CN106434994A - Rapid detecting mycoplasma ovipneumoniae method - Google Patents

Rapid detecting mycoplasma ovipneumoniae method Download PDF

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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
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lamp
reaction
primer
sheep
concentration
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乔军
孟庆玲
田路路
卢海亭
乔梦凡
贡莎莎
陈诚
李重阳
孟丹
于伟伟
陈创夫
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Shihezi University
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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

A kind of method of quick detection mycoplasma ovine pneumoniae
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.
CN201611082202.0A 2016-11-30 2016-11-30 Rapid detecting mycoplasma ovipneumoniae method Pending CN106434994A (en)

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Cited By (4)

* Cited by examiner, † Cited by third party
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

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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
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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

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