CN108796091A - Loop-mediated isothermal amplification is applied to the genotyping detection method and detection kit of more room/Echinococcus Granulosus Cysts - Google Patents
Loop-mediated isothermal amplification is applied to the genotyping detection method and detection kit of more room/Echinococcus Granulosus Cysts Download PDFInfo
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Abstract
The present invention relates to genotyping detection methods and detection kit that a kind of loop-mediated isothermal amplification (LAMP) is applied to more room/Echinococcus Granulosus Cysts, it include the detection designed for the selection and LAMP amplified productions of the LAMP primer, LAMP amplification conditions of distinguishing more room/Echinococcus Granulosus Cysts, the method of the present invention targetedly parting can detect the sample containing more room/Echinococcus Granulosus Cysts, accuracy is high, susceptibility is high, easy to operate, amplification can be visually observed directly.
Description
Technical field
The invention belongs to technical field of biological, and in particular to a kind of loop-mediated isothermal amplification (LAMP) is answered
Method for more rooms/Echinococcus Granulosus Cysts parting detection.
Background technology
Loop-mediated isothermal amplification, i.e. LAMP (Loop-mediated isothermal amplification)
Annealing extension need not be deformed under isothermal conditions by being one kind, it is only necessary to which simplest water-bath can carry out the new of nucleic acid amplification
Technology, design of primers is primarily directed to six different regions of target gene, the area F3c, F2c and Flc held based on target gene 3 '
And 5 ' the different sites in 6, the area Bl, B2 and B3 etc. at end design 4 kinds of primers, including FIP (Forward Inner
Primer)：Upstream internal primer is made of the areas F2 and the regions F1C, and the areas F2 and the regions F2c that target gene 3 ' is held are complementary, the areas F1C
It is identical as the Flc regional sequences that target gene 5 ' is held；F3 primers：Upstream outer primer (Forward Outer Primer), by F3
District's groups at, and with the regions F3c of target gene complementation；BIP primers：Downstream inner primer (Backward Inner Primer), by
The regions B1C and B2 form, and the areas B2 and the regions B2c that target gene 3 ' is held are complementary, the Blc regional sequences that the domains B1C are held with target gene 5 '
It is identical；B3 primers：Downstream outer primer (Backward Outer Primer), is made of and the areas B3c of target gene the regions B3
Domain is complementary.The amplification of LAMP is divided into two stages, and the 1st stage was initial period, complementary portion of any one primer to double-stranded DNA
When position carries out base pairing extension, another chain will dissociate, and become single-stranded.The F2 sequences of upstream internal primers F IP first with
Template F2c is combined, and is extended forward under the action of strand displacement type archaeal dna polymerase and is started strand displacement synthesis.External primers F3 and mould
Plate F3c is combined and is extended, and displaces the complementary single strand of complete FIP connections, the F1c on FIP with this it is single-stranded on Fl be complementation
Structure.Self base pairing forms cyclic structure, using this chain as template, downstream primer BIP and B3 successively start similar to FIP and
The synthesis of F3 forms the single-stranded of dumbbell structure.It is rapidly starting point using itself as template using the Fl sections of 3 ' ends, carries out DNA
Synthesis extends to form stem loop structure, which is the initial structure of LAMP gene magnifications cycle.2nd stage was amplification cycles rank
Section, using stem loop structure as template, FIP is combined with the areas F2c of stem ring.Start strand displacement to synthesize, on the single-chain nucleic acid dissociateed
Also cyclic structure can be formed, rapidly using the B1 sections of 3 ' ends as starting point, using itself as template, carries out DNA synthesis extension and chain
Displacement, forms the DNA of the new stem loop structure of different in size 2, and the B2 on BIP primers is hybrid with it, and starts new round amplification,
And product DNA length doubles.The end product of amplification is the mixing for having different number loop-stem structures, different length DNA
Object, and product DNA is the alternately inverted repetitive sequence for expanding target sequence.Due to the technology have quickly, accurate, hypersensitivity,
Isothermal duplication is not necessarily to the advantages that specialized equipment equipment, in recent years, is widely used in the quick detection of pathogenic microorganism.
Echinococcus (echinococcus) is recognized at least 5 kinds, is Echinococcus granulosus respectively
(E.granulosus), Echinococcus multilocularis (E.mulitilocularis), Fu Shi echinococcus (E.vogeli), save spine less
Ball tapeworm (E.oligarthra) and Shiqu echinococcus (E.shiquicus), wherein Echinococcus moltilocularis and Echinococcus Granulosus Cysts are wide
The general north and southwest for being popular in China.There is presently no using LAMP technology parting detection Echinococcus moltilocularis and particulate spine
The report of the ball larva of a tapeworm or the cercaria of a schistosome mainly uses Microscopical Method For Detection at present for the diagnosis of Echinococcus moltilocularis and Echinococcus Granulosus Cysts.But due to echinococcus
Individual is small, and conventional microscopy has observation morphological differences unobvious, and changes that method takes time and effort and sensitivity is low, has at present
The method that MGB probe real-time quantitative PCR partings detect Echinococcus moltilocularis and Echinococcus Granulosus Cysts, but need special detecting instrument
(fluorescence quantitative PCR instrument) is not suitable for using in base.Therefore, how quickly, accurate, sensitive parting detect more room spine
The ball larva of a tapeworm or the cercaria of a schistosome and Echinococcus Granulosus Cysts, and make testing result is straightforward not need specific apparatus, it has also become urgent problem to be solved.
Invention content
The present invention in view of the shortcomings of the prior art, provides a kind of by designing for more rooms/Echinococcus Granulosus Cysts mitochondrial DNA
The little ND2 gene primers of sequence similarity, are detected Echinococcus moltilocularis and Echinococcus Granulosus Cysts using LAMP partings, are mediated with ring
Isothermal amplification is applied to the genotyping detection method and detection kit of more room/Echinococcus Granulosus Cysts.
In order to achieve the object of the present invention, the present invention adopts the following technical scheme that：A kind of ring mediated isothermal nucleic acid amplification skill
Art is applied to the genotyping detection method of more room/Echinococcus Granulosus Cysts, it is characterised in that：The ring mediated isothermal nucleic acid amplification applied draws
Object to including primer ND2A (Em) and primer ND2A (Eg),
Wherein, the sequence of primer ND2A (Em) is as follows：
ND2A(Em)-F3：TGTCATATTTCTTTGTCGTCTG
ND2A(Em)-B3：CAACACACACACTCAACTT
ND2A(Em)-FIP：
ACCCCCAGAAAAAGTAATACCAAT-ACATTGGTTGTTGCATGTT
ND2A(Em)-BIP：
GTGGTTGTAGCAGATTCAACAGATT-GCATAGATACAGGAGTCACT；
The sequence of primer ND2A (Eg) is as follows：
ND2A(Eg)-F3：CAGGTGTTTGGTGTGGTT
ND2A(Eg)-B3：TGTTAAATCAGAGACGACCG
ND2A(Eg)-FIP：
GCAACAACTAACGTAGAAACAGACG-AGGTTAAGTTTGGAGTATATATGGT
ND2A(Eg)-BIP：
ATAGTGAGACGCAAACTTGTTTTTT-CAAAATAAACAATTCTACACAGCC。
A kind of aforementioned loop-mediated isothermal amplification is applied to the inspection of the genotyping detection method of more room/Echinococcus Granulosus Cysts
Test agent box, it is characterised in that：The kit contains primer ND2A (Em) as described in claim 1 and primer ND2A (Eg).
Further, the kit further includes having 10 × ThermoPol Buffer, MgSO4 (100mM), dNTP Mix
(10mM), Bst DNA polymerase (8000U/ml) and LAMP visible dyes.
Further, the kit further includes having standard positive template.
It is applied to the genotyping detection method of more room/Echinococcus Granulosus Cysts, profit based on aforementioned loop-mediated isothermal amplification
Parting is carried out to Echinococcus moltilocularis and Echinococcus Granulosus Cysts with the detection kit containing primer ND2A (Em) and primer ND2A (Eg)
LAMP is detected.
The loop-mediated isothermal amplification is applied to the genotyping detection method of more room/Echinococcus Granulosus Cysts, including following
Step：
1) total DNA in sample tissue is extracted；
2) using the total DNA in step 1) as template, draw respectively using primer ND2A (Em) and primer ND2A (Eg) as reaction
Object carries out LAMP amplified reactions with different reaction conditions；
3) product with 2% Ago-Gel and LAMP visible dyes analysis DNA after LAMP is expanded.
Further, for respectively using primer ND2A (Em) and primer ND2A (Eg) as the LAMP reactants of reaction primer
System is calculated as with 25 μ L：
Primer ND2A (Em)：10 × ThermoPol Buffer, 2.5ul；MgSO4 (100mM), 1.5ul；dNTP Mix
(10mM)3.4ul；FIP (40umol/L), 1.0ul；BIP (40umol/L), 1.0ul；F3 (5umol/L), 1.0ul；B3
(5umol/L), 1.0ul；Bst DNApolymerase (8000U/ml), 1.0ul；LAMP visible dyes, 1.25ul；DNA moulds
Plate, 2 μ L；DdH2O, 9.35 μ L；Overall reaction system is 25 μ L；
Primer ND2A (Eg)：10 × ThermoPol Buffer, 2.5ul；MgSO4 (100mM), 1.7ul；dNTP Mix
(10mM), 3.5ul；FIP (40umol/L), 1.0ul；BIP (40umol/L), 1.0ul；F3 (5umol/L), 1.0ul；B3
(5umol/L), 1.0ul；Bst DNA polymerase (8000U/ml), 1.0ul；LAMP visible dyes, 1.25ul；DNA
Template, 2 μ L；DdH2O, 9.05 μ L；Overall reaction system is 25 μ L.
Further, for respectively using primer ND2A (Em) and primer ND2A (Eg) as the LAMP reactions of reaction primer
Condition is：Primer ND2A (Em)：64 degrees Celsius of reactions 60min, 82 degrees Celsius of 10min make enzyme-deactivating；Primer ND2A (Em)：61 take the photograph
Family name's degree reacts 60min, and 82 degrees Celsius of 10min make enzyme-deactivating；2% Ago-Gel and the detection LAMP amplifications of LAMP visible dyes
As a result, sequence is：Template is Em DNA, and template is Eg DNA, and template is water, and template is normal human tissue DNA.
The present invention is compared with conventional method and is had the following advantages：
The first, accuracy is high, and the present invention is according to Echinococcus moltilocularis with Echinococcus Granulosus Cysts mtdna sequence without too big phase
LAMP primer is designed like the ND2 genes of degree, accurately parting can detect Echinococcus moltilocularis and Echinococcus Granulosus Cysts mitochondria
DNA；
The second, easy to operate, can directly macroscopic LAMP visible dyes reaction system be added in the present invention, be not required to
Will separately plus detection method can directly observing response as a result, convenient for base use；
Third, susceptibility are high, and the present invention extracts DNA as template, respectively with primer ND2A (Em) and primer from sample
ND2A (Eg) carries out LAMP efficient amplifications as reaction primer can be right since the copy number of LAMP is just higher by 1000 times than PCR
Micro worm sources mitochondrial DNA is expanded and reaches the level of parting detection in sample.
Description of the drawings
2% agarose gel electrophoresis testing result figure of Fig. 1 Echinococcus moltilocularis and the detection of Echinococcus Granulosus Cysts parting, wherein A
For ND2A (Em) primer, 1：Marker, 2：Template is Em DNA, 3：Template is Eg DNA, 4：Ultra-pure water is as reaction template, and 5：
The tissue DNA of normal person is as reaction template；B be ND2A (Eg) primer, 1：Marker, 2：Template is Em DNA, 3：Template is
Eg DNA, 4：Ultra-pure water is as reaction template, and 5：The tissue DNA of normal person is as reaction template；
Fig. 2 is the LAMP visible dyes result figures of Echinococcus moltilocularis and Echinococcus Granulosus Cysts parting detection, wherein A is
ND2A (Em) primer, 1：Template is EmDNA, 2：Template is EgDNA, 3：Ultra-pure water is as reaction template, and 4：The tissue of normal person
DNA is as reaction template；B be ND2A (Eg) primer, 1：Template is EmDNA, 2：Template is EgDNA, 3：Ultra-pure water is as reaction
Template, 4：The tissue DNA of normal person is as reaction template；
Fig. 3 is 2% agarose gel electrophoresis of Echinococcus moltilocularis and the reaction temperature optimization of Echinococcus Granulosus Cysts parting detection
Testing result figure, wherein A be ND2A (Em) primer, 1：Marker, 2：61 DEG C, 3：62 DEG C, 4：63 DEG C, 5：64 DEG C, 6：65℃；B
For ND2A (Eg) primer, 1：Marker, 2：61 DEG C, 3：62 DEG C, 4：63 DEG C, 5：64 DEG C, 6：65℃；
Fig. 4 is 2% agarose gel electrophoresis of Echinococcus moltilocularis and the MgSO4 concentration optimizations of Echinococcus Granulosus Cysts parting detection
Testing result figure, wherein A be ND2A (Em) primer, 1：Marker, 2：1.3×10-4MM, 3：1.4×10-4MM, 4：1.5×10-4MM, 5：1.6×10-4MM, 6：1.7×10-4mM；B be ND2A (Eg) primer, 1：Marker, 2：1.3×10-4MM, 3：1.4×
10-4MM, 4：1.5×10-4MM, 5：1.6×10-4MM, 6：1.7×10-4mM；
Fig. 5 is 2% Ago-Gel of Echinococcus moltilocularis and the dNTP Mix concentration optimizations of Echinococcus Granulosus Cysts parting detection
Electrophoresis detection result figure, wherein A be ND2A (Em) primer, 1：Marker, 2：3.3×10-5MM, 3：3.4×10-5MM, 4：3.5
×10-5MM, 5：3.6×10-5MM, 6：3.7×10-5mM；B be ND2A (Eg) primer, 1：Marker, 2：3.3×10-5MM, 3：
3.4×10-5MM, 4：3.5×10-5MM, 5：3.6×10-5MM, 6：3.7×10-5mM。
Specific implementation mode
Further clear, complete description is done to technical scheme of the present invention below in conjunction with the accompanying drawings.
The present invention is based on the stability of more rooms/Echinococcus Granulosus Cysts mitochondrial DNA, and design is for Echinococcus moltilocularis and particulate spine
The primer of ball larva of a tapeworm or the cercaria of a schistosome mitochondrial DNA ND2 genes, filters out the spy that can distinguish Echinococcus moltilocularis and Echinococcus Granulosus Cysts mitochondrial DNA
Specific primer reuses real LAMP and is expanded, and 2% agarose gel electrophoresis and LAMP visible dyes are carried out to amplified production
Detection and analysis, can reach the level of accurate judgement Echinococcus moltilocularis and Echinococcus Granulosus Cysts DNA.Primer after optimized and
Amplification condition carries out LAMP experiments have shown that Echinococcus moltilocularis and Echinococcus Granulosus Cysts DNA can be identified completely.
Material
1. sample：Sample containing Echinococcus moltilocularis and the sample containing Echinococcus Granulosus Cysts；
2.DNA is extracted：It is extracted using the DNA kits of Tiangeng biochemical technology Co., Ltd；
3.ND2A (Em) and ND2A (Eg), LAMP Bst DNApolymerase；
4.PCR instrument：Eppendrof-6325.
One：The extraction of DNA
1) the echinococcus sample of 1.5mL centrifuge tubes, 2mg adds 200 μ l buffer solution GA, oscillation to suspend to thorough.
2) 20 μ l Proteinase K solution are added, mixing is placed at 56 DEG C, until histolysis, brief centrifugation is to go
Except the droplet of cap wall, then carry out next step.
3) 200 μ l buffer solution GB are added, fully reverse mixing, 70 DEG C of placement 10min, limpid, the brief centrifugation of solution strain
To remove the droplet of cap wall.
4) add 200 μ l absolute ethyl alcohols of people, mixing 15sec is fully vibrated, at this time it is possible that flocculent deposit, brief centrifugation
To remove the droplet of cap wall.
5) previous step acquired solution and flocculent deposit be all added in an adsorption column CB3 (adsorption column is put into collecting pipe
In), 12000rpm (~13400 × g) centrifuges 30sec, outwells waste liquid, adsorption column CB3 is put back in collecting pipe.
6) 500 μ l buffer solutions GD (please first checked whether before use and absolute ethyl alcohol has been added) are added into adsorption column CB3,
12000rpm (~13400 × g) centrifuges 30sec, outwells waste liquid, adsorption column CB3 is put into collecting pipe.
7) 600 μ l rinsing liquids PW (please first checked whether before use and absolute ethyl alcohol has been added) are added into adsorption column CB3,
12000rpm (~13400 × g) centrifuges 30sec, outwells waste liquid, adsorption column CB3 is put into collecting pipe.
Repetitive operation step 7).
Adsorption column CB3 is put back in collecting pipe, 12000rpm (~13400 × g) centrifuges 2min, outwells waste liquid.It will absorption
Column CB3 is placed in 37 DEG C and places several minutes, thoroughly to dry rinsing liquid remaining in sorbing material.
Adsorption column CB3 is transferred in a clean centrifuge tube, 50 μ l elutions are vacantly added dropwise to the intermediate position of adsorbed film
Buffer solution TE, is placed at room temperature for 5min, and 12000rpm (~13400 × g) centrifuges 2min, solution is collected into centrifuge tube.
The obtained solution of centrifugation is added in adsorption column CB3, is placed at room temperature for 2min, 12000rpm (~13400 × g) from
Heart 2min.
Two：Design primer
According to known array (gene accession number：NC_000928.2；AF297617.1 LAMP primer) is designed：
The sequence of ND2A (Em) primer pair is as follows：
ND2A(Em)-F3：TGTCATATTTCTTTGTCGTCTG
ND2A(Em)-B3：CAACACACACACTCAACTT
ND2A(Em)-FIP：
ACCCCCAGAAAAAGTAATACCAAT-ACATTGGTTGTTGCATGTTND2A(Em)-BIP：
GTGGTTGTAGCAGATTCAACAGATT-GCATAGATACAGGAGTCACT
The sequence of ND2A (Eg) primer pair is as follows：
ND2A(Eg)-F3：CAGGTGTTTGGTGTGGTT
ND2A(Eg)-B3：TGTTAAATCAGAGACGACCG
ND2A(Eg)-FIP：
GCAACAACTAACGTAGAAACAGACG-AGGTTAAGTTTGGAGTATATATGGT
ND2A(Eg)-BIP：
ATAGTGAGACGCAAACTTGTTTTTT-CAAAATAAACAATTCTACACAGCC
Primer is synthesized by Sangon Biotech (Shanghai) Co., Ltd..
Three：Echinococcus Granulosus Cysts are reacted with the LAMP that Echinococcus moltilocularis parting detects
Using sample DNA as template, respectively with ND2A (Em) and ND2A (Eg) for primer, LAMP amplifications are carried out.LAMP
Reaction system is：Primer ND2A (Em)：10 × ThermoPol Buffer, 2.5ul；MgSO4 (100mM), 1.5ul；dNTP
Mix (10mM), 3.4ul；FIP (40umol/L), 1.0ul；BIP (40umol/L), 1.0ul；F3 (5umol/L), 1.0ul；B3
(5umol/L), 1.0ul；Bst DNA polymerase (8000U/ml), 1.0ul；LAMP visible dyes, 1.25ul；DNA
Template, 2 μ L；DdH2O, 9.35 μ L；Overall reaction system is 25 μ L.Primer ND2A (Eg)：10 × ThermoPol Buffer,
2.5ul；MgSO4(100mM),1.7ul；DNTP Mix (10mM), 3.5ul；FIP (40umol/L), 1.0ul；BIP(40umol/
L), 1.0ul；F3 (5umol/L), 1.0ul；B3 (5umol/L), 1.0ul；Bst DNApolymerase (8000U/ml),
1.0ul；LAMP visible dyes, 1.25ul；DNA profiling, 2 μ L；DdH2O, 9.05 μ L；Overall reaction system is 25 μ L.Respectively with
ND2A (Em) and the LAMP reaction conditions that ND2A (Eg) is primer are：Primer ND2A (Em)：64 DEG C reaction 60min, 82 degrees Celsius
10min makes enzyme-deactivating；Primer ND2A (Em)：61 DEG C of reactions 60min, 82 degrees Celsius of 10min make enzyme-deactivating.2% Ago-Gel
LAMP amplifications are detected with LAMP visible dyes.2% agarose gel electrophoresis testing result is as shown in Figure 1：ND2A(Em)
Primer only amplifiable Em DNA (only characteristic scalariform bands of swimming lane LAMP positive findings), reaction template Eg
DNA, ultra-pure water, the tissue DNA (there was only primer dimer in swimming lane) neither amplifiable (A in Fig. 1) of normal person；ND2A(Eg)
Primer only amplifiable Eg DNA (only characteristic scalariform bands of swimming lane LAMP positive findings), reaction template Em
DNA, ultra-pure water, the tissue DNA (there was only primer dimer in swimming lane) neither amplifiable (B in Fig. 1) of normal person.LAMP is visible
The results are shown in Figure 2 for photoinitiator dye：Only (LAMP visible dyes become amplifiable Em DNA ND2A (Em) primer from original purple
Blue), reaction template is Eg DNA, and the tissue DNA (LAMP visible dyes are still purple) of ultra-pure water, normal person can not all expand
Increase (A in Fig. 2)；ND2A (Eg) primer only amplifiable Eg DNA (LAMP visible dyes become blue from original purple),
Reaction template is Em DNA, ultra-pure water, tissue DNA (LAMP visible dyes are still purple) neither amplifiable (Fig. 2 of normal person
In B).
Four：Echinococcus moltilocularis and the reaction temperature of Echinococcus Granulosus Cysts parting detection optimize
Within the temperature range of 61-65 DEG C, most suitable Echinococcus moltilocularis is found out, the reaction temperature that Echinococcus Granulosus Cysts expand,
Its 2% agarose gel electrophoresis testing result figure is as shown in Figure 3, wherein A be ND2A (Em) primer, 1：Marker, 2：61 DEG C,
3：62 DEG C, 4：63 DEG C, 5：64 DEG C, 6：65 DEG C, the temperature for being most suitable for Echinococcus moltilocularis amplification is 64 DEG C；B is ND2A (Eg) primer,
1：Marker, 2：61 DEG C, 3：62 DEG C, 4：63 DEG C, 5：64 DEG C, 6：65 DEG C, the temperature for being most suitable for Echinococcus Granulosus Cysts amplification is 61 DEG C.
Five：The MgSO4 concentration optimizations of Echinococcus moltilocularis and the detection of Echinococcus Granulosus Cysts parting
MgSO4 concentration is in 1.3-1.7 × 10-4In the range of mM, most suitable Echinococcus moltilocularis, Echinococcus Granulosus Cysts amplification are found out
MgSO4 concentration, 2% agarose gel electrophoresis testing result figure as shown in figure 4, A be ND2A (Em) primer, 1：Marker,
2：1.3×10-4MM, 3：1.4×10-4MM, 4：1.5×10-4MM, 5：1.6×10-4MM, 6：1.7×10-4MM is most suitable for more rooms
The MgSO4 a concentration of 1.5 × 10 of echinococcus amplification-4mM；B be ND2A (Eg) primer, 1：Marker, 2：1.3×10-4MM, 3：
1.4×10-4MM, 4：1.5×10-4MM, 5：1.6×10-4MM, 6：1.7×10-4MM is most suitable for Echinococcus Granulosus Cysts amplification
A concentration of 1.7 × 10^ of MgSO4-4mM。
Six：The dNTP Mix concentration optimizations of Echinococcus moltilocularis and the detection of Echinococcus Granulosus Cysts parting
DNTP Mix concentration is in 3.3-3.7 × 10-4In the range of mM, most suitable Echinococcus moltilocularis, Echinococcus Granulosus Cysts are found out
The dNTP Mix concentration of amplification, 2% agarose gel electrophoresis testing result figure are as shown in Figure 5：A be ND2A (Em) primer, 1：
Marker, 2：3.3×10-5MM, 3：3.4×10-5MM, 4：3.5×10-5MM, 5：3.6×10-5MM, 6：3.7×10-5MM, most
It is suitble to the dNTP Mix a concentration of 3.4 × 10 of Echinococcus moltilocularis amplification-5mM；B be ND2A (Eg) primer, 1：Marker, 2：3.3×
10-5MM, 3：3.4×10-5MM, 4：3.5×10-5MM, 5：3.6×10-5MM, 6：3.7×10-5MM is most suitable for Echinococcus Granulosus Cysts
The dNTP Mix a concentration of 3.5 × 10 of amplification-5mM。
The present embodiment is merely illustrative of the technical solution of the present invention, rather than its limitations；Although right with reference to the foregoing embodiments
The present invention is described in detail, it will be understood by those of ordinary skill in the art that：It still can be to previous embodiment institute
The technical solution of record is modified, and either carries out equivalent replacement to which part or all technical features；And these are changed
Or it replaces, the range for various embodiments of the present invention technical solution that it does not separate the essence of the corresponding technical solution.Therefore, not inclined
From these modifications or improvements on the basis of spirit of that invention, the scope of protection of present invention is belonged to.
Claims (8)
1. a kind of loop-mediated isothermal amplification is applied to the genotyping detection method of more room/Echinococcus Granulosus Cysts, feature exists
In：The ring mediated isothermal nucleic acid amplification primers applied to including primer ND2A (Em) and primer ND2A (Eg),
Wherein, the sequence of primer ND2A (Em) is as follows：
ND2A(Em)-F3：TGTCATATTTCTTTGTCGTCTG
ND2A(Em)-B3：CAACACACACACTCAACTT
ND2A(Em)-FIP：
ACCCCCAGAAAAAGTAATACCAAT-ACATTGGTTGTTGCATGTT
ND2A(Em)-BIP：
GTGGTTGTAGCAGATTCAACAGATT-GCATAGATACAGGAGTCACT；
The sequence of primer ND2A (Eg) is as follows：
ND2A(Eg)-F3：CAGGTGTTTGGTGTGGTT
ND2A(Eg)-B3：TGTTAAATCAGAGACGACCG
ND2A(Eg)-FIP：
GCAACAACTAACGTAGAAACAGACG-AGGTTAAGTTTGGAGTATATATGGT
ND2A(Eg)-BIP：
ATAGTGAGACGCAAACTTGTTTTTT-CAAAATAAACAATTCTACACAGCC。
2. a kind of point being applied to more room/Echinococcus Granulosus Cysts based on loop-mediated isothermal amplification described in claim 1
The detection kit of type detection method, it is characterised in that：The kit contain primer ND2A (Em) as described in claim 1 and
Primer ND2A (Eg).
3. detection kit as claimed in claim 2, it is characterised in that：The kit further include have 10 ×
ThermoPol Buffer, MgSO4 (100mM), dNTP Mix (10mM), Bst DNApolymerase (8000U/ml) and
LAMP visible dyes.
4. detection kit as claimed in claim 2 or claim 3, it is characterised in that：The kit further includes having standard positive mould
Plate.
5. the parting that loop-mediated isothermal amplification according to claim 1 is applied to more room/Echinococcus Granulosus Cysts is examined
Survey method, it is characterised in that：Using the detection kit containing primer ND2A (Em) and primer ND2A (Eg) to Echinococcus moltilocularis
And Echinococcus Granulosus Cysts carry out parting LAMP detections.
6. the parting that loop-mediated isothermal amplification according to claim 5 is applied to more room/Echinococcus Granulosus Cysts is examined
Survey method, it is characterised in that：This approach includes the following steps：
1) total DNA in sample tissue is extracted；
2) using the total DNA in step 1) as template, respectively using primer ND2A (Em) and primer ND2A (Eg) as reaction primer, with
Different reaction conditions carries out LAMP amplified reactions；
3) product with 2% Ago-Gel and LAMP visible dyes analysis DNA after LAMP is expanded.
7. the parting that loop-mediated isothermal amplification according to claim 6 is applied to more room/Echinococcus Granulosus Cysts is examined
Survey method, it is characterised in that：For respectively using primer ND2A (Em) and primer ND2A (Eg) as the LAMP reactions of reaction primer
System is calculated as with 25 μ L：
Primer ND2A (Em)：10 × ThermoPol Buffer, 2.5ul；MgSO4 (100mM), 1.5ul；dNTP Mix(10mM)
3.4ul；FIP (40umol/L), 1.0ul；BIP (40umol/L), 1.0ul；F3 (5umol/L), 1.0ul；B3 (5umol/L),
1.0ul；Bst DNA polymerase (8000U/ml), 1.0ul；LAMP visible dyes, 1.25ul；DNA profiling, 2 μ L；
DdH2O, 9.35 μ L；Overall reaction system is 25 μ L；
Primer ND2A (Eg)：10 × ThermoPol Buffer, 2.5ul；MgSO4 (100mM), 1.7ul；dNTP Mix
(10mM), 3.5ul；FIP (40umol/L), 1.0ul；BIP (40umol/L), 1.0ul；F3 (5umol/L), 1.0ul；B3
(5umol/L), 1.0ul；Bst DNA polymerase (8000U/ml), 1.0ul；LAMP visible dyes, 1.25ul；DNA
Template, 2 μ L；DdH2O, 9.05 μ L；Overall reaction system is 25 μ L.
8. the parting that loop-mediated isothermal amplification according to claim 6 is applied to more room/Echinococcus Granulosus Cysts is examined
Survey method, which is characterized in that for being reacted respectively using the LAMP of primer ND2A (Em) and primer ND2A (Eg) as reaction primer
Condition be：Primer ND2A (Em)：64 degrees Celsius of reactions 60min, 82 degrees Celsius of 10min make enzyme-deactivating；Primer ND2A (Em)：61
Degree Celsius reaction 60min, 82 degrees Celsius of 10min make enzyme-deactivating；2% Ago-Gel and LAMP visible dyes detection LAMP expand
Increase as a result, sequence is：Template is Em DNA, and template is Eg DNA, and template is water, and template is normal human tissue DNA.
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| CN109234410A (en) * | 2018-11-15 | 2019-01-18 | 海南国际旅行卫生保健中心（海南出入境检验检疫局口岸门诊部） | A method of the detection echinococcosis based on loop-mediated isothermal amplification technique |
| CN111197093A (en) * | 2019-02-28 | 2020-05-26 | 北京市动物疫病预防控制中心 | LAMP (loop-mediated isothermal amplification) detection primer group, kit and method for echinococcus caninum |
| CN111518877A (en) * | 2020-05-12 | 2020-08-11 | 青海大学 | One-tube method nest type real-time quantitative PCR detection kit for detecting echinococcus multilocularis and echinococcus granulosus by parting trace samples |
| CN112226517A (en) * | 2020-09-22 | 2021-01-15 | 杭州医学院 | Primer group for loop-mediated isothermal amplification detection and distinguishing echinococcus granulosus from echinococcus multilocularis |
| CN113512590A (en) * | 2020-04-09 | 2021-10-19 | 中国疾病预防控制中心寄生虫病预防控制所(国家热带病研究中心) | Kit for detecting different echinococcus species based on honeycomb chip and detection method |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109234410A (en) * | 2018-11-15 | 2019-01-18 | 海南国际旅行卫生保健中心（海南出入境检验检疫局口岸门诊部） | A method of the detection echinococcosis based on loop-mediated isothermal amplification technique |
| CN111197093A (en) * | 2019-02-28 | 2020-05-26 | 北京市动物疫病预防控制中心 | LAMP (loop-mediated isothermal amplification) detection primer group, kit and method for echinococcus caninum |
| CN113512590A (en) * | 2020-04-09 | 2021-10-19 | 中国疾病预防控制中心寄生虫病预防控制所(国家热带病研究中心) | Kit for detecting different echinococcus species based on honeycomb chip and detection method |
| CN111518877A (en) * | 2020-05-12 | 2020-08-11 | 青海大学 | One-tube method nest type real-time quantitative PCR detection kit for detecting echinococcus multilocularis and echinococcus granulosus by parting trace samples |
| CN112226517A (en) * | 2020-09-22 | 2021-01-15 | 杭州医学院 | Primer group for loop-mediated isothermal amplification detection and distinguishing echinococcus granulosus from echinococcus multilocularis |
| CN112226517B (en) * | 2020-09-22 | 2023-10-20 | 杭州医学院 | Primer group for detecting and distinguishing echinococcosis granulosa and echinococcosis multifilialis through loop-mediated isothermal amplification |
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