CN106520993A - Florescent real-time quantitative PCR method for rapidly detecting first component content of dermatophagoides farinae allergen in house dust - Google Patents
Florescent real-time quantitative PCR method for rapidly detecting first component content of dermatophagoides farinae allergen in house dust Download PDFInfo
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- CN106520993A CN106520993A CN201611137521.7A CN201611137521A CN106520993A CN 106520993 A CN106520993 A CN 106520993A CN 201611137521 A CN201611137521 A CN 201611137521A CN 106520993 A CN106520993 A CN 106520993A
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Abstract
The method discloses a florescent real-time quantitative PCR method for rapidly detecting a first component content of dermatophagoides farinae allergen in house dust. According to the method, Der f 1 recombinant plasma is taken as a standard substance, a florescent real-time quantitative PCR and a TaqMan probe of the Der f 1 are designed; and by virtue of the TaqMan probe florescent real-time quantitative PCR method, the rapid quantitative detection of the Der f 1 content of the dermatophagoides farinae allergen in the house dust is achieved by constructing a Der f 1 florescent real-time quantitative PCR standard curve. The method provided by the invention detects a same house dust sample respectively by virtue of the environment-friendly and cheap Der f 1 florescent real-time quantitative PCR method and a commercial and expensive ELISA kit, and a correlation coefficient R between the two methods is equal to 0.94, wherein P is less than 0.05, showing that the two methods keep a good linear relation.
Description
Technical field
The present invention relates to Allergic skin test technical field, and in particular to dust mite allergen in a kind of dirt of quick detection room
The method of the fluorescence real-time quantitative PCR of 1 constituent content.
Background technology
At present, China's indoor environment dust mite allergen content detection popularization degree is not high, clinically mainly to the patient that goes to a doctor
Which is diagnosed by vitro method such as specific IgE antibodies in the vivo approaches such as skin test or detection patients serum, or profit
The Allergic skin test related to medical history is carried out to patient with noctovisor scan detecting system.But these means can only all play diagnosis
Effect, can not play a preventive effect.Since eighties of last century eighties, Chinese scholars successively establish radioanaphylaxis
Former adsorption test (Radioallergosorbent Test, RAST) inhibition test, radioimmunoassay
(Radioimmunoassay, RIA), ELISA double antibody sandwich methods are used for quantitative determining the dust mite allergen content in the dirt of room.
RAST inhibition tests determine the content of dust mite allergen in air, but RAST inhibition tests are the inspections to dust mite allergen gross activity
Survey, can not detect the concentration of various allergenic components in dust mite respectively, and its expense costly with there is environmental pollution.Put
Penetrate immunoassay (RIA) method and determine dust mite (Dermatophagoides farina, Der f) and dermatophagoides pteronyssinuses
The total antigen (antigen P1) of (Dermatophagoides pteronyssinus, Der p) I group anaphylactogens, it is right to realize
Dust mite carries out typing, overcome RAST methods can not detect dust mite different groups allergen content defect, but due to it
Equally existing radiosiotope has the shortcomings that half-life and pollution environment.The double antibody sandwich method of ELISA is widely used, but its
Somewhat expensive, detection speed are slow, are used for scientific research, are popularized in sensing chamber's dirt in a large number in being not particularly suited for Epidemiological study
Dust mite allergen content.
The content of the invention
For the deficiencies in the prior art, it is an object of the invention to provide dust mite allergen in a kind of dirt of quick detection room
The method of the fluorescence real-time quantitative PCR of the 1st constituent content, the method detection speed is fast, price is low, environment friendly and pollution-free.
For solving prior art problem, the technical scheme taken of the present invention is：
The method of the fluorescence real-time quantitative PCR of the 1st constituent content of dust mite allergen in a kind of dirt of quick detection room, using Der
f 1（The 1st component of dust mite allergen）Recombiant plasmid as standard substance, design Der f 1 real-time fluorescence quantitative PCR primer,
TaqMan probe, using real-time fluorescence TaqMan probe quantifying PCR method, by constructing 1 real-time fluorescence quantitative PCRs of Der f
Standard curve, realizes the Quantitative detection to 1 contents of dust mite allergen Der f in house dust.
Used as preferably, the parameter of real-time fluorescence TaqMan probe quantifying PCR method is：95 DEG C of denaturations 1min;
95 DEG C of degeneration 15s;60 DEG C of annealing and extension 1min, period 40.
As preferably, the coefficient R of 1 real-time fluorescence quantitative PCR standard curves of Der f2=0.98, amplification efficiency
E=1.216, slope of standard curve are -2.893, and intercept is 32.677, and calibration curve equation is y=-2.893x+32.677.
Beneficial effect
It is convenient that the inventive method is suitable for, fast, dust mite allergen content in Quantitative Monitoring environment, and as a result accurately, bootable crowd
Active avoidance anaphylactogen, so as to preventing dust mite allergy disease has extremely strong practical value.The proposition of the inventive method,
For realize with the dust mite allergen detection by quantitative standardization of products of China's independent intellectual property right, commercialization provide science according to
According to.
Description of the drawings
Real-time fluorescence quantitative PCR standard curves of the Fig. 1 for Der f 1.
Fig. 2 is the comparative result figure that the inventive method and ELISA determine Der f 1.
Specific implementation step
It is prepared by 1 Der f of embodiment, 1 plasmid standards
According to 1 nucleotide sequences of the 1st component Der f of the dust mite allergen design primer that GenBank AB034946 are announced, and
Add BamH I/Xho I restriction enzyme sites, synthetic primer forward primer F：5′-GGATCCATGAAATTCGTTTTGGCCATTG-3′
(SEQ ID NO:1), downstream primer R0：5′-TC GCAAGAGTAGTTGTTTTTAT-3′(SEQ ID NO:2), downstream primer
R：5′-CTCGAGTCACATGATT ACAACATATGGATATT-3′(SEQ ID NO:3).Under anatomical lens, picking dust mite is about
600, after homogenate, Total RNA are extracted with RNAiso Reagent test kits；With Total RNA as template, closed with RT-PCR
Into cDNA；Above-mentioned PCR primer is reclaimed, plus after " A " tail, DNA are refined, after genes of interest fragment is connected with pMD19-T carriers,
With 1 recons of BamH I and Xho I double digestions identification pMD19-T-Der f, the genes of interest that sequencing checking is obtained.With
After TaKaRa DNA Ligation Kit are by genes of interest fragment and pET28a (+) connection, engineering bacteria BL21 (DE3) is proceeded to,
Addition IPTG abduction deliverings are after SDS-PAGE and Western blotting identification recombiant proteins, pure using affinity column chromatography method
Change and obtain recombiant protein.The synthesis of 1 encoding genes of Der f and PCR amplifications is subsequently carried out, 5 ml PCR primer loading electrophoresis is taken, is seen
Examine purpose band.Above-mentioned PCR primer is reclaimed, plus after " A " tail, DNA are refined, genes of interest fragment is connected with pMD19-T simple
After connecing, connection product transformed competence colibacillus are takenE.coliJM109, coats on the LB flat boards containing ampicillin (100 mg/ml),
37 DEG C of overnight incubations.From on the LB flat boards containing ampicillin, 16 white colonies of random picking, are entered using the primer on carrier
Row agarose gel electrophoresis after performing PCR amplification, the bacterium colony containing purpose band are positive colony bacterium.Take positive colony bacterium and be placed in and contain
In the TB culture fluid of ampicillin, 37 DEG C of shaking overnight incubations.Recombinant clone is taken, plasmid DNA is extracted, is usedBamH I
WithXho1 recons of I double digestions identification pMD19T-Der f.The size of endonuclease bamhi is detected with 1.0% agarose gel electrophoresiies.
With recombinant plasmid dna as template, sequencing, recombiant plasmid that is sequencing is correct and correctly can expressing are diluted as plasmid mark
Quasi- product are standby.
The real-time fluorescence quantitative PCR primer of 2 Der f 1 of embodiment, TaqMan probe design：
Using 2.0 softwares of Primer Express and TaqMan probe design principle is followed, according to GenBank (AB034946)
1 fluorescence quantification PCR primers of sequential design Der f and TaqMan probes.It is shown in Table 1.
1 Der f of table, 1 primers and probe sequence.
The real-time fluorescence quantitative PCR standard curve of 3 Der f 1 of embodiment
By sequencing identification be Der f 1 recombinate positive plasmid Plasmid samples on DNA/RNA quantitative instruments determination sample plasmid it is dense
Degree, calculates plasmid copy number according to the computing formula of document report.By Der f 1 recombinate positive plasmid do 10 times be incremented by dilution,
Respectively with 5.0 × 108-5.0×101Copies/ μ L8 gradients are template, according to reactant on real-time fluorescence quantitative PCR instrument
System and response parameter are reacted, and draw standard curve, and the coefficient of determination R of record standard curve2, amplification efficiency E, slope K,
And then set up regression equation.Plasmid copy number（copies/μL）=（Recombiant plasmid concentration ng/ μ L × 10-9） ×6.02×1023/
（660 × recombiant plasmid base number）.
Real-time fluorescence quantitative PCR detects program:In a certain order, toward 0.1ml PCR pipes, sequentially add such as the following group
Part：10 μ L 2X Realtime PCR Master Mix, 1 μ L dilution Der f, 1 plasmid standards, 2 μ L primed probe MIX (
F/ R/ P are each 10 μM), 7 μ L, 0.1% DEPC water, 20 μ L of total system.Real-time fluorescence quantitative PCR journey is implemented using following program
Sequence, 95 DEG C of denaturations 1min;95 DEG C of degeneration 15s;60 DEG C of annealing and extension 1min, period 40.
Using the standard curve that quantitative real time PCR Instrument is drawn according to the template of known copy, as shown in figure 1, obtaining related
Coefficients R2=0.98, amplification efficiency E=1.216, slope of standard curve（Slope）For -2.893, intercept is 32.677.Der f 1
Real-time fluorescence quantitative PCR calibration curve equation is y=-2.893x+32.677.
The real-time fluorescence quantitative PCR repeatability of 4 Der f 1 of embodiment and reliability
1 plasmid standards of Der f do 10 times of doubling dilutions, and each sample repeats 3 batches (repeating between group), each batch weight
Multiple 3 multiple holes (repeating in group), 2 recombiant plasmid concentration of Der f are that 5.0 × 108copies/ μ L and sterilized water are carried out 3 times respectively
Duplicate detection, obtains average and the coefficient of variation of respective ct values respectively, and the repeatability and reliability of standard curve are carried out
Evaluate.The replica test of 1 standard curves of Der f the results are shown in Table 2, in same template difference dilution factor group during replication its
Between 0.92%-1.76%, between group, during replication, its CV value, between 0.73%-1.88%, is below 2%, shows Der CV values
The real-time fluorescence quantitative PCR of f 1 is reproducible.With the positive plasmid and sterilized water of Der f 2 as control, song is not amplified
Line, shows that methods and resultses reliability is high.
2 Der f of table, 1 real-time fluorescence quantitative PCRs repeatability and reliability test result.
The real-time fluorescence quantitative PCR of Der f 1 and ELISA correlation results in 5 Room dirt sample of embodiment
In the dirt extracting solution of room, the concentration of Der f 1 is determined with double antibody sandwich ELISA, and concrete operation step is pressed test kit and said
Bright book carries out (1 ELISA kit of Der f (6A8/4C1, EL-DF1, Indoor company).To gathering room dirt sample application
The real-time fluorescence quantitative PCR of Der f 1 detects respectively that with ELISA kit Fig. 2 shows, two methods testing result phase relation
Number R=0.94,P<0.05, it is seen that two methods linear relationship is good.
It can be seen from the results above that the inventive method Detection results are good, as a result accurately, with good market prospect.
SEQUENCE LISTING
<110>Yancheng Health Vocational ＆ Technical College
<120>The method of the fluorescence real-time quantitative PCR of the 1st constituent content of dust mite allergen in a kind of dirt of quick detection room
<130>
<160> 6
<170> PatentIn version 3.3
<210> 1
<211> 28
<212> DNA
<213>Artificial sequence
<400> 1
ggatccatga aattcgtttt ggccattg 28
<210> 2
<211> 22
<212> DNA
<213>Artificial sequence
<400> 2
tcgcaagagt agttgttttt at 22
<210> 3
<211> 32
<212> DNA
<213>Artificial sequence
<400> 3
ctcgagtcac atgattacaa catatggata tt 32
<210> 4
<211> 20
<212> DNA
<213>Artificial sequence
<400> 4
tcaattcggt taacgttcca 20
<210> 5
<211> 20
<212> DNA
<213>Artificial sequence
<400> 5
cttgcatacg gattggagtg 20
<210> 6
<211> 25
<212> DNA
<213>Artificial sequence
<400> 6
cgcagtgatc gtaaatccaa ttccg 25
Claims (3)
1. in a kind of dirt of quick detection room the fluorescence real-time quantitative PCR of the 1st constituent content of dust mite allergen method, its feature
It is, by the use of 1 recombiant plasmid of Der f as standard substance, to design the real-time fluorescence quantitative PCR primer of Der f 1, TaqMan and visit
Pin, it is using real-time fluorescence TaqMan probe quantifying PCR method, bent by constructing 1 real-time fluorescence quantitative PCR standards of Der f
Line, realizes the Quantitative detection to 1 contents of dust mite allergen Der f in house dust.
2. in the dirt of quick detection room according to claim 1 the 1st constituent content of dust mite allergen fluorescence real-time quantitative
The method of PCR, it is characterised in that the parameter of real-time fluorescence TaqMan probe quantifying PCR method is：95 DEG C of denaturations 1min;
95 DEG C of degeneration 15s；60 DEG C of annealing and extension 1min, period 40.
3. in the dirt of quick detection room according to claim 1 the 1st constituent content of dust mite allergen fluorescence real-time quantitative
The method of PCR, it is characterised in that the coefficient R of 1 real-time fluorescence quantitative PCR standard curves of Der f2=0.98, amplification efficiency
E=1.216, slope of standard curve are -2.893, and intercept is 32.677, and calibration curve equation is y=-2.893x+32.677.
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| CN201611137521.7A CN106520993A (en) | 2016-12-12 | 2016-12-12 | Florescent real-time quantitative PCR method for rapidly detecting first component content of dermatophagoides farinae allergen in house dust |
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| CN106520993A true CN106520993A (en) | 2017-03-22 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113817838A (en) * | 2021-08-31 | 2021-12-21 | 皖南医学院 | Dust mite microsatellite marker, primer and application thereof, and primer acquisition method |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6423837B1 (en) * | 1990-02-13 | 2002-07-23 | Immulogic Pharmaceutical Corp. | Cloning and sequencing of allergens of dermatophagoides house dust mite |
| WO2004005334A2 (en) * | 2002-07-05 | 2004-01-15 | Stallergenes Sa | Nucleic acid molecule encoding an allergen preprotein of a mite of genus dermatophagoides or euroglyphus and use thereof for protecting said allergen in plants |
| CN1597985A (en) * | 2004-07-16 | 2005-03-23 | 深圳大学 | Process for testing gene of allergen in dust mite from dust of screening net in air conditioner |
-
2016
- 2016-12-12 CN CN201611137521.7A patent/CN106520993A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6423837B1 (en) * | 1990-02-13 | 2002-07-23 | Immulogic Pharmaceutical Corp. | Cloning and sequencing of allergens of dermatophagoides house dust mite |
| WO2004005334A2 (en) * | 2002-07-05 | 2004-01-15 | Stallergenes Sa | Nucleic acid molecule encoding an allergen preprotein of a mite of genus dermatophagoides or euroglyphus and use thereof for protecting said allergen in plants |
| CN1597985A (en) * | 2004-07-16 | 2005-03-23 | 深圳大学 | Process for testing gene of allergen in dust mite from dust of screening net in air conditioner |
Non-Patent Citations (1)
| Title |
|---|
| TAKAOMI YASUHARA等: "Cloning and expression of cDNA encoding the complete prepro-form of an isoform of Der f 1, the major group 1 allergen from house dust mite Dermatophagoides farinae", 《BIOSCIENCE, BIOTECHNOLOGY, AND BIOCHEMISTRY》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113817838A (en) * | 2021-08-31 | 2021-12-21 | 皖南医学院 | Dust mite microsatellite marker, primer and application thereof, and primer acquisition method |
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