CN1834655A - Method of detecting etiology by tacteriophage immunity PCR - Google Patents
Method of detecting etiology by tacteriophage immunity PCR Download PDFInfo
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- CN1834655A CN1834655A CN 200610018858 CN200610018858A CN1834655A CN 1834655 A CN1834655 A CN 1834655A CN 200610018858 CN200610018858 CN 200610018858 CN 200610018858 A CN200610018858 A CN 200610018858A CN 1834655 A CN1834655 A CN 1834655A
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Abstract
This invention discloses bacteriophage immune PCR detecting pathogen method. The procedures are that single clone antibody that is coated to sample pathogen antigen on enzyme scale. Then recombined bacteriophage special recognizes pathogen is added. Then it is washed to get sample bacteriophage to use as scale to make PCR reaction. Amplification product is agarose gel electrophoresis detected, and the pathogen is quantifacated by quantificate PCR. The method in this invention is simple,it is propitious to pathogen infection lab diagnosis and blood serum epidemiology investigation.
Description
Technical field
The present invention relates to the detection of bio-terrorism, the particularly detection of cause of disease more specifically relates to a kind of method of detecting etiology by tacteriophage immunity PCR.
Background technology
The large-scale popular propagation of various communicable diseases that pathogenic pathogenic microorganism (comprising pathogenic bacteria, virus) causes is with the national security and the population health of serious harm China.Development is sensitive, cause of disease analytical approach and detection technique be for effective strick precaution of the quick diagnosis of relevant disease and treatment in time, bio-terrorism and sudden public health event with dispose significant fast accurately.The detection method of conventional pathogenic microorganism comprises the evaluation of microorganism separation and Culture, immunological method and based on the molecular detecting method of cause of disease nucleic acid etc.Classical microorganism isolation identification trouble is time-consuming, is difficult to be applied to scene, the fast detecting of pathogenic microorganisms; Immunological method is because make this method have specificity preferably based on antibody to the specific recognition effect of cause of disease, is optimized, improves in sensitivity and aspect such as practical but still need; Detection method based on cause of disease nucleic acid needs complicated nucleic acid extractive process, and this method higher detection sensitivity is given in the amplification of signal effect of PCR process, but occurs false positive easily.The present invention proposes a kind of new cause of disease and detects strategy, is called the immuno-PCR of bacteriophage mediation.
(Immuno-PCR IPCR) the earliest by propositions such as Sano, is a kind of hypersensitive immunologic detection method that development potentiality is arranged very much to immuno-PCR.Its ultimate principle is DNA on covalent cross-linking on the detection monoclonal antibody, the template that makes the latter can be used as pcr amplification is carried out signal and is amplified, fully utilized specific recognition effect and the powerful amplification of signal ability of PCR of antigen, antibody, made testing result have higher specificity and sensitivity concurrently.Develop immuno-PCR detection method, at first need to obtain the monoclonal antibody of considerable specific recognition cause of disease at various cause of diseases.Though it is ripe relatively that hybridoma is produced monoclonal antibody technique, its production run is consuming time, consumption power, requires those skilled in the art to operate, the expense costliness.Simultaneously, the antibody of traditional immuno-PCR process need complexity and DNA covalent cross-linking process, the adding of chemical cross-linking agent causes the forfeiture of partial antibody specific recognition capability sometimes inevitably, influences testing result.Therefore carry out cheap immuno-PCR method simple to operate for the detection of high-sensitive cause of disease with diagnose significant.
Summary of the invention
The objective of the invention is to be to provide a kind of method of detecting etiology by tacteriophage immunity PCR, this method utilize the immuno-PCR of bacteriophage mediation can reach to cause of disease carry out simply, sensitive, detection accurately, and with low cost, be applicable to the laboratory diagnosis and the seroepidemiological survey of pathogen infection.
In order to achieve the above object, the present invention adopts following technical scheme:
The principle of utilizing sandwich ELISA the monoclonal antibody bag of pathogen antigen by on ELISA Plate, through after blockading, add patients serum or pathogen separation nutrient solution, the bacteriophage that adds special recognition pathogen then, after washing, adding the hot cracked phage genome DNA that is attached on the ELISA Plate that makes of distilled water discharges, then lysate is transferred in the PCR pipe as template, the design Auele Specific Primer carries out pcr amplification, has or not to determine whether cause of disease is arranged in the sample through agarose electrophoresis according to the specific amplification band then.Utilize the real-time quantitative PCR instrument again, with the genomic DNA that is attached to the bacteriophage on the ELISA Plate is that template is carried out real-time quantitative PCR, in working sample, measure the pathogen antigen standard solution of the variable concentrations of 10 times of dilutions, the Ct value that reaction finishes to measure with variable concentrations the back is done typical curve with corresponding concentration, can calculate pathogen antigen amount in the sample to the Ct value substitution typical curve of working sample again, reach the purpose of the cause of disease in the sample being carried out detection by quantitative.The fast trace that this method can be widely applied to some potent virus, microorganism and poisonous antigen detects.Because operation is simple fast,, sets up fast detecting as Ebola virus, avian influenza virus, sars coronavirus or the like a new technology platform is provided simultaneously in cause of disease to now increasing newborn communicable disease.
The present invention compared with prior art has following advantage:
1) first bacteriophage being carried out virus as the carrier of immuno-PCR detects, avoided traditional immuno-PCR to need complicated antibody and DNA covalent cross-linking process, this method is simple, the acquisition of sensitivity and bacteriophage is very simple, with low cost, is fit to large-scale promotion application.
2) specificity and highly sensitive, the high specific of this method knot and immunology detection and the high sensitivity of pcr amplification are.
3) can detection by quantitative, utilize real-time quantitative PCR can carry out quantitative test to sample according to typical curve, be applicable to the precious disconnected and blood epidemiology survey in laboratory of pathogen infection.
Description of drawings
Fig. 1 be a kind of bacteriophage immuno-PCR and ELISA detect Hantaan virus nucleoprotein remolding sensitivity.
(a) bacteriophage immuno-PCR test.The bacteriophage that is attached on the microwell plate carries out pcr amplification by thermal cracking release genomic DNA as template, and amplified production is identified by 1.5% agarose gel electrophoresis.Swimming lane 1-7 represents 106 amplified productions to the Hantaan virus nucleoprotein correspondence of 1pg/ml respectively, and swimming lane 8 is the negative control of no nucleoprotein.The M swimming lane is dna molecular amount standard (2000,1000,750,500,250 and 100bp).
(b) remolding sensitivity of bacteriophage immuno-PCR and ELISA.The amplified production of bacteriophage PCR is at 1.5% agarose gel electrophoresis, and is quantitative by computer assisted image analysis system then.The color reaction of ELISA is by anti-M13 the bacteriophage two anti-and tmb substrate generations of horseradish peroxidase-labeled.
Fig. 2 detects the real-time quantitative PCR result of Hantaan virus nucleoprotein for the bacteriophage immuno-PCR.
(a) real-time quantitative PCR detects the quantitative curve of Hantaan virus nucleoprotein.Curve A-F represents the nucleoprotein (10 of 10 times of dilutions
6Pg/ml is to 10pg/m1), the G representative does not have the negative control of purpose antigen.As can be seen, the amount of the detection antigen of adding is big more, and corresponding Ct value is more little, and is linear dependence in certain scope.
(b) real-time quantitative PCR detects the typical curve of Hantaan virus nucleoprotein.In the scope of 2ng/ml, four times of dilution target viral antigens are figure production standard curve, R with the Ct value that produces with corresponding antigen concentration at 2000ng/ml
2=0.96, show that linear relationship is relatively good.
Embodiment
Below in conjunction with accompanying drawing the present invention is further set forth:
The embodiment of " the bacteriophage immuno-PCR detects Hantaan virus " mainly contains three parts and forms: the preparation of specific bacteriophage, specific bacteriophage catch and pcr amplification detects, its step is as follows: the preparation of the recombinant phage (the bacteriophage L13F3 of identification Hantaan virus nucleoprotein) of the specific recognition cause of disease that A, the present invention are used
[the recombinant phage of identification Hantaan virus nucleoprotein (displaying has the single-chain antibody of identification Hantaan virus nucleoprotein) of the recombinant phage of screening specific recognition cause of disease from phage antibody library, or the amplification antibody gene makes up recombinant phage from the hybridoma (hybridoma of the monoclonal antibody of identification Hantaan virus nucleoprotein) of secreting the monoclonal antibody of discerning cause of disease)], select the monoclonal (the recombination bacillus coli TG1 that contains called after bacteriophage L13F3) that is accredited as the positive and activate 2YTAG in 5mL
*Nutrient culture media (1000mL:17g peptone 10g yeast extract 5g sodium chloride 100ug/ml ampicillin 2% glucose), 37 ℃ of 200rpm overnight incubation, the bacterium liquid re-activation of getting the 50uL incubated overnight then is in 5mL 2YTAG nutrient culture media, 37 ℃ of 200rpm cultivate 2-3 hour to exponential phase (OD600 is about 0.5), add helper phage M13K07 10uL (10
11Pfu/mL) rescue continues 37 ℃ of 150rpm and cultivated 1 hour, and 1500g collected thalline in centrifugal 20 minutes, was resuspended in 6mL 2YTAK
*Select in the nutrient culture media (1000mL:17g peptone 10g yeast extract 5g sodium chloride 100ug/ml ampicillin 50ug/ml kanamycins) 30 ℃ of 200rpm overnight incubation.Next day, bacterium liquid 1500g was centrifugal 20 minutes, and supernatant is required bacteriophage.
Catching of B, specific bacteriophage
1) monoclonal antibody of special recognition pathogen (monoclonal antibody of identification Hantaan virus nucleoprotein) is got 100uL bag quilt in ELISA Plate (Costar company) with 1 * PBS dilution, and 4 ℃, the time is 16-24 hour;
2) washing, 1 * PBS (phosphate buffer) washes 3 times;
3) blockade, every hole adds 1 * PBS that 300uL contains 5% skimmed milk power, blockades 2 hours for 37 ℃;
4) washing, 1 * PBS washes 3 times;
5) every hole adds 100uL sample and negative control and positive control, hatches, and places 1-2 hour for 37 ℃;
6) washing, 1 * PBS washes 5 times;
7) add the bacteriophage supernatant of recombinating, hatch, promptly add 100uL bacteriophage L13F3 supernatant (containing 1% skimmed milk power), placed 2 hours for 37 ℃;
8) washing, TBSET[TBS damping fluid (add 8 gram NaCl in 1 liter of distilled water, 0.2 gram KCL and 3 restrains the Tris alkali, and adjust pH is 7.2) comprises 5mM EDTA and 0.1%Tween-20] wash 8 times, distilled water is washed 2 times;
9) every hole adds the 100uL distilled water, and the bacteriophage of (temperature is controlled at 93-95 ℃) cracking combination in 10 minutes is boiled in heating, and the genomic DNA of bacteriophage is released the template as the PCR reaction.
C, PCR detect
Mainly contain two kinds of qualitative detection and detection by quantitative (real-time quantitative PCR)
1. qualitative detection
Lysate with bacteriophage is a template, adds the upstream and downstream primer of design, carries out pcr amplification, each sample is done two repetitions, establish the distilled water that the do not contain template negative control as pcr amplification simultaneously, the PCR product scans with gel imaging system at last through agarose gel electrophoresis;
1) PCR system:
The lysate of bacteriophage (genomic DNA) 10uL
Upstream primer L 13sense (5 '-TTCAGTACCTATGCCATGTCT-3 ') 1uL
Downstream primer L 13anti (5 '-GTAGTCAAGGGGGTTACCTCG-3 ') 1uL
0.1％BSA 5uL
dNTP(2.5mM?each) 5uL
10×PCR?buffer 5uL
DNA polymerase 0.5uL
Distilled water 22.5uL
Each sample is done two repetitions, establishes the distilled water that do not contain any template (phage genome DNA) negative control as pcr amplification simultaneously.
2) PCR reaction conditions:
94 ℃, 10 minutes
94 ℃ 45 seconds, 55 ℃ 45 seconds, 72 ℃ 30 seconds, 30 circulations
72 ℃ 10 minutes.
3) agarose gel electrophoresis
PCR product point sample is in 1.5% Ago-Gel, 100V, and electrophoresis 30 minutes, gel imaging system scanning, this method is more highly sensitive than traditional ELISA method (use single-chain antibody), can detect the Hantaan virus nucleoprotein (Fig. 1) of 10pg/ml.
2. real-time quantitative PCR carries out detection by quantitative
Utilize the lysate of bacteriophage to be template, the upstream and downstream primer and the TaqMan spy battle array that add design are carried out real-time quantitative PCR on the quantitative PCR instrument, in working sample, measure the pathogen antigen standard solution of the variable concentrations of 10 times of dilutions, reaction finishes the back and with the Ct value of various criterion concentration determination concentration is done typical curve, can calculate the Ct value substitution typical curve of working sample the amount of the cause of disease in the sample again.
1) PCR system
The lysate of bacteriophage (genomic DNA) 10uL
Upstream primer L13For (5 '-TCTCACAGTCTCCTCAGCCAAA-3 ') 2uL
Downstream primer L13Rev (5 '-TCTACGCGTGCTTCTGAAAATTC-3 ') 2uL
The TaqMan probe (5 '-CGACACCCCCAAAGCTTGAAGAAGG-3 ') 2uL
0.1％BSA 5uL
dNTP(2.5mM?each) 5uL
10×PCR?buffer 5uL
DNA polymerase 0.8uL
MgCl
2(25mM) 6uL
Distilled water 12.2uL
2) PCR condition
94 ℃, 10 minutes
94 ℃ 45 seconds, 55 ℃ 70 seconds, 50 circulations
72 ℃ 10 minutes.
3) interpretation of result
Real-time quantitative PCR carries out on the PCR instrument (Opticon PCR machine) of MJ Research company.In working sample, measure the Hantaan virus nucleoprotein solution of the variable concentrations of 10 times of dilutions, reaction finishes the Ct value that back system software Opticon Monitor can provide variable concentrations, with the Ct value that variable concentrations is measured concentration is done typical curve (Fig. 2).Based on this typical curve, two concentration 400ng/ml in the quantitative measurement range of linearity and 4ng/ml, quantitative result is respectively 358.01ng/ml and 3.85ng/ml, and the result is in error allowed band (10%).As seen be more accurately with real-time quantitative bacteriophage immuno-PCR detection by quantitative viral antigen.
*The 2YTAG culture medium prescription: the 17g peptone, the 10g yeast extract, 5g sodium chloride, 20g glucose adds the 900ml deionized water, and pH value to 7.0 is regulated with NaOH in the dissolving back fully, adds deionized water to 1L, at 15lbf/in
2(1.034 * 10
5Pa) steam sterilizing 20 minutes under the high pressure.The ampicillin (filtration sterilization) that adds 0.1g during use in advance.
The 2YTAK culture medium prescription: the 17g peptone, the 10g yeast extract, 5g sodium chloride adds the 900ml deionized water, and pH value to 7.0 is regulated with NaOH in the dissolving back fully, adds deionized water to 1L, at 15lbf/in
2(1.034 * 10
5Pa) steam sterilizing 20 minutes under the high pressure.Add 0.1g ampicillin (filtration sterilization) during use in advance, 0.05g kanamycins (filtration sterilization).
1 * PBS (phosphate buffer) prescription: 8g sodium chloride, 0.2g potassium chloride, the 1.44g sodium hydrogen phosphate, the 0.24g potassium dihydrogen phosphate adds distilled water to 800mL, with NaOH adjust pH to 7.4, adds water and is settled to 1L.
TBSET:TBS damping fluid (add 8 gram NaCl in 1 liter of distilled water, 0.2 gram KCL and 3 restrains the Tris alkali, and adjust pH is 7.2) comprises 5mM EDTA and 0.1% Tween-20.
Upstream primer L13sense sequence is: 5 '-TTCAGTACCTATGCCATGTCT-3 '.
Downstream primer L13anti sequence is: 5 '-GTAGTCAAGGGGGTTACCTCG-3 '.
Upstream primer L13For sequence is: 5 '-TCTCACAGTCTCCTCAGCCAAA-3 '.
Downstream primer L13Rev sequence is: 5 '-TCTACGCGTGCTTCTGAAAATTC-3 '.
TaqMan visits the battle array sequence: 5 '-CGACACCCCCAAAGCTTGAAGAAGG-3 '.
Claims (3)
1, a kind of method of detecting etiology by tacteriophage immunity PCR, it comprises the following steps:
The recombinant phage preparation of A, specific recognition cause of disease:
Recombinant phage/the displaying of screening special recognition pathogen has the single-chain antibody of identification pathogen antigen from phage antibody library, or the amplification antibody gene makes up recombinant phage from the hybridoma of secreting the monoclonal antibody of discerning cause of disease, choose through identifying positive monoclonal overnight incubation, re-activation is to exponential phase then, add the helper phage rescue, centrifugal then collection thalline is resuspended in and selects overnight incubation in the nutrient culture media, next day, bacterium liquid was centrifugal, and supernatant is required bacteriophage;
Catching of B, specific bacteriophage the steps include:
1) the monoclonal antibody bag of special recognition pathogen quilt is in ELISA Plate;
2) washing;
3) blockade;
4) washing;
5) every hole adds sample and negative control and positive control, hatches;
6) washing;
7) add the recombinant phage supernatant, hatch;
8) washing;
9) every hole adds distilled water, the bacteriophage of heating pyrolyze combination, and the genomic DNA of bacteriophage is released
Release template as the PCR reaction;
C, PCR detect:
A. qualitative detection:
Lysate with bacteriophage is a template, adds the upstream and downstream primer of design, carries out pcr amplification, each sample is done two repetitions, establish the distilled water that the do not contain template negative control as pcr amplification simultaneously, the PCR product scans with gel imaging system at last through agarose gel electrophoresis;
B. real-time quantitative PCR carries out detection by quantitative:
Utilize the lysate of bacteriophage to be template, the upstream and downstream primer and the TaqMan probe that add design carry out real-time quantitative PCR on the quantitative PCR instrument, in working sample, measure the pathogen antigen standard solution of the variable concentrations of 10 times of dilutions, the Ct value that reaction finishes to measure with normal concentration the back is done typical curve to concentration, can calculate the Ct value substitution typical curve of working sample the amount of the cause of disease in the sample again.
2. the method for a kind of detecting etiology by tacteriophage immunity PCR according to claim 1 is characterized in that the upstream and downstream primer of qualitative detection is:
Upstream primer L13sense sequence is: 5 '-TTCAGTACCTATGCCATGTCT-3 ';
Downstream primer L13anti sequence is: 5 '-GTAGTCAAGGGGGTTACCTCG-3 '.
3. the method for a kind of detecting etiology by tacteriophage immunity PCR according to claim 1 is characterized in that the upstream and downstream primer of detection by quantitative and TaqMan visit battle array and be:
Upstream primer L13For sequence is: 5 '-TCTCACAGTCTCCTCAGCCAAA-3 ';
Downstream primer L13Rev sequence is: 5 '-TCTACGCGTGCTTCTGAAAATTC-3 ';
The TaqMan probe sequence is: 5 '-CGACACCCCCAAAGCTTGAAGAAGG-3 '.
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Cited By (4)
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| CN102936598A (en) * | 2012-11-21 | 2013-02-20 | 江苏省农业科学院 | Coding gene of anti-Cry1Ac toxin single-chain variable fragments (scFv) and immuno-polymerase chain reaction (PCR) detection method |
| CN110231484A (en) * | 2019-06-28 | 2019-09-13 | 扬州大学 | A kind of method and its application of detection expression carcinomebryonic antigen cell |
| CN112458202A (en) * | 2020-10-22 | 2021-03-09 | 福建省农业科学院畜牧兽医研究所 | PCR detection primer and kit for riemerella anatipestifer virulent phage |
| WO2024027083A1 (en) * | 2022-08-01 | 2024-02-08 | 深圳北京中医药大学研究院 | Phage display-mediated immune multiple quantitative pcr method and recombinant phage therefor |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| IT1291913B1 (en) * | 1997-05-22 | 1999-01-21 | Angeletti P Ist Richerche Bio | METHOD INVOLVING THE USE OF BACTERIOPHAGES FOR THE DETECTION OF THE PRESENCE OF MOLECULES OF INTEREST IN BIOLOGICAL SAMPLES |
| GB0303497D0 (en) * | 2003-02-15 | 2003-03-19 | Univ Liverpool | Immuno PCR method |
| WO2005086647A2 (en) * | 2004-02-23 | 2005-09-22 | University Of Maryland, Baltimore | Immuno-pcr method for the detection of a biomolecule in a test sample |
| CN1570118A (en) * | 2004-04-27 | 2005-01-26 | 华东师范大学 | Bacteriophage polypeptide fluorescent protein marking system establishment mentod |
| FI20040768A0 (en) * | 2004-06-04 | 2004-06-04 | Teemu Korpimaeki | Method for stabilizing assay reagents, reagent tank containing stabilized assay reagents and its use |
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| CN102936598A (en) * | 2012-11-21 | 2013-02-20 | 江苏省农业科学院 | Coding gene of anti-Cry1Ac toxin single-chain variable fragments (scFv) and immuno-polymerase chain reaction (PCR) detection method |
| CN102936598B (en) * | 2012-11-21 | 2014-04-16 | 江苏省农业科学院 | Coding gene of anti-Cry1Ac toxin single-chain variable fragments (scFv) and immuno-polymerase chain reaction (PCR) detection method |
| CN110231484A (en) * | 2019-06-28 | 2019-09-13 | 扬州大学 | A kind of method and its application of detection expression carcinomebryonic antigen cell |
| CN112458202A (en) * | 2020-10-22 | 2021-03-09 | 福建省农业科学院畜牧兽医研究所 | PCR detection primer and kit for riemerella anatipestifer virulent phage |
| WO2024027083A1 (en) * | 2022-08-01 | 2024-02-08 | 深圳北京中医药大学研究院 | Phage display-mediated immune multiple quantitative pcr method and recombinant phage therefor |
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