CN106434994A - Rapid detecting mycoplasma ovipneumoniae method - Google Patents
Rapid detecting mycoplasma ovipneumoniae method Download PDFInfo
- Publication number
- CN106434994A CN106434994A CN201611082202.0A CN201611082202A CN106434994A CN 106434994 A CN106434994 A CN 106434994A CN 201611082202 A CN201611082202 A CN 201611082202A CN 106434994 A CN106434994 A CN 106434994A
- Authority
- CN
- China
- Prior art keywords
- lamp
- reaction
- primer
- sheep
- concentration
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to a rapid detecting mycoplasma ovipneumoniae method, according to the gene sequence of the detected mycoplasma ovipneumoniae designing a LAMP primer; establishing a reaction system for LAMP visualization method, and conducting optimizations for LAMP reaction conditions; gathering and embrocating the sheep nasal secretions, preparing DNA template solution, conducting detections using the optimized reaction system and reaction condition. The method targets the shortcomings of requiring professional instruments and labs and the like in detecting mycoplasma ovipneumoniae in the existing methods, which limits the promotion and application of these diagnosis methods in clinics, and establishes a rapid detecting LAMP visualization method to detect MO, the method has very high specificity and sensitivity, and provides a rapid, simple, and accurate detection method for clinical diagnoses of sheep MO infection.
Description
Technical field
The present invention relates to technical field of biological is and in particular to one kind is used for quickly detecting sheep using visual means
The method of mycoplasma pneumoniae.
Background technology
The Mycoplasma ovipneumonia sheep contagious pleuropneumonia that is otherwise known as is by mycoplasma ovine pneumoniae (Mycoplasma
Ovipneumoniae, MO) cause sheep and goat to suffer from chromic fibrous, severe acute respiratory syndrome, apostematosa pneumonia.
Since the reported first on sheep in 1963 such as Mackay, Mycoplasma ovipneumonia is worldwide wide
General popular.MO not only infects sheep and goat, will also result in the infection of other ruminants (as bighorn) simultaneously.In recent years,
In the province that multiple sheep husbandry such as Xinjiang of China, Gansu, the Inner Mongol, Qinghai, Shaanxi, Ningxia, Liaoning, Jiangsu, Sichuan, Yunnan is flourishing
Qu Jun has occurring and popular report of Mycoplasma ovipneumonia, and this disease drastically influence the sound development of sheep husbandry, endangers day
Become to increasing, cause huge economic loss to China's sheep husbandry.
MO belongs to Mollicutes, Mycoplasmas, Mycoplasmataceae, one kind of Mycoplasma.MO is a kind of shortage cell wall, is in
Height pleomorphism, can be by the minimum prokaryotic microorganism of bacteria filter.Because its genome is less, biosynthesiss and metabolism
Limited in one's ability, a lot of nutrient substance that cell needs need, from extraneous picked-up, therefore the separation and Culture condition of this cause of disease to be required
Higher.
Diagnostic method about MO is concentrated mainly on the isolation identification of cause of disease, Serologic detection, molecular biology inspection at present
The aspects such as survey.
However, because the culture of MO is relatively difficult and the speed of growth slow, being separated from pathological material of disease by bacteriological method
Relatively difficult.At present, conventional Serologic detection mainly includes:Complement fixation test (CFT), growth or metabolic inhibition test,
IHAT (IHA), elisa (ELISA) and spot immune percolation test etc..
CFT uses glycolipid antigen, is likely to occur cross reaction with animal tissue, brings difficulty to Differential Diagnosiss, and
Complex operation is seldom applied in clinical diagnosises;Although growth or metabolic inhibition test high specificity, sensitivity are high, be applied to system
Carry out the serological analysis of titration and mycoplasma during standby mycoplasma antiserum, but the method cannot distinguish between IgM's and IgG
Antibody types.
IHA can detect MO antibody, but Sensitivity and Specificity is not all high.The specificity of indirect ELISA, sensitivity, stable
Property and repeatability are higher, but can only detect MO antibody it is impossible to direct detection cause of disease.
PCR method is sensitive, quick but depends on precision instrument costly it is difficult to promote general in the poor basic unit of condition
And use.
In addition, above-mentioned detection mode generally needs special instrument and laboratory, limits these diagnostic methods and is facing
Promoting the use of on bed.Therefore, this area can having compared with hypersensitivity and specific with popularization and application in the urgent need to research and development
Detection mycoplasma ovine pneumoniae method.
Content of the invention
Present invention is primarily intended to provide one kind ring mediated isothermal amplification (LAMP) technology foundation MO infect can
Depending on changing method for quick.
The method of quick detection mycoplasma ovine pneumoniae of the present invention, is realized by procedure below:
A, the gene order design LAMP primer according to detected mycoplasma ovine pneumoniae;
B, the reaction system of establishment LAMP visual method, and LAMP reaction condition is optimized;
Sheep nasal secretion is embrocated in C, collection, and the sheep collecting nasal secretion produces DNA profiling solution, uses
Step B gained reaction system and reaction condition are detected.
LAMP primer designed by step A includes a pair of inner primer and a pair of outer primer, amplifying target genes fragment length
For 188bp.
The sequence of above-mentioned LAMP primer is respectively:
Inner primer:FIP——CCTACCAACTAACTAAAAAATGGGGGTGC
BIP——GATTGAGATACGGCCCGTAGGAG TCTGG
Outer primer:F3——AAGGAGCCTTTAAAGCTC
B3——GCAGCAGTAAGGAATAT
Above-mentioned steps B establish LAMP visual method reaction system concrete operations be:With every 20-30 μ L LAMP reaction
System is part, each 1-1.5 μm of ol/L of inner primer (FIP, BIP), each 0.1-0.3 μm of ol/L of outer primer (F3, B3), dNTP 1.0-
1.5mmoI/L 2.5 μ L, MgSO47-10mmol/L, Betaine 0.7-1mol/L 1-3 μ L, l0 × Bst archaeal dna polymerase delays
Rush liquid, the Bst archaeal dna polymerase 0.8-1.2 μ L of 8U, the MO bacterium solution 1-3 μ L after boiling, complement to 25-30 μ L with pure water, reaction
Condition:In 55-65 DEG C of reaction temperature, it is spaced 1-2 DEG C and is incremented by, each temperature all reacts 0.5-1.5h, last 75-85 DEG C of effect 3-
8min terminating reaction.
Further, after optimizing described in above-mentioned steps B, LAMP reaction condition is:Reaction temperature 60-65 DEG C, Mg2+ concentration is
7-9mmol/L, dNTP concentration is 1.0-1.5mmol/L, glycine betaine Betaine concentration 0.6-1mol/L, inside and outside primer concentration ratio
For 5-7:1, the response time is 30-90min.
Further, the concrete operations of described step C are:Embrocate sheep nasal secretion with sterile cotton swabs, be placed in and go out
In the centrifuge tube of bacterium, more than totally 60 parts of at least 5 sheep raising field sheep nose swab samples of collection altogether, low temperature is transported to laboratory to be carried out
Detection, adds the normal saline of 1ml sterilizing, fully vibration mixing, takes the liquid after process in the centrifuge tube containing sterile cotton swabs
Body is placed in container, and 10000-14000r/min is centrifuged 1-3min, and sterile purified water washes twice above, finally uses 200-400 μ
L TE buffer suspends, and water proof boils 10-20min, and 7000-10000r/min is centrifuged 10-20min, takes supernatant, i.e. DNA profiling
Solution, is detected to clinical sample with the MO visualization LAMP reaction set up, the testing result of statistical detection method.
The present invention passes through to visualize quick detection MO nucleic acid with ring mediated isothermal amplification (LAMP) technology.For MO's
Conservative specific sequence 16S rDNA, designs 4 LAMP primer, to Mg2+Multiple reactions such as concentration, reaction temperature, dNTP concentration
After condition is optimized, establish the reaction system of LAMP visual method.Using the LAMP primer designed by the present invention, difference
To staphylococcus aureuses, escherichia coli, Bacillus subtillis, Listeria monoeytogenes, staphylococcus epidermidiss, sramana
The detection of the various pathogens such as Salmonella and clinical sample it was demonstrated that the MO LAMP visual detection method set up have very high
Specificity and sensitivity, are that the clinical diagnosises of sheep MO infection provide a quick, easy, accurate detection method.
Brief description
Fig. 1 is embodiment of the present invention LAMP visual testing result schematic diagram;
Wherein corresponding DNA fragmentation is respectively:1st, staphylococcus aureuses;2,4th, mycoplasma ovine pneumoniae;3rd, large intestine bar
Bacterium;5th, Salmonella.
Fig. 2 is embodiment of the present invention LAMP specific test result schematic diagram;
Wherein corresponding DNA fragmentation is respectively:1st, staphylococcus aureuses;2nd, escherichia coli;3rd, bacillus subtilises;4、
Listeria monoeytogenes;5th, staphylococcus epidermidiss;6th, Salmonella;7th, mycoplasma ovine pneumoniae.
Fig. 3 is embodiment of the present invention LAMP sensitivity testss result schematic diagram;
Wherein:M represents Protein Marker
1st, represent bacterium solution extension rate 10-1;2nd, represent bacterium solution extension rate 10-2;3rd, represent bacterium solution extension rate 10-3;4、
Represent bacterium solution extension rate 10-4;5th, represent bacterium solution extension rate 10-5;6th, represent bacterium solution extension rate 10-6;7th, represent that bacterium solution is dilute
Release multiple 10-7;8th, represent bacterium solution extension rate 10-8;
Specific embodiment
With Xinjiang Uygur Autonomous Regions pasture sheep as specimen, aseptic culture simultaneously collects sheep Eg cephalomere to the present embodiment, its
His raw material passes through to buy the gained such as test kit.
1st, the design of LAMP primer
According to MO 16S rDNA sequence, carry out sequence alignment with DNAMAN software, filter out the highly conserved sequence of MO
Target sequence as amplification.
According to design of primers principle and the reaction principle of LAMP technology, in Primer ExplorerV4 software system design
The multipair primer pair producing, and filter out suitable primer pair and carry out primer synthesis.The designed primer pair of this research includes one
To inner primer (FIP-BIP) and a pair of outer primer (F3-B3), amplifying target genes fragment length is 188bp, Primer and sequence
Row are shown in Table 1.
Table 1 LAMP specific primer title and sequence
3LAMP and PCR reaction system
In the 25 μ L LAMP reaction systems that this experiment is set up, each 1.2 μm of ol/L of inner primer (FIP, BIP), outer primer
(F3, B3) each 0.2 μm of ol/L, dNTP 1.2mmoI/L 2.5 μ L, MgSO48mmol/L, Betaine 0.8mol/L2 μ L, l0
× Bst DNA polymerase buffer liquid, the Bst archaeal dna polymerase 1 μ L of 8U, the MO bacterium solution 2 μ L after boiling, complement to 25 with ultra-pure water
μL.Reaction condition:In 55-65 DEG C of reaction temperature, it is spaced 1 DEG C and is incremented by, each temperature all reacts 1h, last 80 DEG C of effect 5min are eventually
Only reaction PCR reacts the anti-sensitivity of the LAMP being set up and method contrast reference.It is MO that PCR reacts expanded target gene
16S rDNA, expands forward primer F:5 '-ATGGTAGTTAAAGTTGGTATTAACG-3 ', downstream primer R:5’-
TTATTTAGCGATTTTTGCAAAGTAC-3 ', primer size is in 320bp.PCR reaction system totally 25 μ L, including upstream and downstream
The each 5mmol/L of primer, 2 × Taq MasterMix, 2 μ L template DNA, ultra-pure water complements to 25 μ L.Reaction condition:95 DEG C of pre- changes
Property 5min, 94 DEG C of degeneration 60s, 58 DEG C annealing 60s, 72 DEG C extension 35s, 35 circulation, 72 DEG C extension 10min.10 DEG C of preservations.
The operation sequence of 4LAMP reaction
The LAMP reactant liquor preparing reacts 60min in 63 DEG C of thermostat water bath, subsequently in 80 DEG C of water-bath
Inactivation 5min terminating reaction.After the completion of reaction, only degree change of liquid in pipe is reacted in perusal, and is seen after high speed centrifugation
Examine ttom of pipe and have or not white pyro acid salt precipitation, also can determine whether amplified reaction.Together, amplified production and 6 × loading
Buffer carries out 2% agarose gel electrophoresiies after mixing, and obtains electrophoresis using ultraviolet gel imaging system after 70V voltage 50min
As a result, according to the brightness having or not trapezoid-shaped strips and band it may be appreciated that LAMP reaction amplification efficiency.
The condition optimizing of 5LAMP reaction system
On the basis of conventional LAMP reaction system and reaction condition, for the LAMP that the base of amplification different primers and n is not busy
Amplified reaction, to reaction temperature, Mg2+, dNTP, Betaine concentration, the inside and outside primer concentration when dominant response bar such as response time
Part is optimized, and obtains optimal reaction system.
5.1 optimal Mg2+The selection of concentration
Adjustment MgSO4Addition Mg2+, make the Mg in reaction system2+Concentration respectively 1.0mmo 1/L, 2.0mmol/L,
4.0mmol/L, 6.0mmoI/L, 8.0mmol/L, 10.0mmol/L, 12.0mmol/L (Mg of negative control group2+Concentration is
8.0mmol/L), the electrophoresis result according to amplified production, filters out optimal Mg2+ concentration.
The hoof choosing of 5.2 optimal reaction temperatures
Temperature is designed as successively 55 DEG C, 56 DEG C, 57 DEG C, 58 DEG C, 59 DEG C, 60 DEG C, 61 DEG C, 62 DEG C, 63 DEG C, 64 DEG C, 65
DEG C (the anti-degree of positive control pipe be 64 DEG C) is expanded, is selected the optimum response temperature that amplification efficiency highest temperature is experiment
Degree.Test confirms, trapezoid-shaped strips produced by 63 DEG C the most substantially, brightness maximum, primer dimer is minimum, therefore this selection
The peak optimization reaction temperature of 63 DEG C of LAMP reactions set up as this test.
The screening of 5.3 optimal dNTP concentration
Adjustment LAMP system in dNTP addition, make dNTP concentration in system be followed successively by 0.4mmol/L, 0.6mmoI/L,
0.8mmoI/L, 1.0mmol/L, 1.2mmol/L, 1.4mmol/L, 1.6mmol/L, 1.8mmol/L and 2.0mmol/L are (negative right
The dNTP concentration looked after is 1.4mmol/L) expanded, therefrom it is selected to ensure that the minimum of amplification efficiency highest dNTP needs
The amount of playing.Experiment proves that, the dNTP peak optimization reaction concentration that 1.2mmol/L reacts by set up LAMP.
Inside and outside 5.4, primer concentration is than screening
The concentration of holding outer primer F3/B3 is constant, and final concentration is always 0.2 μm of ol/L, carries out interior as experiment centre plinth
The adjustment of primers F IP/BIP concentration.The addition of inner primer FIP/BIP in adjustment LAMP system, makes inner primer FIP/BIP with outward
The final concentration proportioning of primers F 3/B3 is followed successively by 2:1、4:1、6:1、8:1、10:1、12:1 (the interior outer primer in negative control pipe is dense
Degree ratio is 8:1), select to expand the high interior outer primer matched proportion density in ground effect domain.It is experimentally verified that, it is optimal that set up LAMP reacts
Inside and outside primer concentration ratio is set to 6:1.
The impact that 5.5 glycine betaine Betaine concentration are reacted to LAMP
In reaction system Betaine Concentraton gradient set gradually for 0.0mol/L, 0.2mol/L, 0.4mol/L,
0.6moI/L, 0.8mol/L, 1.0mol/L (in positive control pipe, Betaine concentration is 0.8mol/L), therefrom optimization domain is closed
The working concentration of suitable glycine betaine Betaine.It is experimentally verified that, the glycine betaine Betaine of 0.8mol/L is peak optimization reaction concentration.
The impact that 5.6 response time reacted to LAMP
Amplified reaction reaction is asked and is set as successively:10min、20min、30min、40min、50min、60min、
70min (reaction of positive control is asked as 60min), judges to complete the shortest the asking of LAMP reaction.It is experimentally verified that, the most suitable anti-
It is 60min between seasonable.
The analysis of 6LAMP amplified production
6.1LAMP amplified production observe interpretation of result
After the completion of LAMP amplified reaction, every tube reaction liquid is separately added into 100 × SYBR Green I 1 μ L, visible
Directly detect by an unaided eye under light, reaction result is judged according to the color change of reaction liquid in pipe.If reactant liquor becomes green
It is then that result is positive, it is that result is negative that reactant liquor becomes brown.Use Portable ultraviolet light irradiation, if reaction tube has fluorescence, be judged to
Positive findingses, otherwise for negative (Fig. 1).
The gel imaging analysis of 6.2LAMP amplified production
In the condition optimizing stage of LAMP reaction system, compare, for accurate, the amplification efficiency that under different condition, LAMP reacts,
Need to play and gel electrophoresiss are carried out to LAMP amplified production.Electrophoresis, takes 2LAMP amplified production and 6X loading buffer by 5:1
Ratio all hooks and carries out 2% fine jade tire sugar gel electrophoresiss after mud closes, and is divided according to glue using ultraviolet gel imaging system after 70V electrophoresis 50min
Analysis electrophoresis result.Amplified reaction product, through sepharose electrophoresis, all occurs in that expected staged band.
7LAMP specific test
To staphylococcus aureuses, escherichia coli, bacillus subtilises, Listeria monoeytogenes, epidermis Fructus Vitis viniferae
Ball Tong, Salmonella carry out extracting genome DNA, to extract DNA as template, according to above-mentioned optimum reaction condition, prepare 25 μ L
Visualization LAMP detection system reactant liquor, carries out the specific detection of LAMP method.Result staphylococcus aureuses, large intestine bar
Bacterium, bacillus subtilises, Listeria monoeytogenes, epidermis Fructus Vitis viniferae ball Tong, Salmonella amplification are negative (figure
2) it was demonstrated that the LAMP method set up has very high specificity.
8LAMP sensitivity testss
By the MO of culture to exponential phase, optimal reaction system and reaction condition according to visualization LAMP are expanded
Increase, determine the lowest detection limit.When result is detected using LAMP, bacterial concentration extension rate is 10-7Template still may be used
Obtain stepped band to expand, calculated according to bacterium solution CCU (bacterium solution color changing units), the detection MO LAMP method of foundation
10 CCU/ml (Fig. 3) can be detected.
The detection of 9 clinical samples
Embrocated with sterile cotton swabs and obtain sheep nasal secretion, be placed in the 10ml centrifuge tube of sterilizing, gather Xinjiang altogether
Totally 60 parts of 5, area sheep raising field sheep nose swab sample, low temperature is transported to laboratory and is detected.Containing sterile cotton swabs
The normal saline of 1ml sterilizing, fully vibration mixing is added in centrifuge tube.Take the liquid after process, be placed in Eppendorf, 12
000r/min is centrifuged 2min,
Sterile purified water washes twice, and is finally suspended with 300 μ L TE buffer, water proof boils 15min, 8 000r/min
Centrifugation 15min, takes supernatant, i.e. DNA profiling solution.(use KM2 with the MO visualization LAMP reaction set up, traditional separation and Culture
Culture medium) and PCR method clinical sample is detected, statistics three kinds of detection methods testing result, according to formula calculate
The sensitivity of LAMP visual method and conventional bacteria separation and Culture and PCR method, specificity and coincidence rate.Result is as shown in table 2,
Traditional bacteria distribution culture positive is 20 parts, and feminine gender is 40 parts;PCR detection positive is 22 parts, and feminine gender is 38 parts;
LAMP detection positive is that 22 parts of feminine genders are 38 parts.Through analysis, LAMP detection method with the coincidence rate of antibacterial culturing is
90.91%, be 100% with the coincidence rate of PCR testing result, show LAMP visual detection method have height sensitivity,
Specificity and coincidence rate.
The testing result to sheep clinical sample for table 2 LAMP
The above is only the preferred embodiment of the present invention it is noted that above-mentioned preferred implementation be not construed as right
The restriction of the present invention, protection scope of the present invention should be defined by claim limited range.For the art
For those of ordinary skill, without departing from the spirit and scope of the present invention, some improvements and modifications can also be made also should regard
For protection scope of the present invention.
<110>Shihezi Univ
<120>Echinococcus Granulosus Cysts multi-epitope fusion diagnosis antigen protein, preparation method and applications
<141>
<160> 3
<210> 1
<211> 155
<212>Aminoacid sequence
<213>Echinococcus granulosus(Echinococcus granulosus.)
<220>
<221> misc_feature
<223>Eg-meAg1 protein amino acid sequence
<400> 1
MVVKKRWGELRDFFRNDGPGTGNCDQGKAQSANVTGGPGDDGLTSTSRSVMKMFGEVKYFFERDPLGQKVVDL
LEELGPGTQAITAPDLTTPYPSSGPGRDFFRSDPLGQKGPGKFTEKPKREWRGPGLEEEDEDDLKGPGIETPR
AGKKESTVM
<210> 2
<211> 468
<212>Nucleotide sequence
<213>Eg-meAg1 protein gene(Codon is not optimised)
<220>
<221> misc_feature
<223>Eg-meAg1 protein gene nucleotide sequence
<400> 1
ATGGTGGTAAAAAAAAGATGGGGTGAACTTCGAGACTTCTTTAGAAATGATGGCCCGGGCACTGGCAATTGTG
ATCAAGGAAAAGCACAAAGTGCCAATGTGACAGGAGGCCCGGGCGATGATGGCCTCACCTCGACGTCGAGGAG
TGTGATGAAAATGTTTGGCGAAGTGAAGTACTTCTTCGAACGTGATCCGTTGGGTCAGAAAGTGGTTGACCTC
TTAGAGGAACTGGGCCCGGGCACCCAGGCAATTACTGCTCCAGACCTGACAACCCCATATCCCAGTTCTGGCC
CGGGCCGGGACTTCTTTAGAAGTGATCCACTGGGTCAAAAAGGCCCGGGCAAGTTTACTGAGAAGCCTAAACG
GGAATGGCGCGGCCCGGGCCTGGAAGAAGAAGATGAGGATGATTTAAAGGGCCCGGGCATTGAGACACCGCGC
GCTGGCAAGAAGGAAAGCACTGTAATGTAA
<210> 3
<211> 468
<212>Nucleotide sequence
<213>Eg-meAg1 protein gene(After codon optimization)
<220>
<221> misc_feature
<223>Eg-meAg1 protein gene nucleotide sequence
<400> 1
ATGGTGGTAAAAAAACGTTGGGGTGAACTTCGTGACTTCTTTCGTAATGATGGCCCGGGCACTGGCAATTGTG
ATCAAGGAAAAGCACAAAGTGCCAATGTGACAGGAGGCCCGGGCGATGATGGCCTCACCTCGACGTCGCGTAG
TGTGATGAAAATGTTTGGCGAAGTGAAGTACTTCTTCGAACGTGATCCGTTGGGTCAGAAAGTGGTTGACCTC
TTAGAGGAACTGGGCCCGGGCACCCAGGCAATTACTGCTCCAGACCTGACAACCCCATATCCGAGTTCTGGCC
CGGGCCGGGACTTCTTTCGTAGTGATCCACTGGGTCAAAAAGGCCCGGGCAAGTTTACTGAGAAGCCTAAACG
GGAATGGCGCGGCCCGGGCCTGGAAGAAGAAGATGAGGATGATTTAAAGGGCCCGGGCATTGAGACACCGCGC
GCTGGCAAGAAGGAAAGCACTGTAATGTAA
Claims (6)
1. a kind of method of quick detection mycoplasma ovine pneumoniae is it is characterised in that realized by procedure below:
A, the gene order design LAMP primer according to detected mycoplasma ovine pneumoniae;
B, the reaction system of establishment LAMP visual method, and LAMP reaction condition is optimized;
C, by the sheep collecting nasal secretion, produce DNA profiling solution, with step B gained reaction system and reaction condition
Detected.
2. the method for quick detection mycoplasma ovine pneumoniae as claimed in claim 1 is it is characterised in that designed by step A
LAMP primer includes a pair of inner primer and a pair of outer primer, and amplifying target genes fragment length is 188bp.
3. a kind of method of quick detection mycoplasma ovine pneumoniae as claimed in claim 2 is it is characterised in that described LAMP draws
The sequence of thing is respectively:
Inner primer:FIP——CCTACCAACTAACTAAAAAATGGGGGTGC
BIP——GATTGAGATACGGCCCGTAGGAG TCTGG
Outer primer:F3——AAGGAGCCTTTAAAGCTC
B3——GCAGCAGTAAGGAATAT .
4. the method for the quick detection mycoplasma ovine pneumoniae as described in any one of claims 1 to 3 is it is characterised in that step B
Establish LAMP visual method reaction system concrete operations be:With every 20-30 μ L LAMP reaction system as part, inner primer
(FIP, BIP) each 1-1.5 μm of ol/L, each 0.1-0.3 μm of ol/L of outer primer (F3, B3), dNTP1.0-1.5mmoI/L 2.5 μ L,
MgSO47-10mmol/L, Betaine 0.7-1mol/L 1-3 μ L, l0 × Bst DNA polymerase buffer liquid, the Bst DNA of 8U
Polymerase 0.8-1.2 μ L, the MO bacterium solution 1-3 μ L after boiling, complement to 25-30 μ L, reaction condition with pure water:At 55-65 DEG C
Reaction temperature, 1-2 DEG C of interval is incremented by, and each temperature all reacts 0.5-1.5h, last 75-85 DEG C of effect 3-8min terminating reaction.
5. the method for quick detection mycoplasma ovine pneumoniae as claimed in claim 4 is it is characterised in that optimize described in step B
LAMP reaction condition is afterwards:Reaction temperature 60-65 DEG C, Mg2+ concentration be 7-9mmol/L, dNTP concentration be 1.0-1.5mmol/L,
Glycine betaine Betaine concentration 0.6-1mol/L, inside and outside primer concentration is than for 5-7:1, the response time is 30-90min.
6. any one of Claims 1 to 5 as described in quick detection mycoplasma ovine pneumoniae method it is characterised in that institute
The concrete operations stating step C are:Embrocate sheep nasal secretion with sterile cotton swabs, be placed in the centrifuge tube of sterilizing, gather altogether
More than totally 60 parts of at least 5 sheep raising field sheep nose swab samples, low temperature is transported to laboratory and is detected, containing sterile cotton swabs
Centrifuge tube in add the normal saline of 1ml sterilizing, fully vibration mixing, take the liquid after process to be placed in container, 10000-
14000r/min is centrifuged 1-3min, and sterile purified water washes twice above, finally with the suspension of 200-400 μ L TE buffer, water proof
Boil 10-20min, 7000-10000r/min is centrifuged 10-20min, takes supernatant, i.e. DNA profiling solution, can with set up MO
Depending on changing LAMP reaction, clinical sample is detected, the testing result of statistical detection method.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201611082202.0A CN106434994A (en) | 2016-11-30 | 2016-11-30 | Rapid detecting mycoplasma ovipneumoniae method |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201611082202.0A CN106434994A (en) | 2016-11-30 | 2016-11-30 | Rapid detecting mycoplasma ovipneumoniae method |
Publications (1)
Publication Number | Publication Date |
---|---|
CN106434994A true CN106434994A (en) | 2017-02-22 |
Family
ID=58223774
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201611082202.0A Pending CN106434994A (en) | 2016-11-30 | 2016-11-30 | Rapid detecting mycoplasma ovipneumoniae method |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN106434994A (en) |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN107142331A (en) * | 2017-07-14 | 2017-09-08 | 福建省农业科学院畜牧兽医研究所 | A kind of primer and probe for being used to detect mycoplasma ovine pneumoniae |
CN109666752A (en) * | 2019-02-02 | 2019-04-23 | 广西壮族自治区兽医研究所 | A kind of real-time quantitative LAMP primer group and kit detecting mycoplasma ovine pneumoniae |
CN111118183A (en) * | 2020-01-19 | 2020-05-08 | 石家庄海关技术中心 | Real-time fluorescent RAA primer, probe and kit for detecting mycoplasma ovipneumoniae and using method of kit |
CN112760392A (en) * | 2020-11-17 | 2021-05-07 | 中国农业科学院兰州兽医研究所 | Specific PCR primer for detecting mycoplasma ovipneumoniae and application |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102634602A (en) * | 2012-05-10 | 2012-08-15 | 贵州大学 | Kit for loop-mediated isothermal amplification detection of Mycoplasma ovipneumoniae and preparation and usage methods thereof |
-
2016
- 2016-11-30 CN CN201611082202.0A patent/CN106434994A/en active Pending
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102634602A (en) * | 2012-05-10 | 2012-08-15 | 贵州大学 | Kit for loop-mediated isothermal amplification detection of Mycoplasma ovipneumoniae and preparation and usage methods thereof |
Non-Patent Citations (1)
Title |
---|
张双翔: "绵羊肺炎支原体LAMP检测方法的建立及初步应用", 《中国兽医学报》 * |
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN107142331A (en) * | 2017-07-14 | 2017-09-08 | 福建省农业科学院畜牧兽医研究所 | A kind of primer and probe for being used to detect mycoplasma ovine pneumoniae |
CN109666752A (en) * | 2019-02-02 | 2019-04-23 | 广西壮族自治区兽医研究所 | A kind of real-time quantitative LAMP primer group and kit detecting mycoplasma ovine pneumoniae |
CN111118183A (en) * | 2020-01-19 | 2020-05-08 | 石家庄海关技术中心 | Real-time fluorescent RAA primer, probe and kit for detecting mycoplasma ovipneumoniae and using method of kit |
CN112760392A (en) * | 2020-11-17 | 2021-05-07 | 中国农业科学院兰州兽医研究所 | Specific PCR primer for detecting mycoplasma ovipneumoniae and application |
CN112760392B (en) * | 2020-11-17 | 2021-08-27 | 中国农业科学院兰州兽医研究所 | Specific PCR primer for detecting mycoplasma ovipneumoniae and application |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN100580091C (en) | Zoonosis tuberculosis fluorescence PCR rapid diagnosis kit | |
CN106434994A (en) | Rapid detecting mycoplasma ovipneumoniae method | |
CN101144775B (en) | Bacteria real-time fluorescence quantitative polymerase chain reaction detection reagent kit | |
CN103045761B (en) | For detecting test kit and the detection method of infectious eye disease pathogenic microorganism | |
Sharawi et al. | Isolation, Serological and Real time PCR diagnosis of Peste Des Petites Ruminants virus in naturally exposed Arabian Gazelle in Saudi Arabia. | |
CN104928260B (en) | A kind of infectious bovine rhinotrachetis virus IBRV-JN03 separation strains and its application | |
WO2023045426A1 (en) | Coxsackie virus a16 strain and immunogenic composition and application thereof | |
CN102094080A (en) | Quick molecular detection method for simultaneously detecting three kinds of fusarium toxins and application thereof | |
CN112176080B (en) | Nested PCR primer group, kit and detection method for specifically detecting purple sisal leaf roll disease phytoplasma | |
CN106434935A (en) | Composition and method for identifying pasteurella multocida and/or haemophilus parasuis | |
CN104946753A (en) | Specificity primer pair for cow mycoplasma detection, detection kit, as well as using method and application of detection kit | |
CN104004842A (en) | Multiplex PCR primer set and detection method for simultaneously detecting three pathogenic bacteria causing sepsis of aquatic animals | |
CN101974633B (en) | Method for quantitatively detecting microcystin and specific standard product thereof | |
CN103789442B (en) | A kind of FRET-PCR in real time and nest-type PRC detect and the rickettsial primer of somatotype, probe and test kit | |
CN111690551A (en) | Separation, purification, culture and identification method for brucella | |
CN109811073A (en) | Double PCR early stage quickly detects primer and its application of Streptococcusagalactiae and Streptococcus iniae | |
CN110195096A (en) | It is a kind of for detecting the sample processing method of bloodstream infection pathogenic bacteria | |
CN104313163B (en) | Method and kit for triple PCR detection of porcine infectious actinobacillus pleuropneumonia serum types 2, 3 and 6 and application of porcine infectious actinobacillus pleuropneumonia serum types 2, 3 and 6 | |
CN1952174A (en) | LUX fluorescent primer special for detecting bovine herpes virus type I and process for nucleic acid amplification | |
CN104032000B (en) | The detection method of a kind of bacillus cereus and test kit | |
CN101736088A (en) | Rapid differential diagnosis method of riemerella anatipestifer disease and colibacillosis | |
CN101353699A (en) | Polyase chain reaction detecting method of Campylobacter jejuni | |
CN111304342A (en) | PCR kit for identifying Brucella strain S2 vaccine strain and wild strains of other species and use method thereof | |
CN201107259Y (en) | Bacteria real time fluorescent fixed quantity polyase chain reaction testing reagent box | |
CN104388575B (en) | Kit for identifying nucleic acid of mycobacterium pathogeny through multiple PCR (polymerase chain reaction) |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
C06 | Publication | ||
PB01 | Publication | ||
C10 | Entry into substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
RJ01 | Rejection of invention patent application after publication | ||
RJ01 | Rejection of invention patent application after publication |
Application publication date: 20170222 |