can predict that HCMV is sick sensitively in early days.The conventional Virus culture of HCMV (CCC) often needs 4 weeks just can obtain positive findings, early diagnosis is helped little.HCMV specific antibody IgM, can not occur or postpone occurring at serious immunosuppressed patient.
Summary of the invention
Technical matters to be solved by this invention is to provide a kind of method based on magnetic bead fast detecting PP65.
Technical scheme of the present invention is as follows: a kind of method based on magnetic bead fast detecting PP65, comprises the following steps:
(1) in centrifuge tube, add the carboxyl modified magnetic bead of 0.5～1 μ L, MES cleans three times, adds the EDC of 80～100 μ L0.5g/L, and 37 ℃ activate 2～4 hours;
(2) what directly add 2～5 μ M HEX red fluorescence marks has specific aptamer with human cytomegalovirus PP65 antigen, and room temperature light shaking is hatched 4～6 hours, after magnetic separation, abandons supernatant, and Washing buffer cleans three times; Described aptamer is one of sequence of SEQ ID No.1～SEQ ID No.6;
Figure BDA0000410079410000021
Figure BDA0000410079410000031
(3) with a BSA80～120 μ L37 ℃ sealing of 0.1～0.3%, spend the night, Washing buffer cleans three times;
(4) after magnetic separation, abandon supernatant, add sample to be checked, room temperature light shaking is hatched 10～14 hours;
(5) after magnetic separation, abandon supernatant, Washing buffer cleans three times, adds the PP65 monoclonal antibody of the FITC green fluorescence mark with Binding buffer configuration of 8～12ng, incubated at room 6～10 hours;
(6) after magnetic separation, abandon supernatant, Washing buffer cleans three times, adds the Binding buffer of 30～70 μ L, fluorescence microscopy Microscopic observation; If there is PP65 antigen in sample to be checked, can see under the microscope that red fluorescence can see green fluorescence again; If there is no PP65 antigen in sample to be checked, can only see red fluorescence under the microscope, can not see green fluorescence.
The secondary conformation of aptamer SEQ ID No.1～SEQ ID No.6 in described step (2) respectively is:
Figure BDA0000410079410000041
Figure BDA0000410079410000051
Described Binding buffer is: PBS, 5mM MgCl 2, 0.05%tween-20, pH7.5.
Described Washing buffer is: PBS, 5mM MgCl 2, 0.01%tween-20, pH7.5.
Beneficial effect: the present invention's application immunofluorescence technique detects HCMV PP65 antigen, utilize with PP65 and there is specific aptamer, build magnetic bead-aptamer-antigen-monoclonal antibody complex, and optimize related reagent system and set up corresponding operating flow process, improved PP65 checkability.For traditional detection method, grow, be subject to the problems such as clinical practice restriction, testing cost height detection time, adopt magnetic bead fast detecting PP65, can predict in early days that HCMV is sick.The present invention has the advantages such as flow process is few, simple to operate, detection time is short, be convenient to popularize, and can be Automated inspection theory and practical basis are provided.
Figure of description
Fig. 1 is the schematic diagram that the present invention is based on magnetic bead fast detecting PP65;
Fig. 2 is testing result schematic diagram of the present invention.
Embodiment
Below in conjunction with drawings and Examples, the invention will be further described:
EDC in the present invention is 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide, and Chinese is: 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimine
MES is 2-(4-Morpholino) ethanes μ Lfonic acid, and Chinese is: 2-morpholine second sulphur
FITC is Fluorescein Isothiocyanate, and Chinese is: fluorescein isothiocynate
HEX is 5 '-Hexachlorofluorescein phosphoramidite, and Chinese is: 5 '-chlordene fluorescein phosphoramidate
Embodiment mono-, a kind of method based on magnetic bead fast detecting PP65, complete according to following steps:
(1) in centrifuge tube, add the carboxyl modified magnetic bead of 0.5 μ L, MES cleans three times, adds the EDC of 0.5g/L, and 37 ℃ activate 2 hours;
(2) directly add the aptamer A4 of 5 μ M red fluorescence marks, room temperature light shaking is hatched 4 hours, after magnetic separation, abandons supernatant, and Washing buffer cleans three times.
(3) with L37 ℃ of sealing of BSA100 μ of 0.1%, spend the night, Washing buffer cleans three times.
(4) after magnetic separation, abandon supernatant, add sample to be checked, room temperature light shaking is hatched 12 hours.Sample preparation methods to be checked is: get urine 1.5ml, and the centrifugal 5min of 12000rpm, separated supernatant adds cell pyrolysis liquid PIPA room temperature cracking 2～5min of 100 μ L in sediment urinalysis, and the centrifugal 5min of 12000rpm, gets supernatant and detects.
(5) after magnetic separation, abandon supernatant, Washing buffer cleans three times, adds the PP65 monoclonal antibody of the green fluorescence mark with Binding buffer configuration of 10ng, incubated at room 8 hours.
(6) after magnetic separation, abandon supernatant, Washing buffer cleans three times, adds the Binding buffer of 50 μ L, fluorescence microscopy Microscopic observation.Observations as shown in Figure 2, has detected PP65 antigen in experimental group, can see that red fluorescence can see green fluorescence again under fluorescent microscope.PP65 antigen in control group, do not detected, under fluorescent microscope, can only see red fluorescence, can't see green fluorescence.
The screening technique of embodiment bis-, human cytomegalovirus PP65 antigen aptamer, completes according to following steps:
One, build random single chain DNA (ssDNA) library and primer
Building length is the ssDNA library of 78 bases, and two ends are fixed sequence program, and middle 35 nucleotide are random series, and wherein N represents A, T, and C, any one in G, storage capacity is about 10 15-10 16:
5’-GGGAGCTCAGAATAAACGCTCAA-N35-TTCGACATGAGGCCCGGATC-3’。Upstream primer is 5 '-GGGAGCTCAGAATAAACGCTCAA-3 ', and downstream primer is 5 '-biotin-(CH2)-TTCGACATGAGGCCCGGATC-3 '.Random single-stranded DNA banks and primer Ke You primer Synesis Company are synthetic.
Pcr amplification and the recovery in double-stranded DNA (dsDNA) library:
PCR reaction system
Response parameter Addition (unit: μ l)
10*PCR damping fluid 2.0
MgCl2(25mmol/L) 1.2
dNTP(2.5mmol/L) 1.6
Primer 1 (10pmol/L) 1.0
Primer 2 (10pmol/L) 1.0
Template (ssDNA library) 0.1
TaqDNA polymerase 1.0
ddH2O 12.1
Cumulative volume 20.0
PCR reaction conditions
Temperature (℃) Effect Time (S)
94 Denaturation 300
94 Sex change 30
65 Annealing 30
72 Extend 30
Carry out altogether 18 circulations, last 72 ℃ are extended 5min., the product obtaining carries out next step experiment (or 4 ℃ of preservations) PCR product purification and recovery: toward the sodium acetate of 3mol/L pH5.2 and the ice-cold absolute ethyl alcohol of 2-2.5 times of volume that add 0.1 times of volume in DNA solution, on spiral mixer oscillator, vibration mixes and is placed in-20 ℃ of refrigerators and places 30min, then the centrifugal 5min of 12000rpm, abandon supernatant, 70% ethanol that adds 1ml precooling, put upside down centrifuge tube for several times, the centrifugal 5min of 12000rpm, abandon supernatant, after drying precipitation, (can be placed in baking oven 10min) is dissolved in 20 μ LTE damping fluid (pH8.0) or ddH2O, 4 ℃ of preservations are spent the night.
Pcr amplification and the recovery in single stranded DNA (dsDNA) library:
Adopt asymmetric PCR to increase,
PCR reaction system
Response parameter Addition (unit: μ l)
10*PCR damping fluid 2.0
MgCl2(25mmol/L) 0.2
dNTP(2.5mmol/L) 1.6
Primer (0.4pmol/L) 0.5
Primer 2 (10pmol/L) 2.0
Template (dsDNA library) 0.5
TaqDNA polymerase 0.4
ddH2O 12.8
Cumulative volume 20.0
PCR reaction conditions
Temperature (℃) Effect Time (S)
94 Denaturation 300
94 Sex change 30
65 Annealing 30
72 Extend 45
Carry out altogether 40 circulations, last 72 ℃ are extended 7min., and the product obtaining carries out the recovery in next step experiment (or 4 ℃ of preservations) same double-stranded DNA of recovery method (dsDNA) library.
Agarose gel electrophoresis is observed PCR result
The Ago-Gel of preparation 2%: accurately take 2g electrophoresis level agarose, add 100ml1 * TAE electrophoretic buffer, in micro-wave oven, be heated to dissolve, when gel is cooled to 55 ℃ of left and right, add ethidium bromide (final concentration is 0.5 μ g/mL) to rotate and shake up gently, pour glued membrane into, insert suitable comb, use after allowing gel solution condense.
PCR product electrophoresis: gel is placed in to electrophoresis tank, get 3 μ L PCR products and add 1 μ l6 * loading buffer (0.25% bromophenol blue, 0.25% dimethylbenzene cyanogen, 30% glycerine water solution), loading after mixing, separately get 5 μ L DL Marker, add respectively in Ago-Gel hole, in 1 * TAE electrophoretic buffer, 80V electrophoresis 15-20min observes electrophoresis result on gel imaging instrument.
Two, SELEX screening
1. albumen is coated
With the carbonate buffer solution of pH9.60.05mol/L, PP65 albumen is diluted to desired concn, adds 100 μ l in each hole of each SELEX96 orifice plate (polystyrene board), establish blank hole and anti-sieve control wells simultaneously, 4 ℃ are spent the night.Next day, discards solution in hole, with lavation buffer solution PBST, washes three times.
2. sealing
3% BSA (calf serum), 37 ℃ of sealings are spent the night for 1 hour or 4 ℃.
The combination in 3.ssDNA library
The ssDNA library 100 μ l that add SELEX binding buffer to dilute, first hatch with blank hole (or anti-sieve contrasts empty) 37 ℃, anti-sieve is removed the ssDNA with target protein non-specific binding, then transfer in the coated hole of PP65 albumen 37 ℃ and hatch, with SELEX washing buffer washing 5 times.
4. wash-out
Add SELEX eluting buffer, in 80 ℃ of water-bath effect 10min, under wash-out with protein bound ssDNA.
5. the ssDNA of phenol-chloroform extracting, the combination of ethanol deposition and purification
Toward filling, in the 1.5mL Ep pipe of DNA solution to be purified, add isopyknic phenol: chloroform: isoamylol (25:24:1 volume ratio), on spiral mixer oscillator, vibration mixes, the centrifugal 15s of room temperature 12000rpm.With liquid-transfering gun by the upper strata of containing DNA (water) careful new Ep pipe of immigration, toward the sodium acetate (NaAc) and 2～2.5 times of absolute ethyl alcohols that volume is ice-cold that add the 3mol/L pH5.2 of 0.1 times of volume in ssDNA solution, on spiral mixer oscillator, vibration mixes and is placed in-20 ℃ of 30min, then the centrifugal 5min of 12000rpm, abandon supernatant, 70% ethanol that adds 1mL precooling, put upside down centrifuge tube for several times, the centrifugal 5min of 12000rpm, abandon supernatant, after drying precipitation, be dissolved in 20 μ LTE damping fluid (PH8.0) or ddH20,4 ℃ of preservations.
SsDNA library after being screened by PCR and asymmetric PCR, then carries out electrophoresis detection.Finally carry out altogether 8 take turns screening reach capacity, obtain specificity the highest, the ssDNA library that affinity is the strongest.
Three, Cloning and sequencing
1. reclaiming product connects
The saturated library of screening is connected to pMD18-T Simple Vector(TaKaRa company).
Amplification obtains biotin labeled ssDNA library through asymmetric PCR by the 8th, to take turns the ssDNA library obtaining, by cytomegalovirus PP65 for albumen carbonate (PH9.6) damping fluid be diluted to 10 μ g/ml, get the coated elisa plate of 100 μ l, 4 ℃ are spent the night, PBST (PBS+Tween) washs 4 times, 3min/ time; 3%BSA37 ℃ is sealed 1 hour, PBST washing 4 times, 3min/ time; With the 8th of SELEX binding buffer dilution, take turns biotin labeled ssDNA0.05 μ g/ hole, 37 ℃ of incubation 60min, PBST washing 6 times, 3min/ time; 37 ℃ of the horseradish peroxidases of the marked by streptavidin of dilution in 1: 1000 are hatched 30min, PBST washing 6 times, 3min/ time; Add 37 ℃ of colour developing 15min of tetramethyl benzidine (tetramethylbenzidine, TMB) nitrite ion; 2mol/L concentrated sulphuric acid cessation reaction, microplate reader is measured A value in 450nm place.A value is greater than 0.1, reclaims product, and screening product is connected to pMD18-T simple vector (TaKaRa company).With reference to the pMD18-T Simple Vector of the TaKaRa company description of product, the genes of interest PCR product after purifying is connected with cloning vector pMD18-TSimple Vector, 16 ℃ of connections are spent the night.
2. connect the conversion of product
A: by being connected in the EP pipe that product 20 μ L add the E.coli DH5a competent cell that contains 100 μ L of genes of interest fragment and pMD18-T Simple Vector, ice bath 30min.
B: put into 42 ℃ of water-bath heat shock 1min, will manage fast and take out ice bath 2min.
C: every pipe adds SOC nutrient solution 600 μ l, 37 ℃ of shaking tables, 150rpm, cultivates 1h, makes the antibiotic resistance marker gene of bacteria resuscitation expression plasmid coding.
D: the competent cell that proper volume has been transformed is coated on the LB flat board that contains ampicillin 100 μ g/ml, and flat board is placed in to room temperature until liquid is absorbed.
E: be inverted flat board, in 37 ℃ of constant temperature culture, occur bacterium colony after 12～16h, the several bacterium colony PCR of random picking identify.
F: random 110 monoclonals of picking, add in the 1.5ml EP pipe that fills 600 μ L LB fluid nutrient mediums (containing ampicillin 100 μ g/ml) 37 ℃ of shaking tables, 200rpm, overnight incubation.Add 600 μ L(equivalent next day) 80% autoclaving glycerine, sealing orifice, preserves bacterial classification in-70 ℃.
3. the extraction of recombinant plasmid
Get 50 μ L monoclonal bacterium liquid and be inoculated in 5ml containing the LB nutrient culture media of ampicillin 100 μ g/ml, 37 ℃ of shaking tables, 250rpm, overnight incubation.With plasmid extraction kit, extract recombinant plasmid.
4.PCR identifies
The extraction plasmid of take is template, adds SELEX primer I and primer I I to carry out pcr amplification reaction, and product electrophoresis is determined positive colony, and send the order-checking of order-checking company.
The sequence of human cytomegalovirus PP65 antigen aptamer:
A4
GGGAGCTCAGAATAAACGCTCAAGGCATGGTCCTCGATCGTATGGCTTTGCGGTCGTGTT?CGACATGAGGCCCGGATC
A5
GGGAGCTCAGAATAAACGCTCAATATCCCTATTCCGCTCCATGTTGCGTACCCGTGCCTT?CGACATGAGGCCCGGATC
A6
GGGAGCTCAGAATAAACGCTCAACGGATTAGTCAATAGACTGGTGGCTTTGTGTGGTGTT?CGACATGAGGCCCGGATC
A7
GGGAGCTCAGAATAAACGCTCAATATCCCTATTCCGCTCCATGCTGCGTACCCGTGCCTT?CGACATGAGGCCCGGATC
A10
GGGAGCTCAGAATAAACGCTCAATGTGTTCATTTTTGGGGCTTGCGGGTGGTTCGCTGTT?CGACATGAGGCCCGGATC
A19
GGGAGCTCAGAATAAACGCTCAACGTCCCTCTCGTGTTGCTGCTGCTTTTTCCTGTGGTT?CGACATGAGGCCCGGATC
5. fit compatibility detects
The single clone who selects is obtained to ssDNA after SELEX primer I and the amplification of primer III asymmetric PCR, and by this ssDNA and the protein combination being coated with, enzyme-linked method is measured its compatibility.By the coated hole of every hole 1 μ g ssDNA and 2 μ g albumen in 100 μ Lselex binding buffer 37 ℃ hatch 40min; Selex washing buffer washing 5 times,, add marked by streptavidin horseradish peroxidase 100 μ L, hatch 30min for 37 ℃; Lavation buffer solution washing 5 times, then every hole adds each 50 μ L of substrate nitrite ion A, B, 37 ℃ of colour developing 15min; The 2mol/L concentrated sulphuric acid 50 μ L cessation reactions, microplate reader is measured absorbance (A) value in 450nm place, record fit compatibility as follows:
Numbering OD value Numbering OD value
A4 2.726 A10 1.271
A5 1.054 A19 1.137
A6 1.522 NC 0.067
A7 1.629 ? ?
Note: the negative contrast of NC.

Claims (4)

1. the method based on magnetic bead fast detecting PP65, comprises the following steps:
(1) in centrifuge tube, add the carboxyl modified magnetic bead of 0.5～1 μ L, MES cleans three times, adds the EDC of 80～100 μ L0.5g/L, and 37 ℃ activate 2～4 hours;
(2) what directly add 2～5 μ M HEX red fluorescence marks has specific aptamer with human cytomegalovirus PP65 antigen, and room temperature light shaking is hatched 4～6 hours, after magnetic separation, abandons supernatant, and Washing buffer cleans three times; Described aptamer is one of sequence of SEQ ID No.1～SEQ ID No.6;
(3) with a BSA80～120 μ L37 ℃ sealing of 0.1～0.3%, spend the night, Washing buffer cleans three times;
(4) after magnetic separation, abandon supernatant, add sample to be checked, room temperature light shaking is hatched 10～14 hours;
(5) after magnetic separation, abandon supernatant, Washing buffer cleans three times, adds the PP65 monoclonal antibody of the FITC green fluorescence mark with Binding buffer configuration of 8～12ng, incubated at room 6～10 hours;
(6) after magnetic separation, abandon supernatant, Washing buffer cleans three times, adds the Binding buffer of 30～70 μ L, fluorescence microscopy Microscopic observation; If there is PP65 antigen in sample to be checked, can see under the microscope that red fluorescence can see green fluorescence again; If there is no PP65 antigen in sample to be checked, can only see red fluorescence under the microscope, can not see green fluorescence.
2. a kind of method based on magnetic bead fast detecting PP65 according to claim 1, is characterized in that: the secondary conformation of the No.1～SEQ of aptamer SEQ ID described in step (2) ID No.6 respectively is:
Figure FDA0000410079400000021
Figure FDA0000410079400000031
Figure FDA0000410079400000041
3. a kind of method based on magnetic bead fast detecting PP65 according to claim 1 and 2, is characterized in that: described Binding buffer is: PBS, 5mM MgCl 2, 0.05%tween-20, pH7.5.
4. a kind of method based on magnetic bead fast detecting PP65 according to claim 1 and 2, is characterized in that: described Washing buffer is: PBS, 5mM MgCl 2, 0.01%tween-20, pH7.5.
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CN104946655A (en) * 2015-07-01 2015-09-30 上海市肺科医院 DNA aptamer of mycobacterium tuberculosis standard strain H37Rv and preparation method thereof
CN106226513A (en) * 2016-08-02 2016-12-14 北京乐普医疗科技有限责任公司 A kind of method of immunomagnetic beads detection by quantitative related antigen and application thereof
CN108950060A (en) * 2017-09-06 2018-12-07 武汉中科志康生物科技有限公司 A kind of microorganism detection method
CN111394427A (en) * 2020-04-22 2020-07-10 宁波市博坤生物科技有限公司 Method for extracting trace DNA in soil-containing test material
CN112067802A (en) * 2019-05-25 2020-12-11 首都师范大学 H1N1 influenza virus detection method and kit thereof

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