CN109913562A - Based on PCR-gold magnetic nano immune chromatography mycoplasma pneumoniae detection method - Google Patents
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Abstract
The invention discloses based on PCR-gold magnetic nano immune chromatography mycoplasma pneumoniae detection methods, specifically carry out as steps described below: being modified with the specific primer of digoxin and biotin for the 16S rRNA nucleic acid sequence design of mycoplasma pneumoniae；It extracts the DNA of sample to be tested, using the DNA of sample to be tested as template, PCR amplification is carried out as primer using specific primer and obtains sample to be tested MP target fragment amplification product, the amplified production of acquisition detects golden magnetic immuno-chromatographic test paper strip using mycoplasma pneumoniae and detected.It is easy to operate the present invention is based on the mycoplasma pneumoniae detection method of PCR- gold magnetic nano immune chromatography, it can quickly detect mycoplasma pneumoniae.
Description
Technical field
The invention belongs to molecular biological detection technical fields, are related to a kind of pneumonia of based on PCR-gold magnetic nano immune chromatography
Detection of mycoplasma method.
Background technique
Mycoplasma pneumoniae (Mycoplasma pneumoniae, MP) is a kind of common infection in respiratory system pathogen, is
One of community acquired pneumonia (Community acquired pneumonia, CAP) most important pathogen.Mycoplasma pneumoniae
Infect clinical manifestation and do not have characteristic, be easy and other viral, bacteriums caused by respiratory tract infection mutually obscure, and often with it is other
Pathogen is mixed infection, therefore the clinical detection method for needing one kind sensitive, quick, special is former to make a definite diagnosis pneumonia branch in early days
Body pneumonia reduces so as to timely, correctly taking drugs and prevents drug resistance.
The method of mycoplasma pneumoniae detection at present is mainly separately cultured, serology antibody test and PCR are diagnosed.But lung
Scorching mycoplasma, which is separately cultured when being detected, needs to expend longer time, and condition of culture is harsh, and sensitivity is not high, for facing
Bed diagnostic significance is little；And carrying out serum science antibody test at present mainly has two major classes: one kind is serum pneumonia mycoplasma
Body IgM or IgG detection, one kind are mycoplasma pneumoniae nucleic acid (MP-DNA) detections.Serologic detection is simple and efficient, clinically
To being widely applied.But this method only can provide preferable Testing index to previous infection, and to Current Infection examination
Application value is lower,
Quantitative fluorescent PCR is most common method in MP-DNA detection, but this method is complicated for operation, and there are fluorescence spies
The problems such as needle, expensive instrument price, many clinical units, base cannot still carry out.Key is detected for MP so studying and developing
Technology and coherent detection product have major clinical significance.
Summary of the invention
The object of the present invention is to provide a kind of based on PCR-gold magnetic nano immune chromatography mycoplasma pneumoniae detection method, behaviour
Make simply, can quickly detect mycoplasma pneumoniae.
The technical scheme adopted by the invention is that based on PCR-gold magnetic nano immune chromatography mycoplasma pneumoniae detection side
Method specifically carries out as steps described below:
Step 1, the special of digoxin and biotin is modified with for the 16S rRNA nucleic acid sequence design of mycoplasma pneumoniae
Property primer；Extract the DNA of sample to be tested；
Step 2, using the DNA of sample to be tested as template, PCR amplification is carried out as primer using specific primer and obtains sample to be tested
DNA cloning product；
Step 3, sample to be tested DNA cloning product golden magnetic immuno-chromatographic test paper strip is detected using mycoplasma pneumoniae to examine
It surveys.
The features of the present invention also characterized in that:
Specific primer are as follows:
MP-F:5 '-Digoxin-AAGGACCTGCAAGGGTTGT-3 '；
MP-R:5 '-Biotin-CTCTAGCCATTACCTGCTAA-3 '.
PCR amplification program in step 2 are as follows:
50 DEG C of 2min of UNG enzyme；95 DEG C of initial denaturation 5min；94 DEG C of denaturation 30s, 54 DEG C of annealing 30s, 72 DEG C of extension 1min, into
Row 30 circulations；72 DEG C of fully extended 1min.
When carrying out PCR amplification, according to volume fraction, 10% 10 × Taq buffer, 8% d NTP is added to PCR pipe
(A:U:C:G), 1% HotMaster Taq DNA enzymatic, 1% UNG enzyme, 4%~10% specific primer, 2%~10%
Template and 60%~74% dd H2O。
It includes PVC offset plate that mycoplasma pneumoniae, which detects golden magnetic immuno-chromatographic test paper strip, on PVC offset plate upper surface it is linear successively
It is connected with loading pad, bonding pad, chromatographic film and blotting paper；Loading pad is bonded in conjunction with pad part, bonding pad and chromatography membrane part
Fitting, chromatographic film are bonded with blotting paper part；The coated golden magnetic nanometer particle of DigiTAb, chromatographic film are coated on bonding pad
It is upper to be embedded with detection line T and nature controlling line C, it is coated with Streptavidin on detection line T, sheep anti-mouse igg is coated on nature controlling line C, is examined
Survey line T is located at the bonding pad of immune gold-magnetic particles label.
The material of loading pad and bonding pad is hydrophilic polyester fiber, and chromatographic film is cellulose nitrate film.
It is specific in step 3 to carry out by the following method:
Step 3.1, the sample to be tested DNA cloning product that step 2 obtains is added dropwise on loading pad；
Step 3.2, observation mycoplasma pneumoniae detects the nature controlling line on golden magnetic immuno-chromatographic test paper strip；
If chromogenic reaction occurs for nature controlling line, observe whether detection line occurs chromogenic reaction, if colour developing occurs for detection line instead
It answers, then illustrates that sample to be tested has mycoplasma pneumoniae infection, be as a result positive；If detection line does not develop the color, illustrate that sample to be tested does not have
There is mycoplasma pneumoniae infection, is as a result negative；
If there is no chromogenic reactions for nature controlling line, is detected again to nature controlling line and chromogenic reaction occurs.
The beneficial effects of the invention are as follows
A kind of based on PCR of the present invention-gold magnetic nano immune chromatography mycoplasma pneumoniae detection method, using PCR to MP spy
Anisotropic target gene is identified and is expanded, using amplified fragments label digoxin and biotin respectively with gold-magnetic particles surface
Strepto- parent interaction on DigiTAb and test strips detection line is realized in conjunction with lateral flow technologies to MP target gene
Identification.After completing target gene amplification, directly amplified production point can with the naked eye be sentenced after a few minutes, not needed in loading pad
Any valuableness detecting instrument and complicated operating process.
Detailed description of the invention
Fig. 1 is that mycoplasma pneumoniae (MP) detects golden magnetic immuno-chromatographic test paper strip structural schematic diagram；
Fig. 2 is that the present invention is based on detect in the mycoplasma pneumoniae detection method embodiment 1 of PCR- gold magnetic nano immune chromatography
Result figure；
Fig. 3 is that the present invention is based on the detections of the mycoplasma pneumoniae detection method embodiment 2 of PCR- gold magnetic nano immune chromatography
Result figure.
In figure, 1.PVC offset plate, 2. loading pads, 3. bonding pads, 4. chromatographic films, 5. blotting papers.
Specific embodiment
The following describes the present invention in detail with reference to the accompanying drawings and specific embodiments.
Based on PCR-gold magnetic nano immune chromatography mycoplasma pneumoniae detection method, specifically carries out as steps described below:
Step 1, the special of digoxin and biotin is modified with for the 16S rRNA nucleic acid sequence design of mycoplasma pneumoniae
Property primer:
MP-F:5 '-Digoxin-AAGGACCTGCAAGGGTTGT-3 '；
MP-R:5 '-Biotin-CTCTAGCCATTACCTGCTAA-3 '；
Extract the DNA of sample to be tested；
Step 2, using the DNA of sample to be tested as template, PCR amplification is carried out as primer using specific primer and obtains sample to be tested
DNA cloning product specifically carries out by the following method:
Step 2.1,10% 10 × Taq buffer, 8% d NTP (A:U:C:G), 1% is added to PCR pipe
HotMaster Taq DNA enzymatic, 1% UNG enzyme, 4%~10% specific primer, 2%~10% template and 60%~
74% dd H2O；
Step 2.2, PCR amplification, PCR amplification program are carried out are as follows:
50 DEG C of 2min of UNG enzyme；95 DEG C of initial denaturation 5min；94 DEG C of denaturation 30s, 54 DEG C of annealing 30s, 72 DEG C of extension 1min, into
Row 30 circulations；72 DEG C of fully extended 1min；
Step 3, sample to be tested DNA cloning product step 2 obtained detects golden magnetic immunochromatography using mycoplasma pneumoniae
Test strips are detected:
Wherein, as shown in Figure 1, it includes PVC offset plate 1, PVC offset plate 1 that mycoplasma pneumoniae, which detects golden magnetic immuno-chromatographic test paper strip,
It is that hydrophilic polyester is fine that the loading pad 2, material that material is hydrophilic polyester fiber are linearly successively connected on upper surface
The bonding pad 3 of the immune gold-magnetic particles label of dimension, the chromatographic film 4 and blotting paper 5 that material is cellulose nitrate film；Loading pad 2
It is bonded with 3 part of bonding pad of immune gold-magnetic particles label, the bonding pad 3 and 4 part of chromatographic film that gold-magnetic particles label is immunized paste
It closes, chromatographic film 4 is bonded with 5 part of blotting paper；It is coated that DigiTAb is coated on the bonding pad 3 of immune gold-magnetic particles label
Golden magnetic nanometer particle is embedded with detection line T and nature controlling line C in chromatographic film 4, is coated with Streptavidin, nature controlling line C on detection line T
On be coated with sheep anti-mouse igg, detection line T is located at the bonding pad of immune gold-magnetic particles label.
Golden magnetic immuno-chromatographic test paper strip is detected using mycoplasma pneumoniae and detects target fragment amplification product, specifically according to following sides
Method carries out:
Step 3.1, the sample to be tested DNA cloning product that step 2 obtains is added dropwise on loading pad 2；
Step 3.1, the sample to be tested DNA cloning product that step 2 obtains is added dropwise on loading pad；
Step 3.2, observation mycoplasma pneumoniae detects the nature controlling line on golden magnetic immuno-chromatographic test paper strip；
If chromogenic reaction occurs for nature controlling line, observe whether detection line occurs chromogenic reaction, if colour developing occurs for detection line instead
It answers, then illustrates that sample to be tested has mycoplasma pneumoniae infection, be as a result positive；If detection line does not develop the color, illustrate that sample to be tested does not have
There is mycoplasma pneumoniae infection, is as a result negative；
If there is no chromogenic reactions for nature controlling line, is detected again to nature controlling line and chromogenic reaction occurs.
In the mycoplasma pneumoniae detection method chromatographed the present invention is based on PCR- gold magnetic nano immune, target segment is expanded
When increasing, if there are mycoplasma pneumoniae target segments in sample to be tested, to be directed to the 16S rRNA nucleic acid sequence of mycoplasma pneumoniae
The primer of design enables to the target segment in sample to be tested to carry out PCR amplification, to obtain being modified with digoxin and biotin
Sample to be tested DNA cloning product；
And the sample to be tested DNA cloning product for being modified with digoxin and biotin is immune using the golden magnetic of mycoplasma pneumoniae detection
When chromatograph test strip is detected, digoxin in sample to be tested DNA cloning product can in chromatographic film 4 be modified with ground
The nanoparticle of digoxin antibody (Anti-Digoxin antibody) forms compound, and liquid phase is reached by chromatographic flow and detected
When at line T, the pneumonia branch that one end is marked with Biotin by fixed Streptavidin (streptavidin, SA) at detection line is former
Body trapping nucleic acids make a part of golden magnetic Nano Composite Particles (Biotin-DNA-Digoxin-Anti-Digoxin antibody-
Nanometer gold magnetic particle) it rests at detection line, red band is generated, to detect to contain mycoplasma pneumoniae in sample to be tested
Target segment；
The IgG on nature controlling line C in chromatographic film 4 can capture extra nanometer gold magnetic particle, so that nature controlling line C is shown
Red stripes, to carry out Quality Control to test strips.
If mycoplasma pneumoniae is not present in sample to be tested, to be designed for the 16S rRNA nucleic acid sequence of mycoplasma pneumoniae
Primer cannot smoothly complete PCR amplification, to be unable to get the mycoplasma pneumoniae target gene for being modified with digoxin and biotin
Amplified production；When reusing mycoplasma pneumoniae and detecting golden magnetic immuno-chromatographic test paper strip and detected, nanometer gold magnetic particle can not stop
Detection line is stayed in, chromogenic reaction does not occur for detection line T.And the extra golden magnetic nano-probe (Jin Cina of coating DigiTAb
Rice particle) finally captured by the sheep anti-mouse igg on nature controlling line C, so that nature controlling line C is equally developed the color.
It is the nanometer being combined with gold and magnetic material using nanometer gold magnetic particle as marker material in the present invention
Composite material, kernel are superparamagnetic material, and surface covering material is gold.It combines colloidal gold can efficient conjugated biological molecules
With the double dominant of magnetic nano-particle superparamagnetism, it is not necessarily to covalent coupling reagent, using Electrostatic Absorption by anti digoxin antibody
It is coupled to its surface and forms the immune gold-magnetic particles of anti digoxin antibody.
The present invention establishes based on PCR-gold magnetic nano immune chromatography mycoplasma pneumoniae detection method, in conjunction with PCR amplification
Technology and nano immune probe identification technology, realize that the nucleic acid of MP quickly detects.Golden magnetic nanometer is coated with using DigiTAb to visit
The distinctive visualization characteristic of needle, " biotin-Streptavidin " and " digoxin-DigiTAb " effect, to PCR amplification
Product completes visualizing monitoring.After the PCR amplification for completing clinical sample, without purifying, amplified production is dripped in chromatographic apparatus
Detection can be completed without 5-10 minutes in loading wells, did not needed any expensive detecting instrument and complicated operating process.And carry out
Preliminary clinical application research establishes certain basis for the epidemiological survey and basic hospital physical examination of mycoplasma pneumoniae, is
Clinician's rational use of medicines provides guidance.
Embodiment 1
1. patient's sputum sample that sample range is suitable for Diagnosis of Suspected Pneumonia mycoplasma infection.Use appropriate 10:1TE buffer
(PH 8.0) liquefaction sputum, vibrates 30S, and 10000rpm is centrifuged 10min, discards supernatant liquid, extracts template with DNA extraction kit
DNA or addition 20-40 μ L tri-distilled water boil 15min, and 10000-13000rpm is centrifuged 5min, takes mould of the supernatant as augmentation detection
Plate liquid is spare.
2. taking PCR centrifuge tube, each reagent is added according to following table
3. PCR pipe is put into PCR amplification instrument, expanded according to the procedure below:
50 DEG C of 2min of UNG enzyme；95 DEG C of initial denaturation 5min；94 DEG C of denaturation 30s, 54 DEG C of annealing 30s, 72 DEG C of extension 1min, into
Row 30 circulations；72 DEG C of fully extended 1min.
4 are added drop-wise to the amplified production of PCR pipe on the loading pad of test strips, as a result as shown in Figure 2.Middle Quality Control according to fig. 2
The interpretation that develops the color occurs for line and detection line, which is that mycoplasma pneumoniae is positive.
Embodiment 2
1. patient's throat swab sample that sample range is suitable for Diagnosis of Suspected Pneumonia mycoplasma infection.It is buffered using appropriate 10:1TE
Liquid (PH 8.0) impregnates throat swab cotton swab, and cotton swab is discarded after repeatedly extruding, and vibrates 30S, and 10000rpm centrifugation 10 is extracted with DNA
Kit, which extracts template DNA or 20-40 μ L tri-distilled water is added, boils 15min, and 10000-13000rpm is centrifuged 5min, supernatant is taken to make
It is spare for the template liquid of augmentation detection.
2. taking PCR centrifuge tube, each reagent is added according to following table
3. PCR pipe is put into PCR amplification instrument, expanded according to the procedure below:
50 DEG C of 2min of UNG enzyme；95 DEG C of initial denaturation 5min；94 DEG C of denaturation 30s, 54 DEG C of annealing 30s, 72 DEG C of extension 1min, into
Row 30 circulations；72 DEG C of fully extended 1min.
4 are added drop-wise to the amplified production of PCR pipe on the loading pad of test strips, as a result as shown in Figure 3.It is aobvious according to T line in Fig. 3
Color interpretation, the sample are that mycoplasma pneumoniae is negative, illustrate do not have mycoplasma pneumoniae in this sample.
All DNA sample in embodiment 1 and embodiment 2 and blank control are subjected to the species specific 16S rRNA gene of MP
PCR amplification carries out electrophoresis or gene sequencing is confirmed, the pneumonia branch all chromatographed with PCR- gold magnetic nano immune of the invention is former
Body detecting method result is consistent.
Claims (7)
1. based on PCR-gold magnetic nano immune chromatography mycoplasma pneumoniae detection method, which is characterized in that specifically according to following steps
It is rapid to carry out:
Step 1, the specificity for being modified with digoxin and biotin for the 16S rRNA nucleic acid sequence design of mycoplasma pneumoniae is drawn
Object；Extract the DNA of sample to be tested；
Step 2, using the DNA of sample to be tested as template, PCR amplification is carried out as primer using the specific primer and obtains sample to be tested
DNA cloning product；
Step 3, the sample to be tested DNA cloning product golden magnetic immuno-chromatographic test paper strip is detected using mycoplasma pneumoniae to examine
It surveys.
2. based on PCR according to claim 1-gold magnetic nano immune chromatography mycoplasma pneumoniae detection method, feature
It is, the specific primer are as follows:
MP-F:5 '-Digoxin-AAGGACCTGCAAGGGTTGT-3 '；
MP-R:5 '-Biotin-CTCTAGCCATTACCTGCTAA-3 '.
3. based on PCR according to claim 1-gold magnetic nano immune chromatography mycoplasma pneumoniae detection method, feature
It is, PCR amplification program in the step 2 are as follows:
50 DEG C of 2min of UNG enzyme；95 DEG C of initial denaturation 5min；94 DEG C of denaturation 30s, 54 DEG C of annealing 30s, 72 DEG C of extension 1min carry out 30
A circulation；72 DEG C of fully extended 1min.
4. based on PCR according to claim 3-gold magnetic nano immune chromatography mycoplasma pneumoniae detection method, feature
Be, when carrying out PCR amplification, according to volume fraction, to PCR pipe be added 10% 10 × Taq buffer, 8% d NTP (A:
U:C:G), 1% HotMaster Taq DNA enzymatic, 1% UNG enzyme, 4%~10% specific primer, 2%~10%
Template and 60%~74% dd H2O。
5. based on PCR according to claim 1-gold magnetic nano immune chromatography mycoplasma pneumoniae detection method, feature
It is, it includes PVC offset plate (1) that the mycoplasma pneumoniae, which detects golden magnetic immuno-chromatographic test paper strip, PVC offset plate (1) upper surface
It is upper to be linearly successively connected with loading pad (2), bonding pad (3), chromatographic film (4) and blotting paper (5)；The loading pad (2) and knot
It closes pad (3) part to be bonded, the bonding pad (3) is bonded with chromatographic film (4) part, the chromatographic film (4) and blotting paper (5) part
Fitting；It is coated with the coated golden magnetic nanometer particle of DigiTAb on the bonding pad (3), is embedded with inspection on the chromatographic film (4)
It is coated with Streptavidin on survey line T and nature controlling line C, the detection line T, sheep anti-mouse igg, the inspection are coated on nature controlling line C
Survey line T is at the bonding pad (3) of immune gold-magnetic particles label.
6. with the based on PCR described in claim 5-gold magnetic nano immune chromatography mycoplasma pneumoniae detection method, feature exists
In the material of the loading pad (2) and bonding pad (3) is hydrophilic polyester fiber, and the chromatographic film (4) is cellulose nitrate
Plain film.
7. based on PCR according to claim 5-gold magnetic nano immune chromatography mycoplasma pneumoniae detection method, feature
It is, specific in the step 3 to carry out by the following method:
Step 3.1, the sample to be tested DNA cloning product that step 2 obtains is added dropwise on loading pad；
Step 3.2, observation mycoplasma pneumoniae detects the nature controlling line on golden magnetic immuno-chromatographic test paper strip；
If chromogenic reaction occurs for nature controlling line, observe whether detection line occurs chromogenic reaction, if chromogenic reaction occurs for detection line,
Illustrate that sample to be tested has mycoplasma pneumoniae infection, is as a result positive；If detection line does not develop the color, illustrate that sample to be tested does not have pneumonia
As a result mycoplasma infection is negative；
If there is no chromogenic reactions for nature controlling line, is detected again to nature controlling line and chromogenic reaction occurs.
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