CN107142329A - A kind of upland rice cyst roundworm LAMP quick determination methods - Google Patents
A kind of upland rice cyst roundworm LAMP quick determination methods Download PDFInfo
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Abstract
The present invention relates to a kind of upland rice cyst roundworm LAMP quick determination methods.By comparing upland rice cyst roundworm ribosomes rDNA ITS sequences, screening obtains specific position, and design filters out 5 LAMP primer HE F3, HE B3, HE FIP, HE BIP and HE LB.The present invention extracts upland rice cyst roundworm genomic DNA, pass through ring mediated isothermal amplification, amplified production is developed the color and observed again, so as to quick detection and identify upland rice cyst roundworm, special from 14 kinds of Plant nematodes, 22 colonies it can detect upland rice cyst roundworm, detection threshold value is 1/20000 cephalo capsule, and sensitivity is high 10 times compared with regular-PCR method.The detection method of the present invention has that high specificity, sensitivity be high, easy to operate, efficient quick, and as a result naked eyes be observable, with very high application value in terms of upland rice cyst roundworm quick detection and early stage are with field diagnosis.
Description
Technical field
The present invention relates to a kind of upland rice cyst roundworm LAMP quick determination methods, belong to biological technical field.
Background technology
Upland rice cyst roundworm (Heterodera elachista) is a kind of main plant nematode for infecting paddy rice, most
Earlier than being found (Ohshima Y.Heterodera elachista n sp.an upland in the rice field of Tochigi prefecture,Japan
Rice cyst nematode from Japan [J] .Japanese Journal of Nematology, 1974 (4):51-
56.).Current rice terrace of the nematodiasis in multiple counties of Hunan Province (city) hilly country find first (Ding Z,
Namphueng J, He X F.First report of the cyst Nematode (Heterodera elachista) on
rice in Hunan province,China[J].Plant Disease,2012,96(1):151).Upland rice cyst roundworm one
As colonize in host root, symptom caused by the imbalance of its hazard symptoms and liquid manure is extremely similar, except the nutrition that absorbs host and right
Plant root damages outer, also reduces utilization ratio of the paddy rice to water, then influence host growth and development (in fourth,
The upland rice cyst roundworm history of life such as Namphueng Janthathang, He Xu peaks and infectivity [J] rice in China science,
2012,26(6):746-750), (Bridge J, Luc M, the Plowright R A.Plant of the paddy rice underproduction 7%~19% are caused
Parasitic Nematodes in Tropical and Subtropical Agriculture[M].Wallingford：
CAB International,1990:69–108).Current upland rice cyst nematode disease occurs mainly in middle and lower reach of Yangtze River Rice Cropping
Area, and harm be on the rise, it is possible in the Rice Production as China it is another it is serious threaten, while the hair of the nematodiasis
Cloth estranged is not also very clear and definite, and it is that current wheat crops production urgent need solution is asked that fast and accurately identification is made to the nematode
Topic.
Upland rice cyst roundworm belongs to Cyperi groups, morphologically with H.oryzae, H.sacchari and
H.leuceilyma is very close.It is with H.sacchari and H.leuceilyma clearest differences is that in the case where sporangiocyst is elongated
Lack the projection of finger-type on bridge, and compared with H.oryzae, sporangiocyst is smaller, second instar larvae length is slightly short.Current upland rice sporangiocyst line
The identification of worm is most of to still rely on traditional Morphological Identification method, but is due to that Morphological Identification is time-consuming, it is very ripe to need
Based on experienced technology, and need to specialize in the personnel of classification to complete, when these reasons make traditional authentication method
It is commonly present certain error and is not suitable for large-scale sample detection.
In recent years, with the fast development of molecular biology, the Fast Detection Technique such as PCR widely applies to plant
In the quick detection and identification of nematode.Applying to the detection technique of cyst roundworm at present mainly includes ITS-RFLP, PCR, real
Time PCR etc..
Amiri etc. by H.betae, H.ciceri, H.glycines and H.medicaginis, H.schachtii,
H.trifolii ITS sequence is analyzed, and filters out specific primer SHF6, by this primer and AB28 (or rDNA2) primer
With reference to, construct the one-step dual PCR of beet cyst roundworm one detection method (Amiri S., Subbotin S.A., Moens M.,
An efficient method for identification of the Heterodera schachtii sensu
stricto group using PCR with specific primers.Nematol.medit.2001,29:241-246)。
The method that Subbotin S A, Peng D L, Moens M. detect soy bean cyst roundworm with double PCR, in a PCR reaction
Universal primer D3A and D3B and specific primer GlyF1 and rDNA2 are used in system simultaneously, from 52 soy bean cyst roundworm groups
Two DNA segments (181bp and 345bp) are amplified in body, the susceptibility of detection is up to single sporangiocyst, the age of single head two children
Minim DNA (Subboton S.A., Peng D L, Moens M., the A rapid method for the of worm
identification of the soybean cyst nematode Heterodera glycines using duplex
PCR.Nematology 2001,vol.3(4):365-371)。
Upland rice cyst roundworm PCR Fast Detection Technique researchs have been related to it both at home and abroad.Peng Deliang etc. is according to upland rice sporangiocyst
Nematode specificity RAPD amplified fragments, design specific SCAR label primer HeF1 and HeR1, it is amplifiable go out 434bp specificity
Fragment (patent of invention：Upland rice cyst roundworm upland rice cyst roundworm specific RAPD and SCAR mark rapid molecular detection method-
CN201210200140.4)；Fourth is medium to develop specific detection upland rice cyst roundworm according to upland rice cyst roundworm ITS sequence
PCR method, the specific primer can go out 281bp fragment in upland rice cyst roundworm colony specific amplification, and its sensitivity is up to 1/
256 2 instar larvaes (PCR method-CN201210322051.7 of patent of invention specific detection upland rice cyst roundworm).
Circulation constant temperature amplification technique (loop-mediated isothermal amplification of DNA, LAMP)
It is a kind of New Cycle constant temperature nucleic acid amplification technology of Rong Yan Co., Ltd. of Japan Notomi in 2000 et al. exploitations.(Notomi
T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N and Hase T.Loop-
Mediated isothermal amplification of DNA [J] .Nucleic Acids Res, 2000,28 (12):
E63.), the technology is by recognizing on target sequence the specific primer of 6 specific regions and utilizing the Bst with strand displacement function
Archaeal dna polymerase, under constant temperature can special, Gao Min, rapidly expand target sequence.Japanese Scientists are utilized first within 2008
LAMP technology establishes directly method (Kikuchi T, the Aikawa T, Oeda of detection pine wood nematode LAMP detections from diseased wood
Y,Karim N,Kanzaki N.A Rapid and Precise Diagnostic Method for Detecting the
Pinewood Nematode Bursaphelenchus xylophilus by Loop-Mediated Isothermal
Amplification.Nematology,2009,12:1365-1369).Then establishing Meloidogyne incognita, enterolobium cyclocarpum root
The LAMP detection techniques of the important pathogen nematodes such as tie lines worm, M hapla, radopholus similes thorne, citrus Tylenchulus Semipenetrans
(Niu J H,Guo Q X,Jian H,Chen C L,Yang D,Liu Q,Guo Y D.Rapid detection of
Meloidogyne spp.by LAMP assay in soil and roots.Crop Protection,2011,8:1063-
1069；Niu J H,Jian H,Guo Q X,Chen C L,Wang X Y,Liu Q,Guo Y D.Evaluation of
loop-mediated isothermal amplification(LAMP)assays based on 5S rDNA-
IGS2regions for detecting Meloidogyne enterolobii.Plant Pathology,2011,
02562.x；Peng H,Peng D L,Hu X Q,He X F,Wang Q,Huang W K,He W T.Loop-mediated
isothermal amplification for rapid and precise detection of the burrowing
Nematode, Radopholus similis, directly from diseased plant tissues.Nematology,
2012,14(8):977-986.) detection of remolding sensitivity Standard PCR is high 10-100 times.LAMP detection techniques are in upland rice cyst roundworm
On there is no relevant report, the present invention establishes upland rice cyst roundworm LAMP quick determination methods first.
The content of the invention
The purpose of the present invention is that the LAMP for setting up circulation constant temperature amplification technique (LAMP) detection upland rice cyst roundworm is quickly examined
Survey method, the features such as this method has high sensitivity, high specificity, it is adaptable to the use of work is detected under various experiment conditions,
It also is adapted in the not enough outdoor detection of experiment condition.
A kind of upland rice cyst roundworm LAMP quick determination methods, it is characterised in that：Wherein used in LAMP reaction systems
Primer is：
1)HE-F3：5’-GCTCTCTGTCCATGTCAGGA-3’
2)HE-B3：5’-CCGCAACTAGCCCAATGC-3’
3)HE-FIP：5’-GCAGGACAGCTGCCCATGTGGCGGATCGTTCGGGAGAA-3’
4)HE-BIP：5’-GCGCAGCTTGGGATGCTTTTACCACAGGCTTACACTTGTG-3’
5)HE-LB：5’-AGGTTGGAGCTGGGATGCG-3’
HE-FIP therein is marked using fluorophor, and 5` sections use biotin labeling.
LAMP reaction systems therein include：
(1) primer mixed liquor：Outer primer HE-F3 and HE-B3 each 0.2 μm of ol/L, inner primer HE-FIP and HE-BIP each 1.4
μm ol/L, ring primer HE-LB are 0.4 μm of ol/L；
(2) reaction mixture：3.2mmol/L dNTP,20mmol/L Tris-HCl(pH 8.8),10mmol/L KCl,
3mmol/L MgS04,10mmol/L(NH4)2S04, 0.1%Triton x-100,8U Bst DNA Large fragment polymerases；
(3) 1 μ l DNA profilings；
Plus sterilizing double distilled water completion is to 25 μ l.
Wherein LAMP reaction conditions are as follows：1 μ l DNA moulds are added after primer mixed liquor and reaction mixture are well mixed
Plate, 61~65 DEG C of incubations 30~90min, 85 DEG C of insulation 5min.
Also include the reacted authentication steps of LAMP, the authentication step includes colour developing identification or electrophoretogram identification.
It is 100 times of SYBR Green I that the colour developing, which is accredited as the developer added in amplified reaction product, with naked eyes
Observation colour developing result, show green fluorescence for upland rice cyst roundworm.
The electrophoretogram be accredited as electrophoretogram be shown as trapezoid-shaped strips for upland rice cyst roundworm.
The electrophoretogram is accredited as adds probe in amplified reaction product, after hybridization, inserts LFD test paper, finds simultaneously
There is calibration tape and control band, the sequence of the probe is：
HE-probe：5-CGCCGAGGTTGGAGCTGGGATGC-3`,
Described probe 5` sections are marked using FITC.
Any of the above-described detection method early diagnoses and aided in upland rice cyst roundworm to apply in identification.
The present invention sets up the LAMP detection method for upland rice cyst roundworm using circulation constant temperature amplification technique.This method has
There is the amplification of a plurality of primer, and form at two ends the cyclic structure with primer function, this many primers combine and can be self-produced
The features such as principle of raw primer makes it have high sensitivity, high specificity, because LAMP operations step is simple and reaction
As a result product can directly be judged after fluorescence developing by naked eyes, it is adaptable to the use of detection work under various experiment conditions,
It is particularly suitable in the not enough outdoor detection of experiment condition.
The solution of the present invention specific implementation step is as follows：
1.DNA extraction
The single upland rice sporangiocyst of picking is put into equipped with 10 μ l ddH2In O 0.2mL centrifuge tubes, liquid nitrogen frozen is placed in after taking-up
On ice, ice-out is turned in centrifuge tube with sterile glass bar, sporangiocyst is broken, discharge ovum, add 10 times of 8 μ l
PCR buffer solutions, 2 μ l 600 μ g/ml Proteinase K Solutions, then freeze 30min at -80 DEG C.Centrifuge tube is taken out, at 65 DEG C
Lower incubation 90min, supernatant is directly used in LAMP and PCR as nematode DNA profiling and reacted after 95 DEG C of reaction 10min processing.
2. upland rice cyst roundworm LAMP primer is designed
According to upland rice cyst roundworm ITS-rDNA sequence (Genbank accession numbers in NCBI：JN_257082,KC_618466,
KF_430213and KM_017067) and its nearly source kind Heterodera oryzicola (AF_274387), H.cyperi (AF_
274388), the ITS sequence such as H.mothi (AF_498392), after comparing, selection upland rice cyst roundworm specific regions are used
PrimerExplorer V4(http://primerexplorer.jp/elamp4.0.0/index.html) online softwares set
Meter, testing sieve are selected following 5 Non-specific strong, and the high primer of stability, sequence is as follows:
1)HE-F3:5’-GCTCTCTGTCCATGTCAGGA-3’
2)HE-B3:5’-CCGCAACTAGCCCAATGC-3’
3)HE-FIP5’-GCAGGACAGCTGCCCATGTGGCGGATCGTTCGGGAGAA-3’
4)HE-BIP5’-GCGCAGCTTGGGATGCTTTTACCACAGGCTTACACTTGTG-3’
5)HE-LB 5’-AGGTTGGAGCTGGGATGCG-3’
3.LAMP reaction systems are configured
Outer primer HE-F3 and HE-B3 each 1.4 μm of ol/L of each 0.2 μm of ol/L, inner primer HE-FIP and HE-BIP, ring primer
HE-LB is 0.4 μm of ol/L；Reaction mixture：3.2mmol/L dNTP,20mmol/L Tris-HCl(pH 8.8),10mmol/L
KCl,3mmol/L MgSO4,10mmol/L(NH4)2SO4, 0.1%Triton x-100,8U Bst DNA Large fragment polymerases；1
μ l DNA profilings；Plus sterilizing double distilled water completion is to 25 μ l.
LAMP reaction conditions are as follows：1 μ l DNA profilings, 61 are added after primer mixed liquor and reaction mixture are well mixed
~65 DEG C of incubations 30~90min, 85 DEG C of insulation 5min.
LAMP results result can use following three kinds of detection methods：
1) 1 μ l developer will be added in the complete system of above-mentioned reaction.Light shake mixes, you can observation；
2) take 2 μ l amplified productions electrophoresis in 2% agarose gel electrophoresis that trapezoid belt can be observed；
3) amplified production kind is added after the probe (10 μM) of 1 μ l FITC marks, 65 DEG C of insulation 5min, naturally cools to room
Temperature, takes 10ul hybridization solutions, is added in 100 μ l of BPS buffer solutions, inserts a LFD test paper, stands 5-15min observations.4.
As a result observe
Under optimal reaction system and reaction condition, the gel electrophoresis of upland rice cyst roundworm positive reaction is able to observe that allusion quotation
The stepped band of type.In addition, adding after SYBR Green I coloring agents, naked eyes are observed that LAMP products become green,
And in lateral chromatography monitoring, there is band in p-wire and control line.And carrying out LAMP using other kind of nematode DNA can not obtain
To positive findings, show that the LAMP method degree of accuracy is high；LAMP detection architectures of the present invention are able to detect that 1,/20 000 upland rice spores
Capsule nematode, than sensitive 10 times of Standard PCR detection, with high sensitivity.
In summary, the present invention than existing detection upland rice cyst roundworm method have higher specificity, sensitivity and
Portability.Field application detection can be carried out in actual production.The technology can be applied to upland rice cyst roundworm field soil
The generation early stage rapid molecular detection of sample and Wheat cyst nematode, with actual application value.
Brief description of the drawings
Fig. 1 is upland rice cyst roundworm LAMP primer design diagram,
Fig. 2 is the detection of upland rice cyst roundworm LAMP method,
A is to add detection of fluorescent dyes result, 1,2：Positive findings, with green fluorescence, 3：Negative control, is brown.
B testing results are electrophoretogram, M:100bp DNA standard molecular weights；1,2：Positive findings, is that LAMP expands trapezoidal bar
Band, 3：Negative control, no amplified production.
C testing results are electrophoretogram, 1,2：Positive findings has control line and p-wire simultaneously；3：Negative control, only
Control line.
Fig. 3 is upland rice cyst roundworm sensitivity technique result
A is that LAMP detects electrophoretogram, M：1000DNA standard molecular weights, single head sporangiocyst, 10-1、10-2、10-3、10-4、10-5、
10-6With negative control (ck).
B is that regular-PCR method detects electrophoretogram, M：100DNA standard molecular weights, 10-1、10-2、10-3、10-4Individual sporangiocyst and the moon
Property control (ck).
Fig. 4 is upland rice cyst roundworm LAMP specific detection results
1-7 is upland rice cyst roundworm (He01, He02, He03, He04, He05, He06, He07), and 8-22 is respectively south
Root-knot nematode (Mi01 and Mi02), Meloidogyne enterolobii (Me01), Meloidogyne graminicola (Mg01 and Mg02),
M hapla M.hapla, javanese root knot nematode (Mj), peanut root-knot nematode (Ma), Hirschmanniella Oryzae (Ho01 and
), Ho02 H.mucronata, cereal cyst nematode (Ha), Philips's cyst roundworm (Hf), corn lesion nematode (Pz) and
Tylenchorhynchus annulatus(Ta)；23-24 is negative control..
Embodiment
Below by embodiment, the present invention is described in further detail.
The reagent is commercially available, and there is preservation in used test nematode the applicant laboratory, freely can be sent out to the public
Put.
Main agents：Taq archaeal dna polymerases are purchased from Tiangeng company；DNA marker are purchased from TaKaRa companies；Primer is by upper
Hai Shenggong Bioisystech Co., Ltd synthesizes；PGEM-T Easy Vector are purchased from U.S. promega Pro K (Proteinase K) purchases
From Roche companies；Bst archaeal dna polymerases large fragment is purchased from New England Biolabs companies；SYBR green I are purchased from
Invitrogen companies.
The upland rice cyst roundworm DNA of embodiment 1 extraction, ITS amplifications and sequence analysis
1.1 upland rice cyst roundworm DNA extraction
The single upland rice sporangiocyst of picking is put into equipped with 10 μ l ddH2In O 0.2mL centrifuge tubes, liquid nitrogen frozen is placed in after taking-up
On ice, ice-out is turned in centrifuge tube with sterile glass bar, sporangiocyst is broken, discharge ovum, add 10 times of 8 μ l
PCR buffer solutions, 2 μ l 600 μ g/ml Proteinase K Solutions, then freeze 30min at -80 DEG C.Centrifuge tube is taken out, at 65 DEG C
Lower incubation 90min, supernatant is directly used in LAMP and PCR as nematode DNA profiling and reacted after 95 DEG C of reaction 10min processing.
1.2 upland rice cyst roundworm ITS fragment amplifications and sequence analysis
Upland rice cyst roundworm ITS fragments are expanded using ITS universal primers TW81 and AB28.Pcr amplification reaction system is 50 μ
L, PCR reaction system are that 10 × Buffer (contains Mg2+) 5 μ l, 10mM dNTP 4 μ l, primer TW81 and AB28 (10 μm of ol/L) each 1
μ l, Taq enzyme (5U/ μ l, Takara) 0.5 μ l, μ l of template DNA 5, sterilize ddH2O complements to 50 μ l.PCR amplification conditions are：95
DEG C pre-degeneration 5min, 55 DEG C of annealing 30sec, 72 DEG C of extension 1.5min；35 circulations；72 DEG C extend 10min, 4 DEG C of preservations again.
After PCR amplifications, 5 μ l amplified productions plus 1 μ l the sample loading buffers electrophoresis on 1.5% Ago-Gel, EB dyeing, in uviol lamp are taken
Lower observation and recovery of taking a picture, clone and sequencing, sequencing are completed by Beijing six directions Hua Da Gene Tech. Company Limited.
The foundation of the upland rice cyst roundworm LAMP of embodiment 2 detections
2.1LAMP design of primers
According to the sequencing result of upland rice cyst roundworm ITS regiospecificity fragments, design and screen following LAMP primers (see
Fig. 1), primer hands in the synthesis of marine growth engineering services Co., Ltd.Primer sequence is as follows：
1)HE-F3:5’GCTCTCTGTCCATGTCAGGA-3’
2)HE-B3:5’-CCGCAACTAGCCCAATGC-3’
3)HE-FIP5’-GCAGGACAGCTGCCCATGTGGCGGATCGTTCGGGAGAA-3’
4)HE-BIP5’-GCGCAGCTTGGGATGCTTTTACCACAGGCTTACACTTGTG-3’
5)HE-LB 5’-AGGTTGGAGCTGGGATGCG-3’
2.2LAMP reaction systems are configured：
Outer primer HE-F3 and HE-B3 each 1.4 μm of ol/L of each 0.2 μm of ol/L, inner primer HE-FIP and HE-BIP, ring primer
HE-LB is 0.4 μm of ol/L；Reaction mixture：3.2mmol/L dNTP,20mmol/L Tris-HCl(pH 8.8),10mmol/L
KCl, 3mmol/L MgS04,10mmol/L (NH4) 2S04,0.1%Triton x-100,8U Bst DNA Large fragment polymerases；
1 μ l DNA profilings；Plus sterilizing double distilled water completion is to 25 μ l.
2.3LAMP reacts amplification condition：Liquid mixed above is placed in 65 DEG C of water bath with thermostatic control isothermal amplification 45min, 85 DEG C
10min is incubated, reaction adds observation result (Fig. 2-A) after the developer mixing that 1 μ l are prepared after terminating.2 μ l products are taken 2%
Electrophoresis on Ago-Gel, EB dyeing, observe and take a picture under uviol lamp, it can be seen that have LAMP characteristic trapezoid belt (Fig. 2-
B), add after probe hybridization, insert LFD test paper, find there is calibration tape and control band (Fig. 2-C) simultaneously.
The upland rice cyst roundworm LAMP sensitivity techniques of embodiment 3
The general sporangiocyst DNA profiling of single head upland rice is diluted to 1~1.0 × 10 by 10 times-76 concentration, respectively take 1 μ l DNA to do
Template, after above-mentioned primer mixed liquor and reaction mixture are well mixed, is carried out by 2.3 reaction conditions, after reaction terminates, taken
3 μ l products electrophoresis on 2% Ago-Gel, EB dyeing, observes under uviol lamp and takes a picture (Fig. 4-A), seen in 2-5 swimming lanes
LAMP characteristic trapezoid belt is observed, illustrates that DNA is diluted after 1000 times, detection still is able to, detection sensitivity is 1,/20 000
Nematode.Simultaneously using above-mentioned dilution DNA as template, using outside primers F 3 and B3 as primer, Standard PCR detection is carried out, system is as follows：
2.5 μ 10 × PCR of l Buffer (contain Mg2+), 2.5 μ l 10mM dNTPs, 1 μ l F3 and B3 (10uM), 0.3 μ l Taq DNA gather
Synthase (5U/ul), 1 μ l template DNAs, sterilize ddH2O complements to 25 μ l, uses no nematode DNA profiling for negative control.PCR expands
Increasing condition is：95 DEG C of pre-degenerations 5min, 52 DEG C of annealing 30sec, 72 DEG C of extension 1.5min；35 circulations；72 DEG C extend again
10min, 4 DEG C of preservations.After PCR amplifications, 5 μ l amplified productions plus 1 μ l the sample loading buffers electrophoresis on 1.5% Ago-Gel are taken,
After EB dyeing, gel electrophoresis observation result (Fig. 4-B).As a result show to be diluted to 10 in DNA-2Times when, be able to observe that amplification bar
Band, 10-3Standard PCR can not then be detected during lower concentration.
It these results suggest that：Above-mentioned LAMP detection architectures are able to detect that 1,/20 000 upland rice cyst roundworms, than routine
Sensitive 10 times of PCR detections, with high sensitivity.
The upland rice cyst roundworm LAMP specific detections of embodiment 4
Collect upland rice cyst roundworm, cereal cyst nematode, Philips's cyst roundworm, javanese root knot nematode, Roots of Peanut tie lines
22 populations (being shown in Table 1) such as worm, M hapla, Meloidogyne enterolobii, latent root nematode Pratylenchidae, extract it respectively
DNA carries out LAMP detections as template together with upland rice cyst roundworm DNA profiling, to detect upland rice cyst roundworm LAMP detection sides
The specificity of method.
Other Plant nematode population sample codes and source of the table 1 for examination
After above-mentioned primer mixed liquor and reaction buffer mixture are well mixed, 1 μ l template DNAs are added, by 2.3 reaction bar
Part is carried out, and reaction is added after terminating after the developer mixing that 1 μ l are prepared, and observes color change.As a result it is upland rice to show 1-7 pipes
Cyst roundworm DNA, it is observed that green fluorescence, other plant nematode and negative control are bronzing for the reaction of template
(Fig. 4).As a result show that above-mentioned LAMP primer and reaction system being capable of specific detection upland rice cyst roundworms.
SEQUENCE LISTING
<110>Plant Protection institute, Chinese Academy of Agricultral Sciences
<120>A kind of upland rice cyst roundworm LAMP quick determination methods
<130>PP17088-ZWB
<160> 6
<170> PatentIn version 3.3
<210> 1
<211> 20
<212> DNA
<213>HE-F3 sequences
<400> 1
gctctctgtc catgtcagga 20
<210> 2
<211> 18
<212> DNA
<213>HE-B3 sequences
<400> 2
ccgcaactag cccaatgc 18
<210> 3
<211> 38
<212> DNA
<213>HE-FIP sequences
<400> 3
gcaggacagc tgcccatgtg gcggatcgtt cgggagaa 38
<210> 4
<211> 40
<212> DNA
<213>HE-BIP sequences
<400> 4
gcgcagcttg ggatgctttt accacaggct tacacttgtg 40
<210> 5
<211> 19
<212> DNA
<213>HE-LB sequences
<400> 5
aggttggagc tgggatgcg 19
<210> 6
<211> 23
<212> DNA
<213>HE-probe sequences
<400> 6
cgccgaggtt ggagctggga tgc 23
Claims (10)
1. a kind of upland rice cyst roundworm LAMP quick determination methods, it is characterised in that：Wherein draw used in LAMP reaction systems
Thing is：
1)HE-F3：5 '-GCTCTCTGTCCATGTCAGGA-3 ',
2)HE-B3：5 '-CCGCAACTAGCCCAATGC-3 ',
3)HE-FIP：5 '-GCAGGACAGCTGCCCATGTGGCGGATCGTTCGGGAGAA-3 ',
4)HE-BIP：5 '-GCGCAGCTTGGGATGCTTTTACCACAGGCTTACACTTGTG-3 ',
5)HE-LB：5’-AGGTTGGAGCTGGGATGCG-3’.
2. detection method according to claim 1, LAMP reaction systems therein include：
(1) primer mixed liquor：Outer primer HE-F3 and HE-B3 each 0.2 μm of ol/L, each 1.4 μ of inner primer HE-FIP and HE-BIP
Mol/L, ring primer HE-LB are 0.4 μm of ol/L；
(2) reaction mixture：3.2mmol/L dNTP,20mmol/L Tris-HCl(pH 8.8),10mmol/L KCl,
3mmol/L MgSO4,10mmol/L(NH4)2SO4, 0.1%Triton x-100,8U Bst DNA Large fragment polymerases；
(3) 1 μ l DNA profilings；
Plus sterilizing double distilled water completion is to 25 μ l.
3. detection method according to claim 2, wherein LAMP reaction conditions are as follows：Primer mixed liquor and reaction are mixed
1 μ l DNA profilings, 61~65 DEG C of incubations 30~90min, 85 DEG C of insulation 5min are added after liquid is well mixed.
4. detection method according to claim 3, including the reacted authentication steps of LAMP, the authentication step include aobvious
Color is identified or electrophoretogram identification.
5. detection method according to claim 4, the colour developing is accredited as the developer added in amplified reaction product
For 100 times of SYBR Green I, to visually observe colour developing result, show green fluorescence for upland rice cyst roundworm.
6. detection method according to claim 4, the electrophoretogram be accredited as electrophoretogram be shown as trapezoid-shaped strips for drought
Rice cyst roundworm.
7. detection method according to claim 4, the electrophoretogram is accredited as adds probe in amplified reaction product, miscellaneous
After friendship, LFD test paper is inserted, finds there is calibration tape and control band simultaneously, the sequence of the probe is：HE-probe：5’-
CGCCGAGGTTGGAGCTGGGATGC-3’。
8. detection method according to claim 7, HE-FIP therein is marked using fluorophor, 5 ' sections use biotin
Mark.
9. detection method according to claim 7, the section of probe 5 ' is marked using FITC.
10. any detection method described in claim 1-9 early diagnoses and aided in upland rice cyst roundworm to apply in identification.
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