CN106048029A - LAMP (Loop-mediated Isothermal Amplification) detection primer set, detection kit and detection method of mycoplasma hyopneumonia - Google Patents
LAMP (Loop-mediated Isothermal Amplification) detection primer set, detection kit and detection method of mycoplasma hyopneumonia Download PDFInfo
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Abstract
The invention discloses an LAMP (Loop-mediated Isothermal Amplification) detection primer set, a detection kit and a detection method of mycoplasma hyopneumonia. The detection primer set comprises 6 specific primers. The detection kit comprises primer liquid, reaction liquid, DNA polymerase and a contrast; the kit can also contain a developing agent or a fluorescence indicator. The detection method of the mycoplasma hyopneumonia comprises the steps of extracting DNA of mycoplasma hyopneumonia to be detected and amplifying a sample DNA template at 60-65 DEG C by adopting the 6 specific primers and the DNA polymerase with chain displacement activity, wherein the amplification efficiency can reach 10<9>-10<10> copies in short time; and the identification is to determine whether a sample to be detected contains the mycoplasma hyopneumonia or not through adopting the changes of turbidity of sediment in a reaction pipe, adding a developing agent and observing the changes of colors in the reaction pipe by utilizing a real-time fluorescence detector. The LAMP detection primer set, the detection kit and the detection method of the mycoplasma hyopneumonia have the advantages of rapidness and high efficiency, simplicity in operation, high specificity, high sensitivity, simplicity in identification, applicability to field detection and the like, and are suitable for popularizing and applying.
Description
Technical field
The present invention relates to technical field of molecular biology, relate to the method for quick turning mycoplasmal pneumonia of swine, specifically
LAMP detection primer group, detection kit and the detection method thereof of a kind of mycoplasma hyopneumoniae.
Background technology
The present invention relates to technical field of molecular biology, relate to the method for quick turning mycoplasmal pneumonia of swine, specifically
LAMP detection primer group, detection kit and the detection method thereof of a kind of mycoplasma hyopneumoniae.
Summary of the invention
It is an object of the invention to provide the LAMP detection primer group of a kind of mycoplasma hyopneumoniae, detection kit and based on
The constant temperature gene amplification detection method of the mycoplasma hyopneumoniae of above-mentioned detection primer group and detection kit, to solve above-mentioned background
The problem proposed in technology.
For achieving the above object, the present invention provides following technical scheme:
The LAMP detection primer group of a kind of mycoplasma hyopneumoniae, including outer primer 1 and outer primer 2, inner primer 1 and inner primer 2, just
Ring primer LF and anti-ring primer LB, its nucleotide sequence is the most as follows:
Outer primer 1:GTATTTGCCTCGGTCATTT SEQ ID No:1；
Outer primer 2:CCTTTGCTGATTATACCCAG SEQ ID No:2；
Inner primer 1:TCCCTGAATCTCTAACTCGTGAGGGGAGATATTATCAAGTTCC SEQ ID No:3；
Inner primer 2:TCGGCACGAGTTTCAAATCTTAAGAATGGATGTCGATCTTG SEQ ID No:4；
Positive ring primer LF:TCATTATTCGCCAAGTACCG SEQ ID No:5；
Anti-ring primer LB:TCGATCTTAGCCAAGAAAGTC SEQ ID No:6.
A kind of LAMP detection kit of mycoplasma hyopneumoniae, including following component:
(1) primer liquid: containing the primer sets described in claim 1, concentration is respectively outside 48～52 μMs of outer primers 1,48～52 μMs
Primer 2,1,48～52 μMs of inner primers 2 of 48～52 μMs of inner primers；
(2) reactant liquor: containing 12 mM dNTP, 10 × Isothermal Amplification reaction buffer, 150 mM
MgSO4 aqueous solution, the volume ratio of three is 7～9:4～6:2；
(3) archaeal dna polymerase: concentration is 7～9U/ μ l；
As the further scheme of the present invention: containing 50 μMs of outer primers, 2,50 μMs of inner primers of 1,50 μMs of outer primers in described primer liquid
1,50 μMs of inner primers 2.
As the present invention further scheme: described archaeal dna polymerase is Bst archaeal dna polymerase, concentration is 8U/ μ l.
As the present invention further scheme: in described reactant liquor, 12mM dNTP:10 × Isothermal
Amplification reaction buffer: the volume ratio of 150mM MgSO4 is 8:5:2.
As the present invention further scheme: possibly together with developer, described developer is SYBR green/HNB dyestuff.
The method of the detection kit detection mycoplasma hyopneumoniae of described mycoplasma hyopneumoniae, comprises the steps:
1) extraction of measuring samples DNA: use boiling method extraction purification sample DNA；
2) Constant Temperature Detection reaction: preparation reaction system in PCR pipe: primer liquid 1.8 μ l, reactant liquor 15.2 μ l, archaeal dna polymerase 1 μ
L, DNA 1～6 μ l to be checked, with DNase/RNase-Free Distilled Water polishing to 25 μ l；It is anti-that positive control is set
At once, the DNA with mycoplasma hyopneumoniae or the e. coli plasmid dna containing genes of interest substitute DNA to be checked, arrange negative control
During reaction, substitute DNA to be checked with the reaction mixture without genes of interest；It is centrifuged after the PCR pipe mixing that will prepare, and in 60
～65 DEG C of reactions 45～90min, and continue 2min at 80 DEG C；
3) result judges: judge amplification by observing the turbidity change of precipitation in PCR pipe, or naked eyes developer or
Fluorescence curve method judges.
As the present invention further scheme: the LAMP detection kit detection hyopneumoniae of described mycoplasma hyopneumoniae
The method of mycoplasma, the boiling method described in step 1) extracts the method for mycoplasma hyopneumoniae and is:
A () will be equipped with 1.5 mL centrifuge tubes of 100 uL liquid samples, 12000 rpm, centrifugal 1min, remove supernatant；
B () adds 0.15 g bead, and 200 μ L 5% Chelex100 in 1.5 mL centrifuge tubes；
(c) concussion instrument maximum (top) speed vortex 5200rpm, vortex concussion 3min, after brief centrifugation 100 DEG C, 5min, 12000 rpm
Centrifugal 2min；
D supernatant is transferred in new centrifuge tube by (), standby as template.
The present invention is based on loop-mediated isothermal amplification technique (LAMP method), according to six primer energy specificitys of target gene design
Identify six isolated areas on target-gene sequence, start endless-chain displacement reaction, start complementary strand synthesis in target region of DNA,
Result is gone round and begun again the stem-circular DNA mixture of the Brassica oleracea L. var. botrytis L. structure being formed with a lot of ring on same chain.Use 4 specificitys
Primer and a kind of archaeal dna polymerase with strand-displacement activity, carry out amplified reaction at 60～65 DEG C to nucleic acid, and reaction need to be at constant temperature
Under the conditions of carry out, the response time according to template DNA mass change, generally 90 min or less, in the short time of 45～90min
Interior amplification efficiency can reach 109～1010Individual copy.Add template DNA, after 60～65 DEG C of reactions 45～90min, at 80 DEG C
Insulation 2min, terminates reaction.In the reaction, when having nucleic acid to synthesize in a large number, the pyrophosphate ion separated out from dNTP is molten with reaction
Mg ions binding in liquid, produces the white precipitate of by-product magnesium pyrophosphate.
Compared with prior art, the invention has the beneficial effects as follows:
(1) rapidly and efficiently: whole amplification only can complete with 45～90min, amplification yield is up to 109～1010Individual copy；
(2) easy and simple to handle: to need not the instrument of complexity, it is not necessary to special reagent, it is not necessary to carry out the degeneration etc. of double-stranded DNA in advance
Tedious steps, it is only necessary to a real-time fluorescence detector just can react and detect, and condition is gentleer；
(3) high specific: the present invention, according to devising six specific primers, applies above-mentioned six primers, the 6 of amplification target sequence
Individual region, has the strongest strain specificity, and highly stable, forms primer dimer probability low, it is ensured that reaction smooth
Carry out；
(4) high sensitivity: the lowest detection limit can reach 10 copies；
(5) qualification is easy: can be by observing addition color change, whether there is magnesium pyrophosphate precipitation or whether occur that " S " type expands
Increase curve to judge whether to expand, it is not necessary to other any analytical procedures such as electrophoresis, be suitable for Site Detection.
Accompanying drawing explanation
Fig. 1 is sensitivity test and the gel electrophoresis figure one of mycoplasma hyopneumoniae LAMP primer in embodiment 2.
Fig. 2 is sensitivity test and the gel electrophoresis figure two of mycoplasma hyopneumoniae LAMP primer in embodiment 2.
Fig. 3 is that in embodiment 3, the amplification under the MH1 primer system of mycoplasma hyopneumoniae LAMP specific detection curve chart is bent
Line chart.
Fig. 4 is the amplification under MH 2 primer system of mycoplasma hyopneumoniae LAMP specific detection curve chart in embodiment 3
Curve chart.
Fig. 5 is that in embodiment 3, the amplification under the MH3 primer system of mycoplasma hyopneumoniae LAMP specific detection curve chart is bent
Line chart.
Fig. 6 is that in embodiment 3, the amplification under the MH4 primer system of mycoplasma hyopneumoniae LAMP specific detection curve chart is bent
Line chart.
Fig. 7 is the MH5 of mycoplasma hyopneumoniae LAMP specific detection curve chart in embodiment 3, under 6,8-12 primer system
Amplification curve diagram.
Fig. 8 is the amplification under MH 7 primer system of mycoplasma hyopneumoniae LAMP specific detection curve chart in embodiment 3
Curve chart.
Fig. 9 is naked eyes coloring reaction system application drawing in mycoplasma hyopneumoniae LAMP detection system.
Detailed description of the invention
Below in conjunction with the accompanying drawing in the embodiment of the present invention, the technical scheme in the embodiment of the present invention is carried out clear, complete
Describe, it is clear that described embodiment is only a part of embodiment of the present invention rather than whole embodiments wholely.Based on
Embodiment in the present invention, it is every other that those of ordinary skill in the art are obtained under not making creative work premise
Embodiment, broadly falls into the scope of protection of the invention.
Embodiment 1
The LAMP detection kit of mycoplasma hyopneumoniae specifically used
The LAMP detection kit of mycoplasma hyopneumoniae, including primer liquid, reactant liquor, archaeal dna polymerase, comparison and developer:
(1) primer liquid: containing 1,50 μMs of outer primers of 50 μMs of outer primers, 1,50 μMs of inner primers 2 of 2,50 μMs of inner primers, four primers are:
Outer primer 1:GTATTTGCCTCGGTCATTT(SEQ ID No:1)；
Outer primer 2:CCTTTGCTGATTATACCCAG(SEQ ID No:2)；
Inner primer 1:TCCCTGAATCTCTAACTCGTGAGGGGAGATATTATCAAGTTCC(SEQ ID No:3)；
Inner primer 2:TCGGCACGAGTTTCAAATCTTAAGAATGGATGTCGATCTTG(SEQ ID No:4).
Positive ring primer LF:TCATTATTCGCCAAGTACCG (SEQ ID No:5)
Anti-ring primer LB:TCGATCTTAGCCAAGAAAGTC (SEQ ID No:6)
(2) reactant liquor: containing 12mM dNTP, 10 × Isothermal Amplification reaction buffer, 150mM
MgSO4 aqueous solution, the volume ratio of three is 8:5:2；
(3) archaeal dna polymerase:BstArchaeal dna polymerase, concentration is 8U/ μ l；
(4) comparison: the e. coli plasmid dna containing genes of interest, negative control is the reaction mixture without genes of interest；
(5) developer: fluorescent dye or naked eyes developing dye.
In the present embodiment, positive naked eyes colour developing is sky blue, and feminine gender and blank colour developing are purple, and sky blue shows
Testing sample is mycoplasma hyopneumoniae, it is judged that for positive findings.
Embodiment 2
The comparison of mycoplasmal pneumonia of swine LAMP reaction method sensitivity:
According to mycoplasmal pneumonia of swine LAMP sensitivity experiment scheme, mycoplasmal pneumonia of swine type strain genomic DNA is diluted respectively
To 0.4 × 10-1 ng/μL、0.4×10-2 ng/μL, 0.4×10-3 ng/μL, 0.4×10-4 ng/μL, 0.4×10-5
Ng/ μ L, 0 ng/ μ L 6 gradients altogether, each dilution factor takes 2 L respectively and carries out LAMP experiment, replaces DNA profiling with ddH2O
As negative control, using artificial constructed positive plasmid as positive control, carrying out LAMP amplification, 63 DEG C are run 60 min, knot
Really shown in Fig. 1-2.
The mycoplasmal pneumonia of swine sample using variable concentrations gradient dilution carries out sensitivity test research.Result is as follows:
System is carried out the stability test under lowest detectable limit simultaneously, test result indicate that and have good stability.
Under the amplification system filtering out LAMP, in sensitivity test, stable detection can go out 0.4 × 10-4Ng/ μ L concentration
Mycoplasma hyopneumoniae.6 passages are blank, and 1 passage is 0.4 × 10-1ng/μL；2 passages are 0.4 × 10-2Ng/ μ L, 3
Passage is 0.4 × 10-3Ng/ μ L, 4 passages are 0.4 × 10-4Ng/ μ L, 5 passages are 0.4 × 10-5Ng/ μ L target pathogen.
Embodiment 3
The specificity experiments of mycoplasma hyopneumoniae specificity LAMP primer
The reaction system of optimizing application and the mycoplasma hyopneumoniae 12 of design overlap LAMP primer, carry out LAMP primer screening and
The assessment of reaction effect.Result is as shown in figures 3-8.
In 12 set primers, primer 1,2,3,4 have amplification；Other are because of undesirable without amplification or expanding effect, Qi Zhongyin
Objects system 1 has the best expanding effect, can carry out sensitivity as follow-up alternative primer and specificity is further
Test.
Embodiment 4
The test kit of developer and detection method thereof:
As it is shown in figure 9, study according to concrete naked eyes developer.This project uses HNB (4 mM, 25 ul)+Sybr Green
(500 ×, 50 ul) nitrite ion, carry out the colour developing research of LAMP mycoplasmal pneumonia of swine naked eyes.2 uL meat are added in reaction system
Accent color liquid is on reaction tube lid, and 63 DEG C are run 60 min, and after reaction, reverse mixing carries out naked eyes colour developing, and sentence read result is such as
Under.Positive naked eyes colour developing is sky blue, and feminine gender and blank colour developing are purple, and yin and yang attribute judges obvious, Ke Yizuo
Carry out for conventional naked eyes colour developing, keep concordance with fluorescence curve and electrophoresis detection in sensitivity simultaneously.
1 is mycoplasma hyopneumoniae standard plasmid；2 concentration are 0.4 × 10-2 ng/ μ L；3 concentration are 0.4 × 10-3 ng/ μ
L；4 concentration are 0.4 × 10-4 ng/ μ L；5 passages are 0.4 × 10-5 ng/ μ L；6 is blank.
It is obvious to a person skilled in the art that the invention is not restricted to the details of above-mentioned one exemplary embodiment, Er Qie
In the case of the spirit or essential attributes of the present invention, it is possible to realize the present invention in other specific forms.Therefore, no matter
From the point of view of which point, all should regard embodiment as exemplary, and be nonrestrictive, the scope of the present invention is by appended power
Profit requires rather than described above limits, it is intended that all by fall in the implication of equivalency and scope of claim
Change is included in the present invention.Should not be considered as limiting involved claim by any reference in claim.
Although moreover, it will be appreciated that this specification is been described by according to embodiment, but the most each embodiment only wraps
Containing an independent technical scheme, this narrating mode of description is only that for clarity sake those skilled in the art should
Description can also be formed those skilled in the art through appropriately combined as an entirety, the technical scheme in each embodiment
May be appreciated other embodiments.
SEQUENCE LISTING
<110>Technical Center for Animal, Plant and Food Inspection and Quarantine, Shanghai Entry-Exit Inspection and Quarantine Bureau
<120>LAMP detection primer group, detection kit and the detection method thereof of mycoplasma hyopneumoniae
<130> 2016
<160> 6
<170> PatentIn version 3.3
<210> 1
<211> 19
<212> DNA
<213>artificial sequence
<400> 1
gtatttgcct cggtcattt 19
<210> 2
<211> 20
<212> DNA
<213>artificial sequence
<400> 2
cctttgctga ttatacccag 20
<210> 3
<211> 43
<212> DNA
<213>artificial sequence
<400> 3
tccctgaatc tctaactcgt gaggggagat attatcaagt tcc 43
<210> 4
<211> 41
<212> DNA
<213>artificial sequence
<400> 4
tcggcacgag tttcaaatct taagaatgga tgtcgatctt g 41
<210> 5
<211> 20
<212> DNA
<213>artificial sequence
<400> 5
tcattattcg ccaagtaccg 20
<210> 6
<211> 21
<212> DNA
<213>artificial sequence
<400> 6
tcgatcttag ccaagaaagt c 21
Claims (8)
1. the LAMP detection primer group of a mycoplasma hyopneumoniae, it is characterised in that include outer primer 1 and outer primer 2, inner primer
1 and inner primer 2, positive ring primer LF and anti-ring primer LB, its nucleotide sequence is the most as follows:
Outer primer 1:GTATTTGCCTCGGTCATTT SEQ ID No:1；
Outer primer 2:CCTTTGCTGATTATACCCAG SEQ ID No:2；
Inner primer 1:TCCCTGAATCTCTAACTCGTGAGGGGAGATATTATCAAGTTCC SEQ ID No:3；
Inner primer 2:TCGGCACGAGTTTCAAATCTTAAGAATGGATGTCGATCTTG SEQ ID No:4；
Positive ring primer LF:TCATTATTCGCCAAGTACCG SEQ ID No:5；
Anti-ring primer LB:TCGATCTTAGCCAAGAAAGTC SEQ ID No:6.
2. the LAMP detection kit of mycoplasma hyopneumoniae, it is characterised in that include following component:
(1) primer liquid: containing the primer sets described in claim 1, concentration is respectively outside 48～52 μMs of outer primers 1,48～52 μMs
Primer 2,1,48～52 μMs of inner primers 2 of 48～52 μMs of inner primers；
(2) reactant liquor: containing 12 mM dNTP, 10 × Isothermal Amplification reaction buffer, 150 mM
MgSO4 aqueous solution, the volume ratio of three is 7～9:4～6:2；
(3) archaeal dna polymerase: concentration is 7～9U/ μ l.
The LAMP detection kit of mycoplasma hyopneumoniae the most according to claim 2, it is characterised in that in described primer liquid
Containing 1,50 μMs of outer primers of 50 μMs of outer primers, 1,50 μMs of inner primers 2 of 2,50 μMs of inner primers.
The LAMP detection kit of mycoplasma hyopneumoniae the most according to claim 2, it is characterised in that described DNA is polymerized
Enzyme isBstArchaeal dna polymerase, concentration is 8U/ μ l.
The LAMP detection kit of mycoplasma hyopneumoniae the most according to claim 2, it is characterised in that described reactant liquor
In, 12mM dNTP:10 × Isothermal Amplification reaction buffer: the volume ratio of 150mM MgSO4 is 8:5:
2。
6. according to the LAMP detection kit of the arbitrary described mycoplasma hyopneumoniae of claim 2-5, it is characterised in that possibly together with
Developer, described developer is SYBR green/HNB dyestuff.
7. the LAMP detection kit detection mycoplasma hyopneumoniae of the mycoplasma hyopneumoniae as described in claim 2-5 is arbitrary
Method, it is characterised in that comprise the steps:
1) extraction of measuring samples DNA: use boiling method extraction purification sample DNA；
2) Constant Temperature Detection reaction: preparation reaction system in PCR pipe: primer liquid 1.8 μ l, reactant liquor 15.2 μ l, archaeal dna polymerase 1 μ
L, DNA 1～6 μ l to be checked, with DNase/RNase-Free Distilled Water polishing to 25 μ l；It is anti-that positive control is set
At once, the DNA with mycoplasma hyopneumoniae or the e. coli plasmid dna containing genes of interest substitute DNA to be checked, arrange negative control
During reaction, substitute DNA to be checked with the reaction mixture without genes of interest；It is centrifuged after the PCR pipe mixing that will prepare, and in 60
～65 DEG C of reactions 45～90min, and continue 2min at 80 DEG C；
3) result judges: judge amplification by observing the turbidity change of precipitation in PCR pipe, or naked eyes developer or
Fluorescence curve method judges.
The method of the LAMP detection kit detection mycoplasma hyopneumoniae of mycoplasma hyopneumoniae the most according to claim 7,
It is characterized in that, the boiling method described in step 1) extracts the method for mycoplasma hyopneumoniae and is:
A () will be equipped with 1.5 mL centrifuge tubes of 100 uL liquid samples, 12000 rpm, centrifugal 1min, remove supernatant；
B () adds 0.15 g bead, and 200 μ L 5% Chelex100 in 1.5 mL centrifuge tubes；
(c) concussion instrument maximum (top) speed vortex 5200rpm, vortex concussion 3min, after brief centrifugation 100 DEG C, 5min, 12000 rpm
Centrifugal 2min；
D supernatant is transferred in new centrifuge tube by (), standby as template.
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| CN108486262A (en) * | 2018-06-06 | 2018-09-04 | 上海速创诊断产品有限公司 | A kind of LAMP primer composition object, kit and its method of detection mycoplasma pneumoniae |
| CN112831578A (en) * | 2020-11-12 | 2021-05-25 | 上海奥普生物医药股份有限公司 | Primer group, kit and method for detecting mycoplasma pneumoniae |
| CN112831578B (en) * | 2020-11-12 | 2022-10-18 | 上海奥普生物医药股份有限公司 | Primer group, kit and method for detecting mycoplasma pneumoniae |
| CN113088578A (en) * | 2021-04-15 | 2021-07-09 | 厦门健康工程与创新研究院 | LAMP primer group, kit and method for detecting mycoplasma pneumoniae |
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