CN105755158A - LAMP detection primer for Nosema ceranae and detection kit employing LAMP detection primer - Google Patents
LAMP detection primer for Nosema ceranae and detection kit employing LAMP detection primer Download PDFInfo
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- CN105755158A CN105755158A CN201610312738.0A CN201610312738A CN105755158A CN 105755158 A CN105755158 A CN 105755158A CN 201610312738 A CN201610312738 A CN 201610312738A CN 105755158 A CN105755158 A CN 105755158A
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Abstract
The invention discloses an LAMP detection primer for Nosema ceranae and a detection kit employing the LAMP detection primer. The LAMP detection primer comprises outer primers Nosc-F3/Nosc-B3 and inner primers Nosc-FIP/Nosc-BIP, of which the sequences are shown in SEQ ID NO.1-4. The LAMP detection method for Nosema ceranae and the detection kit employing the LAMP detection primer are established by the primer, and the detection kit comprises a primer group, a reaction buffer solution, a positive reference substance, a negative reference substance, a color development solution, BstDNA polymerase and sterile deionized water. The detection kit can quickly and flexibly detect Nosema ceranae, is simple and convenient to operate, low in cost, free from special complicated instruments, easy in reaction result determination and strong in specificity, can better meet the crying needs for illness monitoring, on-site emergency and production sample detection and is easy for wide-range popularization.
Description
Technical field
The invention belongs to biological technical field.LAMP detection more particularly, to a kind of Eastern bee microsporidian is drawn
Thing and detection kit thereof.
Background technology
Eastern bee microsporidian is that a kind of important bee fungus disease is former, extensively parasitizes Apis midgut tissue, causes
Apis, especially Apis mellifera suffer from microsporidiosis, not only shorten adult honeybee individual life span but also weaken bee colony group's gesture, also shadow
Ring bee product yield.Meanwhile, the most still without the medicine of a kind of effective preventing and treating Nosema apis parasitosis.
In scientific research and beekeeping, infect the behavior after microsporidian and midgut tissue metamorphosis by Apis
Diagnose with the method for microscope inspection, result all relatively lag behind and unreliable, verification and measurement ratio is low and is difficult to distinguish two kinds of spores
(as obscured mutually with yeast cells in middle intestinal during microscopy), Electronic Speculum authentication method needs higher specialized operations technology and precision
Instrument, PCR detection needs instrument costly and detection time longer, and LAMP testing cost is cheap and without complex instrument.
Current PCR be detected on two kinds of Nosema apis parasitosis cause of diseases be mostly for 16S rRNA(SSU) region sequence (Higes M,
Martin R, Meana A. Nosema ceranae, a new microsporidian parasite in honeybees
in Europe. J.Inver.Pat., 2006, 92(2): 93-95；Chen, Yanping, Evans, Jay D.,
Smith, I. Bart, Pettis, Jeffery S. Nosema ceranae is a long-present and wide-
spread microsporidian infection of the European honey bee (Apis mellifera) in
The United States. J.Inver.Pat., 2008,97 (2): 186-188.), but Gisder etc. (Gisder S,
Genersch E. Molecular differention of Nosema apis and Nosema ceranae based on
species-specific sequence differences in a protein coding gene. J.Inver.Pat.,
2013,113 (1): 1-6.) report with 16S rRNA for knot during extension increasing sequence two kinds of Nosema apis parasitosis aggregate samples of detection
The unreliability of fruit and low positive rate, and (Erler S, Lommatzsch S, the Lattorff H M. such as Erler
Comparative analysis of detection limits and specificity of molecular
diagnostic markers for three pathogens(Microsporidia, Nosema spp.) in the key
pollinators Apis mellifera and Bombus terrestris. Parasitol Res, 2012, 110
(4): 1403-1410.) have studied forefathers' report detects micro-spore (Nosema with ITS, SSU or 16S rRNA for amplification region
The specificity of PCR primer spp.) and susceptiveness, part primer is Nosema bombi, N. apis and N. ceranae three
Between specificity exist intersect.RPB1 gene (Eastern bee microsporidian: Genbank accession number: XM_002995356.1) is target
The PCR primer detection Eastern bee microsporidian of sequential design there is no non-specific band and false positive results, has also reached very
Good detection sensitivity.But use the PCR primer of this gene design for Eastern bee microsporidian detection operating procedure relatively
Many, and the time of cost is longer, is unsuitable for grass-roots unit's detection, is also unfavorable for that produce is widely popularized and uses.
Ring mediated isothermal amplification (loop-mediated isothermal amplification, LAMP) method is Japanology
Person Notomi etc. invention a kind of novel in vitro isothermal duplication specific nucleic acid technology (Notomi T, Okayama H,
Masubuchi H, Yonekawa T, Watanabe K, Amino N, Hase T. Loop-mediated
Isothermal amplifictaion of DNA. Nucleic Acids Res., 2000,28 (12): e63).Along with
The development of LAMP technology, it is widely used in agricultural herding and makes outstanding achievements.At present, LAMP technology has been applied to detection disease
The detection of the cause of diseases such as poison, antibacterial and fungus (Li Qiming, Ma Xuejun, high and cold spring, Zhou Rui, Kuang Zhizhou, Hou Yunde. reverse transcription loop is situated between
Lead the isothermal amplification (RT-LAMP) use in H5N1 bird flu virus gene test. virus journal, 2008,24
(03): 178-184; Gao, SY, Li DD, Liu, Y, Zha EH, Wang S, Li YG, Zhou TZ, Yue
XQ. Development and evaluation of a RT-LAMP assay for rapid detection of
hepatitis E virus from shellfish INT J FOOD MICROBIOL, 2016, 220: 1-5; Lee J
S, Yong S J, Lim H Y, Yoon B S, A Simple and Sensitive Gene-Based Diagnosis
of Aspergillus flavus by Loop-Mediated Isothermal Amplific-
Ation in Hoeybee. Journal of Apiculture, 30 (1): 53-59.).LAMP method has specificity
By force, quickly, efficiently, the advantage such as sensitivity is high, easy and simple to handle, naked eyes get final product judged result, have both simplified detection process, the most significantly
Shorten the detection time.
Summary of the invention
The technical problem to be solved is the deficiency overcoming prior art Eastern bee microsporidian detection technique,
LAMP detection primer and the detection kit thereof of a kind of Eastern bee microsporidian are provided.Utilize described detection kit, can be accurate
Really, sensitive, be quickly detected from Eastern bee microsporidian.
It is an object of the invention to provide the LAMP detection primer group of one group of Eastern bee microsporidian.
It is a further object of the present invention to provide the application of described LAMP detection primer.
Another object of the present invention is to provide a kind of micro-spore of Eastern bee utilizing the establishment of above-mentioned LMAP detection primer vertical
Worm detection method and test kit.
To achieve these goals, the present invention is achieved by the following technical solutions: a kind of Eastern bee microsporidian
LAMP visual quick detection kit, including pair of inside primer Nosc-F3/Nosc-B3 and pair of outside primer Nosc-
FIP/Nosc-BIP, primer sequence is as shown in SEQ ID NO.1～4.
Further, test kit also comprises genomic DNA lysis solution, LAMP reactant liquor, positive reference substance, negative controls
And nitrite ion；
Described LAMP reactant liquor removes containing primer described above possibly together with 2 × LAMP reaction buffer, Bst archaeal dna polymerase (8
U/ μ L), sterile deionized water.
Preferably, described 2 × LAMP reaction buffer contains 40 mM Tris-HCl(pH8.8)；0.2% Tween20；
20 mM KCl；20 mM (NH4)2SO4；8 mM MgSO4；2.0 mM dNTPs.
Preferably, described lysate contains 2% (W/V) CTAB, 1.4 M NaCl, 2% 2-ME, 100 mM Tris-HCl
(pH8.0), 10% SDS.
Preferably, described positive reference substance is Eastern bee microsporidian genomic DNA or Eastern bee microsporidian
The recombiant plasmid of RPB1 gene, negative controls is normal Eastern bee microsporidian DNA.
Preferably, described nitrite ion is calcein.
The reaction system of described test kit is as follows: when the judgement of testing result uses staining or agarose gel electrophoresis method
Time, reaction system is:
2 × reaction buffer 12.5 μ L；
Primer Nosc-F3/Nosc-B3 0.5 μ L；
Primer Nosc-FIP/Nosc-BIP 3.5 μ L；
Bst DNA polymerase 8U/ μ L 1.0 μ L；
Sample DNA 1.0 μ L；
Deionized water 2.5 μ L；
Or when the judgement of testing result uses real-time fluorescence method, reaction system is:
2 × reaction buffer 12.5 μ L；
Primer Nosc-F3/Nosc-B3 0.5 μ L；
Primer Nosc-FIP/Nosc-BIP 3.5 μ L；
Bst DNA polymerase 8U/ μ L 1.0 μ L；
Sample DNA 1.0 μ L；
Deionized water 1.5 μ L；
Luciferase assay reagent 1.0 μ L；
In reaction system, the concentration of primer can determine according to concrete reaction, primer Nosc-F3 and Nosc-B3 in preferred version
It is 5 pmol；Primer Nosc-FIP and Nosc-BIP is 35 pmol.
The LAMP reaction condition of described test kit is 55～61 DEG C of isothermal reactions 40～70 min；Then put at 80～90 DEG C
Put 5 min inactivation BstDNA polymerase termination reactions.
A kind of method with test kit described above detection Eastern bee microsporidian, comprises the steps:
S1. testing sample DNA is extracted；
S2. in reaction tube, prepare reaction system；
S3. in reaction system, add nitrite ion；
S4. reaction: by reaction tube 55～61 DEG C of isothermal reaction 40～70 min；Then 5 min inactivations are placed for 80～90 DEG C
BstDNA polymerase termination reacts.
S5. result judges: result interpretation has development process and observes the sedimentation method, agarose gel electrophoresis method.
(1) observing color change, reactant liquor color becomes light green color, illustrates that testing sample contains the micro-spore of Eastern bee
Worm；Product becomes orange, then do not contain Eastern bee microsporidian；The method can be sunk in conjunction with observing magnesium pyrophosphate simultaneously
The presence or absence formed sediment, positive findings has magnesium pyrophosphate to precipitate, and negative findings is without precipitation.
(2) agarose gel electrophoresis method: take the 5 reacted products of μ L, mixes with 1 μ L 6 × Loading buffer,
In 1.8% agarose gel electrophoresis (buffer is 1 × TBE)；If trapezoid-shaped strips occurs, illustrate that testing sample contains Eastern bee
Microsporidian；If without the numerous trapezoid-shaped strips differed in size, illustrating that testing sample does not contains Eastern bee microsporidian.
The visualization quick detection kit of the present invention, applies 4 to design based on east microsporidian RPB1 gene order
LAMP primer, it is possible to detect Eastern bee microsporidian specifically.
East of the present invention microsporidian LAMP visual quick detection kit, can use two kinds of methods quickly to take out
Carrying microsporidian DNA, be not required to add glycine betaine and just can complete detection in the system of optimization, therefore cost is relatively low；Utilize special
Property primer and the reaction system of optimization, whole course of reaction only needs water-bath to carry out, and east detected at about 57 DEG C reaction about 1 h
Side's Nosema apis worm DNA concentration reaches 0.136 × 10-6 μg∙μL-1, and can be according to agarose gel electrophoresis scalariform band
Or reactant liquor occurs when being light green color or low-speed centrifugal that white precipitate all can be identified as positive findings after addition calcein, negative
Result is to be brownish red or without white precipitate without scalariform band or reactant liquor；So easy and simple to handle, with low cost, reaction result is easy
In judgement, high specificity, carry out the early monitoring of Eastern bee microsporidian for application LAMP technology and prevent to provide important
Method and technical support, can preferably meet disease surveillance, on-the-spot emergent and beekeeping pattern detection in the urgent need to, easily
In popularization and application on a large scale.
Accompanying drawing explanation
The target sequence of tetra-LAMP primer designs of Fig. 1 and primer location schematic diagram.
In Fig. 2 Eastern bee microsporidian LAMP method and Apis body encountered pathogenic agarose gel (concentration is 1.8%,
Its following concentration is all identical with this) electrophoresis result figure.Wherein M is 100 bp marker, and swimming lane 1 is negative control, swimming lane 2
～9:N. ceranae, N. apis, A. apis, M. pluton, the amplification of DWV, SBV, BQCV, IAPV genome, only
Having N. ceranae to have scalariform band (shown in swimming lane 2), other are without band.
Fig. 3 east microsporidian LAMP method and the dyestuff figure of encountered pathogenic in Apis body.Wherein 1 is negative control, 2～9
It is respectively the amplification colour developing of N. ceranae, N. apis, A. apis, M. pluton, DWV, SBV, BQCV, IAPV genome
As a result, only N. ceranae is light green color (No. 2 pipes) as the reactant liquor that template is corresponding, and other each cause of disease nucleic acid are as template
Corresponding reaction tube color is brownish red.
Fig. 4 sensitivity technique figure agarose gel result figure.Wherein M is 100 bp marker, and 1 is negative control, nothing
Scalariform band, 2～10:DNA concentration are respectively 0.136,0.136 × 10-1、0.136×10-2、0.136×10-3、0.136×
10-4、0.136×10-5、0.136×10-6、0.136×10-7、0.136×10-8 μg·μl-1Swimming lane 2～8 has scalariform band,
Swimming lane 9 and 10 is without scalariform band.The dyestuff result figure of Fig. 5 sensitivity technique.Wherein, pipe 1 be negative control, reactant liquor color
For brownish red, the reactant liquor color of pipe 2～8 is light green color, and the reactant liquor color of pipe 9 and 10 is brownish red.
The agarose gel result figure of Fig. 6 clinical sample detection.Wherein M is 100 bp marker, swimming lane 1 be negative
Comparison, swimming lane 7,8,12,13,15,17 and 26 are without scalariform band, for negative findings；Other sampling test groups all have scalariform band
For positive findings.
The dyestuff figure of Fig. 7 clinical sample detection.Wherein 1 is negative control, and reactant liquor color is brownish red；Pipe numbers 7,8,
12,13,15,17 and 26 reactant liquor colors are brownish red, and for negative findings, other pipe reactant liquors are light green color, tie for the positive
Really.
Detailed description of the invention
The present invention is further illustrated below in conjunction with the accompanying drawings with specific embodiment.Test method used in following embodiment
If no special instructions, it is conventional method；The material that used, reagent etc., if no special instructions, for commercially obtaining
Reagent and material.
Embodiment 1
The preparation method of the LAMP visual quick detection kit of Eastern bee microsporidian, concrete grammar step is as follows:
S1. from GenBank data base, retrieval obtains Eastern bee microsporidian (N. ceranae) and Nosema apis (N.
Apis) the RPB1 sequence of worm, by DNAMAN 8 its sequence of software comparison, then uses online software: PrimerExplore V4
(http://primerexplorer.jp/elamp4.0.0/index.html) design primer authorized company's synthesis LMAP inspection
The primer sets surveyed；Primer sets includes inner primer Nosc-B3:5 ＇-ATGGAATTCCTGTAAAACGAA-3 ＇, and primer sequence is such as
Shown in SEQ ID NO.1；Nosc-F3:5 ＇-TATGCACCCTTTCACCAT-3 ＇, primer sequence is as shown in SEQ ID NO.2；Outward
Primer Nosc-BIP:5 ＇-AAGGAATGGGACTTGTTGCGT AAAATAACTTTTCCATCACTGTC-3 ＇, primer sequence is such as
SEQ ID NO.3；Nosc-FIP:5 ＇-ACAGGCTGTTTATTTCCACATCC-AAGTGTTTGTGAGGGAGAA-3 ＇, primer sequence
Row are such as SEQ ID NO.4.
The target-gene sequence of primer and positional information are as shown in Figure 1.
Two kinds of preparation methoies of the template DNA that S2. Nosema apis parasitosis is former:
(1) manual extraction process: take intestinal in measuring samples Apis, in 1.5 mL sterile centrifugation tube, be fully ground；Add aseptic going
Ionized water, then 12000 rpm are centrifuged 5min, abandon supernatant, repeat supreme clear bright；Careful reject upper strata midgut tissue, obtains
The spore precipitation of lower floor；In advance by lysate (2% CTAB, 1.4 M NaCl, 2% 2-ME, 100 mM Tris-HCl, pH8.0)
It is placed in 65 DEG C of preheatings, adds 2 μ L Protein K and 40 μ L SDS 10% in 400 μ l lysates in the spore liquid to be checked of preparation
In, 65 DEG C hatch 1 h after be cooled to room temperature, then add equal-volume phenol: chloroform: isoamyl alcohol (25:24:1), 12000 rpm from
The heart 5 min, shifts supernatant, extracts 2 times, adds isopyknic chloroform: isoamyl alcohol (24:1), 12000 rpm are centrifuged 5 min, transfer
Supernatant, then adds the NaAC of 3 M of 1/10 volume, mixes gently, adds the isoamyl alcohol of 0.6 times of volume, and 12000 rpm are centrifuged 5
Min, abandons supernatant, adds 500 μ L 70% ethanol rinse precipitations, and 10000 rpm are centrifuged 2 min, abandon supernatant, repeat to rinse once, open
Lid room temperature is inverted 5～10 min, adds 20～50 μ L deionized water dissolving, is put in-20 DEG C of preservations.
(2) fungal gene group extraction agent box method: add 400 μ L Buffer in the above-mentioned spore liquid to be checked prepared
Digestion and 4 μ L 2-ME, concussion mixing.65 DEG C of water-bath 1 h crack completely to cell；Add 200 μ L Buffer PF,
The most reverse mixing, 5 min placed by-20 DEG C of refrigerators；Room temperature 10000 rmp is centrifuged 5 min, and supernatant is transferred to new 1.5
In mL centrifuge tube；Adding isopyknic isopropanol, overturn and be allowed to for 5～8 times fully mix, room temperature places 2～3 min, room temperature
10000 rmp are centrifuged 5 min, abandon supernatant；Adding 600 μ L 75% ethanol, reverse rinsing 1～3 min, 10000 rpm are centrifuged 2
Min, abandons supernatant, repeats to rinse once；Room temperature of uncapping is inverted 5～10 min and is volatilized completely to ethanol, adds 20～50 μ L TE
Buffer or deionized water dissolving ,-20 DEG C of preservations.
Suggestion: when using test kit, it should be ensured that dirty without Eastern bee microsporidian DNA in equipment, reagent and operating environment
Dye, in order to avoid causing false positive, adds reaction tube, then mixing, low speed brief centrifugation after fully mixing reaction caching liquid in addition
To remove the bubble in reactant liquor.
S3. primer sets Nosc-F3/B3 is outer primer, and Nosc-FIP/BIP is inner primer, and LMAP reactant liquor includes as follows
Component: 40 mM Tris-HCl(pH8.8)；0.2% Tween20；20 mM KCl；20 mM(NH4)2SO4；8 mM MgSO4；
2.0 mM dNTPs every kind；Primer Nosc-F3/B3 5 pmol and primer Nosc-FIP/BIP 35 pmol；Deionized water；Aobvious
Color liquid；Mentioned reagent is assembled into box and is Eastern bee microsporidian LAMP visual quick detection kit.Test kit
Detection preparation system is as follows.
2 × reaction buffer 12.5 μ L
Inner primer Nosc-F3/Nosc-B3 0.5 μ L
Outer primer Nosc-FIP/Nosc-BIP 3.5 μ L
Bst DNA polymerase 8U/ μ L 1.0 μ L
Sample DNA 1.0 μ L
Deionized water 2.5 μ L
Cumulative volume 25 μ L
Embodiment 2
The Eastern bee microsporidian LAMP visual quick detection kit prepared with reference to embodiment 1 is applied to examine
Survey, specifically comprise the following steps that
S1. nucleic acid samples to be detected is prepared:
From University Of Agriculture and Forestry In Fujian, teaching bee farm takes 30 groups of apis mellifera bee colonies at random, and every case captures 15 works at random on nest doorway
Honeybee, is then prepared by embodiment 1.
S2. reaction system is set up
Operate by embodiment 2 reaction system component.
Suggestion: to N number of sample, the reactant liquor that should prepare (N+3) times volume (includes that blank, negative control, the positive are right
According to each one, in order to avoid subpackage expends causes error), it is ensured that each reaction subpackage is uniform.
S3. sample-adding: fully mix reactant liquor, adds the deionized water of 2 μ L to reaction tube (blank right the most successively
According to), negative control (Apis normal structure DNA), positive control (Eastern bee microsporidian DNA or the weight of RPB1 genetic fragment
Group plasmid)；Being subsequently adding nitrite ion, lid covered tightly, after mixing, instantaneous low-speed centrifugal is to eliminate the bubble in reactant liquor.Respectively
With Nosema apis worm (Nosema apis), Ascosphaera apis (Ascosphaera apis), Nidus Vespae coccus
(Melissococcus pluton), Apis vestigial wing virus (Deformed wing virus, DWV), Apis metacercoid virus
(Sacbrood virus, SBV), black Beequeen table virus (Black queen cell virus, BQCV) and the acute fiber crops of Israel
Numbness virus (Israel acute paralysis virus, IAPV) genome is the template of specific reaction.
S4. result judges: (5000 rpm are centrifuged 1 with low-speed centrifugal to observe by the naked eye color change under natural light
Min) generate with or without white magnesium pyrophosphate precipitation after；Reactant liquor color becomes light green color (observing color with UV is green fluorescence),
Illustrate that testing sample contains Eastern bee microsporidian, see Fig. 3 (pipe 2), Fig. 5 (pipe 2～8) and Fig. 7 (pipe 2～6,9～11,14,
16,18～25,27～31) the reactant liquor color in；Product becomes orange (observing color redgreen fluorescence with UV), the most not
Containing Eastern bee microsporidian, see Fig. 3 (pipe 1,3～9), Fig. 5 (pipe 1,9,10) and Fig. 7 (pipe 1,7,8,12,13,15,17,
26) the reactant liquor color in；Magnesium pyrophosphate can be had to sink in conjunction with observing the presence or absence that magnesium pyrophosphate precipitates, i.e. positive findings simultaneously
Forming sediment, negative findings is without precipitation, and the result figure with dye method is corresponding；Agarose gel electrophoresis (concentration is 1.8%, buffer is 1 ×
TBE) band is positive findings in gradient, see Fig. 2 (swimming lane 2), Fig. 4 (swimming lane 2～8) and Fig. 6 (swimming lane 2～6,9～11,14,
16,18～25,27～31), it is negative findings without band, sees Fig. 2 (swimming lane 1,3～9), Fig. 4 (swimming lane 1,9,10) and Fig. 6 (swimming
Road 1,7,8,12,13,15,17,26).
The foregoing is only presently preferred embodiments of the present invention, all impartial changes done according to scope of the present invention patent with
Modify, all should belong to the covering scope of the present invention.
SEQUENCE LISTING
<110>University Of Agriculture and Forestry In Fujian
<120>LAMP detection primer of a kind of Eastern bee microsporidian and detection kit thereof
<130> 4
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 21
<212> DNA
<213>artificial sequence
<400> 1
atggaattcc tgtaaaacga a 21
<210> 2
<211> 18
<212> DNA
<213>artificial sequence
<400> 2
tatgcaccct ttcaccat 18
<210> 3
<211> 44
<212> DNA
<213>artificial sequence
<400> 3
aaggaatggg acttgttgcg taaaataact tttccatcac tgtc 44
<210> 4
<211> 42
<212> DNA
<213>artificial sequence
<400> 4
acaggctgtt tatttccaca tccaagtgtt tgtgagggag aa 42
Claims (8)
1. the LAMP detection primer of an Eastern bee microsporidian, it is characterised in that: described primer includes a pair outer primer
Nosc-F3/Nosc-B3 and a pair inner primer Nosc-FIP/Nosc-BIP, the nucleotide sequence of described primer is respectively such as SEQ
Shown in ID NO.1～4.
2. the LAMP detection primer group of Eastern bee microsporidian is examined at preparation Eastern bee microsporidian as claimed in claim 1
Application in test agent box.
Apply the most as claimed in claim 2, it is characterised in that described test kit also includes 2 × reaction buffer, positive control
Product, negative controls, nitrite ion, Bst archaeal dna polymerase, sterilizing deionized water.
Application the most according to claim 3, it is characterised in that the component of described 2 × reaction buffer includes the following: 40
mM、pH8.8 Tris-HCl；0.2wt.% Tween20；20 mM KCl；20 mM(NH4)2SO4；8 mM MgSO4；2.0 mM
dNTPs。
Application the most according to claim 3, it is characterised in that described positive reference substance is Eastern bee microsporidian DNA
Or the recombiant plasmid containing Eastern bee microsporidian RPB1 genetic fragment；Described negative controls is normal Apis intestinal tissue
DNA。
Application the most according to claim 3, it is characterised in that described nitrite ion is calcein.
7. according to the arbitrary described application of claim 2～6, it is characterised in that the reaction system of described test kit is as follows: work as inspection
When surveying judgement employing staining or the agarose gel electrophoresis method of result, reaction system is:
2 × reaction buffer 12.5 μ L；
Primer Nosc-F3/Nosc-B3 0.5 μ L；
Primer Nosc-FIP/Nosc-BIP 3.5 μ L；
Bst DNA polymerase 8U/ μ L 1.0 μ L；
Sample DNA 1.0 μ L；
Deionized water 2.5 μ L；
Or when the judgement of testing result uses real-time fluorescence method, reaction system is:
2 × reaction buffer 12.5 μ L；
Primer Nosc-F3/Nosc-B3 0.5 μ L；
Primer Nosc-FIP/Nosc-BIP 3.5 μ L；
Bst DNA polymerase 8U/ μ L 1.0 μ L；
Sample DNA 1.0 μ L；
Deionized water 1.5 μ L；
Luciferase assay reagent 1.0 μ L；
Wherein, described primer Nosc-F3/Nosc-B3 is 5 pmol, and primer Nosc-FIP/Nosc-BIP is 35 pmol.
8. according to the arbitrary described application of claim 2～6, it is characterised in that the LAMP reaction condition of described test kit is: 57
DEG C isothermal reaction 40～70 min；Then place 5 min inactivation Bst archaeal dna polymerases at 80～90 DEG C and terminate reaction.
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