CN106702021A - Duck plague and escherichia coli dual PCR diagnostic kit - Google Patents
Duck plague and escherichia coli dual PCR diagnostic kit Download PDFInfo
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Abstract
The invention discloses a duck plague and escherichia coli dual PCR diagnostic kit. The two kinds of viruses including DPV and E.coli can be simultaneously detected in the same reaction system, and the detection amount of DPV and the detection amount of E.coli are 0.2 ng/mL and 0.1 ng/mL respectively. Through sensibility is lower than that of established single virus (average) PCR, the sensibility is higher than that of a traditional serology and Elisa detection method and meets the requirement of clinical detection; it is displayed by specificity verification results for the diagnostic kit that the amplification results of duck pasteurella multocida, duck salmonella, riemerella anatipestifer and healthy duck tissue DNA are negative, which indicates that the diagnostic kit is high in specificity and is capable of fast and accurately judging single or mixed infection of DPV and E.coli in clinic samples and capable of meeting the requirement of fast detecting samples in large batches and being used and popularized in China basic layer animal prevention and control centers or breeding enterprises.
Description
Technical field
Diagnosed the present invention relates to home poultry raising and disease prevention and cure field, especially a kind of duck plague and Escherichia coli double PCR
Kit.
Background technology
Duck plague (Duck Plague), also known as duck viral enteritis (Duck Viral Enteritis, DVE), are by duck plague
Viral (Duck Plague Virus, DPV) causes a kind of acute septic infectious disease of the aquatic birds such as duck, goose and swan, Yin Qifa
Sick rate and the death rate are higher, often cause heavy economic losses to aviculture.Escherichia coli (Escherichia coli, E coil)
It is one of most common bacterial disease of poultry, draws its disease for locally or systemically infecting, including it is e. coli septicemia, big
Enterobacteria granuloma, airsac disease, fowl cellulitis, swollen head sydrome, peritonitis, salpingitis, synovitis, panophthalmitis and ovum
Yellow capsule infection.
As aquaculture large-scale degree is improved constantly, the situation generally existing of various disease mixed infections in duck group is
The diagnosis of disease brings no small difficulty.Colibacillosis often causes Secondary cases office when avian host defence capability declines
Portion or general infection, and duck plague, colibacillosis and dfuck cholera all deposit at aspects such as clinical symptoms, pathological change and epidemiology
Many similar[3].Therefore, conventional diagnostic method is difficult quickly and accurately to make diagnosis, therefore sets up duck plague and Escherichia coli
The molecular biology method of sick antidiastole is very necessary.
Multiplex PCR is advantageous in that：It is capable of achieving to detect multiple pathogens, and specificity simultaneously in portion sample
By force, sensitiveness is high, detection time is short, testing cost is low, is adapted to cause of disease in a large amount of clinical samples (particularly mixed infection sample)
The quick diagnosis of body.
The content of the invention
The purpose of the present invention is：A kind of duck plague and Escherichia coli double PCR diagnostic kit are provided, it can be simultaneously to facing
Bed sample carries out the double check of DPV and E.coli, and easy to operate, quick and sensitivity and accuracy are good, existing to overcome
There is the deficiency of technology.
What the present invention was realized in：Duck plague and Escherichia coli double PCR diagnostic kit, including Taq
Polymerase, 2 × Buffer of buffer solution, ultra-pure water, mix primer and positive control；Taq polymerase concentration is 1U/
ML, 2 × Buffer of buffer solution concentration are 0.5mol/L, mix primer concentration is 10mmol/L, and positive control concentration is duck plague base
Because of a group DNA, each 10ng/L of genome of E.coli DNA, negative control is ultra-pure water；Wherein, mix primer includes primer DPV-F
5 '-GCGTGATTCAGTGCCTAT-3 ', primer DPV-R 5 '-GTCATCTCGGTATTGTATTGG-3 ', primer E.coli-F
- GTTAC the CTTGTTACGACTTCAC-3 ' of 5 '-AGAGATGAGAATGTGCCTTC-3 ', primer E.coli-R 5 '.
Described positive control is duck plague genomic DNA, genome of E.coli DNA.
In order to verify technique effect of the invention, tests below has been carried out：
1 materials and methods
1.1 test materials
The DPV live vaccines (chicken embryo low virulent strain) of this experiment are purchased from Nanjing Tian Bang Bioisystech Co., Ltd, are write from memory in pest of duck
Family name bacillus reference culture is purchased from Chinese veterinary microorganism preservation administrative center (CVCC), and DPV GZ plants by Guizhou University's animal science and technology
Institute give；Mo Shi Pasteurellas (serum 1 in DPV virus CHv velogen strains, E.coli (serum O78, O11, O88 type), pest of duck
Type), duck pasteurella multocida by this laboratory separate identification after preserve.
Gold view, Taq Polymerase, nuclease free ddH2O、Tris、HCl、KCl、MgCl2、dNTPs、RNase
free dH2O, DL2000, DL500, DNAiso Reagant etc. is purchased from precious biology (Dalian) Engineering Co., Ltd；50 × TAE etc.
Purchased from Sangon Biotech (Shanghai) Co., Ltd.；Primer is synthesized by precious biology (Dalian) Engineering Co., Ltd.
1.2 test methods
1.2.1 DPV TK genes (DQ640611.1) during design of primers is included according to Genbank, Escherichia coli 16
SRNA genes (NC_018658.1) sequence, devises 2 pairs of primers, and for amplifying target genes fragment, primer sequence is shown in Table one.
The primer sequence of table 1
1.2.2 it is each the disease sample such as brain, lung, the heart, liver, intestines, blood, excrement to be taken respectively after viral nucleic acid extraction dissection sick duck
50mg, the extraction of virus (bacterium) DNA is carried out according to DNAiso Reagant specifications, and the DNA sample that will be extracted is in -20 DEG C of guarantors
Deposit.
1.2.3 the RT-PCR reactions of the single virus of single virus PCR increasing are carried out in 25 μ L systems, upstream and downstream primer
Each 1 μ L (10 μM), Taq Polymerase 1.3U, the μ L of 2 × Buffer 12.5, template 1 μ L, nuclease free ddH2O is supplemented to
25 μ L, response procedures：94 DEG C of 5min, 94 DEG C of 30s, 58 DEG C of 30s, 72 DEG C of 30s, 30 circulations, 72 DEG C of extension 5min.Take 5 μ L
Pcr amplification product carries out electroresis appraisal in 20g/L Ago-Gels.
1.2.4 the optimization of multi-PRC reaction condition is to multi-PRC reaction condition, including annealing temperature (54,55 DEG C, 56
DEG C, 57 DEG C, 58 DEG C, 59 DEG C), lower sense primer concentration (2 μM, 5 μM, 10 μM, 15 μM, 20 μM), Taq Polymerase (15U,
25U, 30U, 35U, 40U), dNTPs concentration (5 μM, 10 μM, 20 μM, 25 μM, 30 μM) optimize, to determine optimum response bar
Part, while with nuclease free ddH2O is used as blank.Multiplex PCR is carried out in 50 μ L reaction systems.Reaction condition is 94 DEG C
3min；94 DEG C of 30s, annealing temperature (54,55 DEG C, 56 DEG C, 57 DEG C, 58 DEG C, 59 DEG C, 60 DEG C) 30s, 72 DEG C of 30s, 30 circulations；
Last 72 DEG C of extensions 5min.Take 5 μ L amplified productions carries out electroresis appraisal in 20g/L Ago-Gels.
1.2.5 after virus (bacterium) DNA that sensitivity tests will be extracted determines concentration with protein nucleic acid instrument, 2 μ g are taken
DPV and E.coli DNA mix, and are used for multiplex PCR through 10 times of viral nucleic acids being serially diluted respectively；2μg DPV、2μg
E.coli concentration is used to be reacted for single PCR through 10 times of viral nucleic acids being serially diluted respectively, to determine the sensitivity of multiplex PCR
Property；
1.2.6 specific test respectively with DPV (vaccine strain, Gz plant, CHv velogen strains), E.coli (serum O78, O11,
O88 types), the DNA of Riemerellosis Anatipestifer and Salmonella anatis be template, multi-PRC reaction is carried out, to determine the spy of multiplex PCR
The opposite sex.
1.2.7 multiple PCR method, the single virus (bacterium) of duplicate detection DPV, E.coli are set up in replica test application
DNA or hybrid dna sample 3 times, the reliability of assay.
1.2.8 the multiple PCR method composition that the composition of multiple PCR reagent kit and the detection application to clinical sample are set up
Kit, including：(1)Taq polymerase；(2) buffer solution (2 × Buffer)；(3) nuclease free ddH2O；(4) mixing is drawn
Thing；(5) positive control.Using kit to Guiyang City, Guizhou Province, Kaili City, Duyun City, Xingyi City, Tianzhu County, Sansui County ground
187 points of pathological material of diseases of several scale duckeries of area and ducks specialized household collection are detected, while result is detected with single PCR
Result is contrasted.
2 results and analysis
The identification of 2.1 multi-PRC reactions and product
Multi-PRC reaction optimum reaction condition in 50 μ L reaction systems is 58 DEG C of annealing temperature, DNA Polymerase
The μ L of 2U, 2 × Buffer 25.0,15 μm of ol/L of primer concentration effectively amplify purpose fragment, DPV amplified production 312bp,
E.coli amplified production 499bp, (Fig. 1) is produced without specific fragment.The multiplexed PCR amplification fragment of DPV, E.coli is returned through cutting glue
After receipts, send precious biology (Dalian) Engineering Co., Ltd to be sequenced, as a result show, amplified fragments are respectively the special of two viruses (bacterium)
Property band.
2.2 sensitivity tests
Research find, it is single virus PCR reaction in, the minimum detection of nucleic acids amounts of DPV be 0.02ng/mL (Fig. 2),
E.coli is 0.01ng/mL (Fig. 3)；After two kinds of virus (bacterium) mixing, in multi-PRC reaction, DPV is minimum, and detection of nucleic acids amount is
0.2ng/L, E.coli are 0.1ng/L (Fig. 4).
2.3 specific tests
Organized with DPV, E.coli, Riemerellosis Anatipestifer, duck pasteurella multocida, Salmonella anatis, healthy duck respectively
DNA and water are template, carry out multi-PRC reaction, DPV (vaccine strain, Gz plant, CHv velogen strains), E.coli (O11, O78, O88 blood
Clear type) single and hybrid dna can amplify corresponding specific band, and with killing property Pasteur Riemerellosis Anatipestifer, duck more
Bacillus, Salmonella anatis, healthy duck tissue DNA and water are template, fail to amplify band (Fig. 5-6).
2.4 replica tests
Using the multiple PCR method set up, single virus (bacterium) DNA of duplicate detection DPV, E.coli or hybrid dna sample 3
It is secondary, it is as a result consistent.
Detection of 2.5 multiple PCR reagent kits to clinical sample
Using develop DPV and E.coli multiple PCR reagent kits 200 parts of pathological material of diseases are detected, as a result in have 33 parts of diseases
Material DPV is positive, and positive rate is 16.5%；57 parts of E.coli positives, positive rate is 28.5%.Wherein DPV and E.coli are positive
16 parts, positive rate is 8%.Its result is 100% with the coincidence rate of single virus (bacterium) PCR testing results.
3 discuss
1980, Wang Yongkun et al. had found goose reproductive organs E.coli diseases in first in China, with section's aquaculture not
Disconnected development, the disease has been presented universal Infection trend, and statistics shows, the incidence of disease of fowl E.coli is 11% -69%, and the death rate is
3.8-72.9%, fatal rate is 40.2%-90.3%.Now there are some researches show E.coli antigens mainly have O (somatic antigen), K
(capsular antigen), H (flagellar antigen), four kinds of F (fimbrial antigen), it has been determined that O antigens have 173 kinds, 80 kinds of K antigens, H antigens
56 kinds, F antigens are no less than 17 kinds.E.coli serotype complexity is various, and conventional laboratory conditions are difficult to while detect all serotypes,
Also pointing out us needs then seeks molecular biological testing.Duck plague is a kind of acute septic infectious disease, spread speed
Hurry up, the incidence of disease, the death rate are high, the development of serious threat China duck culturing industry.DPV only has One serotype, the conventional inspection of current DPV
Survey method includes：Virus purification, agar gel diffusion test, Elisa etc., these methods not only take, and sensitiveness is relatively low, are unfavorable for
Quick, the Accurate Diagnosis of disease.Duck plague and colibacillosis all exist at aspects such as clinical symptoms, pathological change and epidemiology
It is many similar.Therefore, conventional diagnostic method is difficult quickly and accurately to make diagnosis.
Round pcr is so that its is quick, sensitive, high specificity the advantages of be used widely in animal epidemic detection, into
One of important method for animal pathogenic detection.Conventional PCR detection method generally only detects a kind of cause of disease, and this research is same first
When detect DPV and E.coli, simplify detection program, cost-saving synergistic, for clinical detection provides new method.16S rRNA bases
Because being prevalent in bacterium and participating in the protein building-up process of bacterium, contain highly conserved sequence area, 16S rRNA bases
Because having turned into the basis of various bacteria diagnostic method.Research display, thymidine kinase (Thymidine kinase, TK) gene belongs to
One of main virulence gene of herpesviral, is also the first-selected target gene for building hsv gene deletion of vaccine.With
The foreign scholars such as Plummer etc. and Hansen successively succeed using PCR detections DPV, Liu Fei of China etc. and Cheng Anchun etc.,
Many scholars such as Meng Zeng etc., Wei Xuetao successively establish the PCR method of detection DPV, obtain good effect.Tang Cheng etc.
Establish the SYBR Green I real time fluorescence quantifying PCR methods of detection DPV, stone Jianping etc. and Xu Yang successively establishes detection
The TaqMan real time fluorescence quantifying PCR methods of DPV, realize to DPV DNA from the detection of qualitative to quantitative.But be worth noting
, real time fluorescent quantitative complex operation, and China major part basic animal pre-control center and plant be not yet equipped with fluorescence determine
Amount PCR instrument.
As a result of above technical scheme, compared with prior art, the present invention can be same in same reaction system again
When detects DPV and E.coli, and both are viral, and detection limit is respectively DPV 0.2ng/mL, E.coli 0.1ng/mL, although sensitive
Property compared with set up single virus (equal) PCR it is low, but be higher than traditional serology and Elisa detection methods, clinic can be met
The need for detection；Meanwhile, the specificity verification result for this diagnostic kit shows：It is husky in duck pasteurella multocida, duck
The amplification of door Salmonella and riemerella anatipestifer and healthy duck tissue DNA is feminine gender, shows the spy of this diagnostic kit
It is different in nature good, the independent or mixed infection of DPV in clinical sample and E.coli can fast and accurately be diagnosed, can expire
The quick detection of sufficient batch samples needs, and can be promoted the use of in China's basic animal pre-control center or breeding enterprise.
Brief description of the drawings
Fig. 1 is DPV and E.coli multiplex PCR results；
M:DL2000Marker；1:DPV and E.coli multiple PCR products；2:The single PCR primers of E.coli；3:DPV is single
PCR primer；4:Blank (water)；
Fig. 2 is the single PCR sensitivity techniques of DPV；
M:DL2000Marker；1:200ng；2:20ng；3:2ng；4:0.2ng；5:0.02ng；6:2pg；
Fig. 3 is the single PCR sensitivity techniques of E.coli；
M:DL2000Marker；1:100ng；2:10ng；3:1ng；4:0.1ng；5:0.01ng；6:1pg；
Fig. 4 is DPV, E.coli multiplex PCR sensitivity technique；
M:DL2000Marker；1:DPV/E.coli:0.02/0.01ng；2:DPV/E.coli:0.2/0.1ng；3:DPV/
E.coli:2/1ng；4:DPV/E.coli:20/10ng；5:DPV/E.coli:2/1μg；6:DPV/E.coli:20/10μg
Fig. 5 is DPV, E.coli multiplex PCR specific detection；
M:DL2000 Marker；1:Riemerella anatipestifer；2:Duck pasteurella multocida；3:Salmonella anatis；4:
E.coli；5:DPV；6:Water；7:Healthy duck；8:DPV and E.coli；
Fig. 6 is duck plague and fowl enteropathogenic E. Coli multiplex PCR specific detection result；
M:DL500 Marker；1:E.coli serum O78 types；2:E.coli serum O11 types；3:E.coli serum O88 types；
4:DPV vaccine strains；5:DPV CHv plants；6:DPV GZ plants；7:Duck pasteurella multocida；8:Mo Shi Pasteurellas in pest of duck:9:
Pasteurella multocida；10:Healthy duck tissue.
Specific embodiment
Embodiments of the invention：Duck plague and Escherichia coli double PCR diagnostic kit, including it is T aq polymerase, slow
2 × Buffer of fliud flushing, ultra-pure water, mix primer and positive control；Taq polymerase concentration be 1U/mL, buffer solution 2 ×
Buffer concentration is 0.5mol/L, mix primer concentration is 10mmol/L, and positive control concentration is duck plague genomic DNA, large intestine
Each 10ng/L of vaccae genomic dna, negative control is ultra-pure water；Wherein, mix primer include primer DPV-F 5 '-
GCGTGATTCAGTGCCTAT-3 ', primer DP V-R 5 '-GTCATCTCGGTATTGTATTGG-3 ', primer E.coli-F 5 '-
- GTTACCTT the GTTACGACTTCAC-3 ' of A GAGATGAGAATGTGCCTTC-3 ', primer E.coli-R 5 '.
Described positive control is duck plague genomic DNA, genome of E.coli DNA.
SEQUENCE LISTING
Sequence table
<110>Guizhou Farming Animal Science and Veterinary Research Institute
<120>Duck plague and Escherichia coli double PCR diagnostic kit
<130> nm:
<160> 4
<170> PatentIn version
<210> 1
<211> 23
<212> DNA
<213>Artificial sequence
<220>
<223>DPV TK genes in Genbank（DQ640611.1）Sequence, using online PrimerExplorer
4.0 Software for Design, expand with PCR.
<400> 1
GCGTG ATTCA GTGCC TAT 23
<210> 2
<211> 21
<212> DNA
<213>Artificial sequence
<220>
<223>DPV TK genes in Genbank（DQ640611.1）Sequence, using online PrimerExplorer
4.0 Software for Design, expand with PCR.
<400> 2
GTCAT CTCGG TATTG TATTG G 21
<210> 3
<211> 20
<212> DNA
<213>Artificial sequence
<220>
<223>The sRNA genes of Escherichia coli 16 in Genbank（NC_018658.1）Sequence, using online
The Software for Design of PrimerExplorer 4.0, expands with PCR.
<400> 3
AGAGA TGAGA ATGTG CCTTC 20
<210> 4
<211> 21
<212> DNA
<213>Artificial sequence
<220>
<223>The sRNA genes of Escherichia coli 16 in Genbank（NC_018658.1）Sequence, using online
The Software for Design of PrimerExplorer 4.0, expands with PCR.
<400> 4
GTTAC CTTGT TACGA CTTCA C 21
Claims (2)
1. a kind of duck plague and Escherichia coli double PCR diagnostic kit, it is characterised in that：Including Taq polymerase, buffering
2 × Buffer of liquid, ultra-pure water, mix primer and positive control；Taq polymerase concentration be 1U/mL, buffer solution 2 ×
Buffer concentration is 0.5mol/L, mix primer concentration is 10mmol/L, and positive control concentration is duck plague genomic DNA, large intestine
Each 10ng/L of vaccae genomic dna, negative control is ultra-pure water；Wherein, mix primer include primer DPV-F 5 '-
GCGTGATTCAGTGCCTAT-3 ', primer DPV-R 5 '-GTCATCTCGGTATTGTATTGG-3 ', primer E.coli-F5 '-
- GTTAC the CTTGTTACGACTTCAC-3 ' of AGAGATGAGAATGTGCCTTC-3 ', primer E.coli-R 5 '.
2. duck plague according to claim 1 and Escherichia coli double PCR diagnostic kit, it is characterised in that：Described sun
Property control be duck plague genomic DNA, genome of E.coli DNA.
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| WEI B等: "Development and application of a multiplex PCR assay for rapid detection of 4 major bacterial pathogens in ducks", 《POULTRY SCIENCE》 * |
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