CN103045751A - Rapid detection method of heterodera avenae wollenweber LAMP and application of detection method - Google Patents
Rapid detection method of heterodera avenae wollenweber LAMP and application of detection method Download PDFInfo
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Abstract
The invention relates to a rapid detection method of heterodera avenae wollenweber LAMP and application of the detection method. Five LAMP primers, namely HA5-F3, HA5-B3, HA5-FIP, HA5-BIP and HA5-LB are designed and screened according to a heterodera avenae wollenweber random amplified polymorphic dna (RAPD) sequence obtained by cloning; and an LAMP reaction system is configured and optimized. By performing DNA extraction, loop-mediated isothermal amplification, amplified product developing and observation on the heterodera avenae wollenweber, the heterodera avenae wollenweber can be rapidly detected. The detection method is high in specificity, low in cost and convenient to operate, and has high application values on rapid detection of the heterodera avenae wollenweber and early and field diagnosis of the heterodera avenae wollenweber diseases.
Description
Technical field
The present invention relates to a kind of cereal cyst nematode LAMP method for quick and application, belong to biological technical field.
Background technology
Cereal cyst nematode (Heterodera avenae Wollenweber, 1 924), namely Cereal cyst nematode (be called for short CCN, have another name called Heterodera avenae) is the cereal crop important pathogen nematode that distributes in the worldwide.This nematode from 1874 since Germany is found, all there is occurrence and harm in more than 40 country in the whole world at present, wherein the Cereal Cyst Nematode in the areas such as China, Australia, Europe, India, Middle East harm is serious and suffered huge financial loss.For example the hazard area at Australian cereal cyst nematode has reached 2,000,000 hectares, general production loss 23-50%, loss 73-89% when serious, year financial loss is 7,200 ten thousand Australian Dollars (Brennan J P approximately, Murray G M.Aust ralian w heat disease assessing their economic import ance[J] .Agric.S ci., 1988,52 (2): 176-183).The main cereal crop growing spots in Europe, nearly plot more than 50% is subject to infecting of cereal cyst nematode, annual approximately 3,000,000 Euros (Alexander H M of financial loss that caused by cereal cyst nematode, Julie M N, Nematode parasites of cereals.Plant parasitic nematodes in subtropical and tropical agriculture[M] .Second edition.CAB international 2005:1 3 1-1 9 1); In India, cereal cyst nematode causes Wheat and barley " Molya " disease, wheat yield loss reaches 47.2%, barley loses up to 87.2% (RivoalR., Cook, R., Nematode pests of cereals.In:Plant Parasitic Nematodes in Temperate Agriculture.[B] .Evaan K.et al., Eds.Walling ford Press, UK, 1993,259-303).
In China, cereal cyst nematode was from [Chen Pinsan since finding first in the short yellow wheat strain in Tianmen, Hubei county in 1989, Wang Mingzu, Peng Deliang. China's wheat cearal cyst nematode (Heterodera avenae wollenweber) identification research [J]. Plant Pathology, 1992,22 (4): 339-343], 16 provinces, municipalities and autonomous regions such as present Hebei in China wheat main producing region, Henan, Beijing, Shanxi, the Inner Mongol, Qinghai, Hubei, Anhui all have distribution, and hazard area reaches 4,000,000 hm
2Above, and the gesture that spreads is gradually arranged, become at present harm China wheat crops the important pathogen nematode (Peng Deliang etc., the kainogenesis Distribution Area of China's wheat cyst roundworm. Chinese nematology research volume Two, 2008,2:344-345; Huang Wenkun, Ye Wenxing, Wang Gaofeng, Longhai City's ripple, Ou Shiqi, Peng Deliang, the occurrence and distribution of Ningxia, China cereal cyst nematode. Hua Zhong Agriculture University's journal, 2011,30 (1): 74-77).According to investigations; but general sick field underproduction 20%-40%; the serious plot underproduction reaches [Peng Deliang more than 70%; Zhang Dongsheng; Qi Shuhua, Chen Pin is third-class, China's wheat cyst roundworm (Heterodera avenae) occurrence and distribution zone and prevent and treat preliminary study. the plant protection progress; 1995, p53-56. China Science Tech Publishing House].Cereal cyst nematode can parasitic grass 27 belong to 34 kinds, to the harm of the cereal crops such as wheat, barley, oat, rye serious [Liu Weizhi. nosophyte nematology [M]. Beijing: Chinese agriculture press, 1998.294-303.].Cereal Cyst Nematode of Wheat has been done to produce to the wheat class of China and has been consisted of serious threat at present, is seriously restricting the development that wheat crops is produced, and this nematode is made identifying fast and accurately it is that current wheat crops is produced urgent problem.
Traditional cyst roundworm detection method is morphologic detection.Cereal cyst roundworm group (Heterodera avenae group) comprises 12 cyst roundworm kinds, wherein cereal cyst nematode (H.avenae) and Philips's cyst roundworm (H.filipjevi) morphological specificity difference are less, and Main Differences is significantly lower bridge (underbrige) structure for Philips's cyst roundworm has.Traditional Morphological Identification, consuming time more and need very abundant expertise, be difficult to satisfy the requirement of present high-throughput rapid detection.
Along with molecular biological development, the Fast Detection Technique such as PCR extensively shipping use in the rapid detection of Plant nematode.The detection technique that applies at present cyst roundworm mainly comprises ITS-RFLP, PCR, real time PCR etc.
Zheng etc. (2000) cut European H.avenae(A type with Hinf I enzyme) all not identical with Chinese cereal cyst nematode with result after the India colony (Type B), be called " C type " (Zheng, J., Subbotin S.A., Waeyenberge L., Moens M., Molecular characterization of Chinese Heterodera glycines and H.avenae populations based on RFLPs and sequences of rDNA-ITS regions.Russian Journal ofNematology2000,8:109-113.).Peng De very waits (2003) increased the Internal Transcribed Spacer of ribosomal gene of Wheat in China cereal cyst nematode colony, cut the ITS amplified production with AluI and RsaI enzyme and prove that Chinese cereal cyst nematode ITS belongs to " Type B ", the HinfI enzyme is cut to disclose between China and the Morocco cereal cyst nematode ITS and is had notable difference (Peng Deliang, Subbotin S.A., M.Moens. ribosomal gene (rDNA) restriction fragment length polymorphism of wheat cearal cyst nematode (Heterodera avenae) research. Plant Pathology, 2003,33 (4): 323-329).Guiping Yan and Richard W. Smiley(2011) method using rDNA-ITS zone RFLP combining form to learn to identify detects the cereal cyst nematode of Some Areas of USA, the method adopts 6 kinds of restriction enzyme endonuclease capables to distinguish cereal cyst nematode and Philips's cyst roundworm (Yan, G. P., SmileyR.W., Distinguishing Heterodera filipjevi and H.avenae Using Polymerase Chain Reaction-Restriction Fragment Length Polymorphism and Cyst Morphology.Nematology2010,3:216-224.).Fu etc. have carried out ITS and rflp analysis (Fu Bo to the cereal cyst nematode of China Huang-Huai-Hai Mai Qu, Ian T.Riley, Li Honglian, et al.Molecular characterisation of cereal cyst nematodes in winter wheat on the Huang-Huai floodplain of China using RFLP and rDNA-ITS sequence analyses.Australasian Plant Pathol2011,40:277285).
In the detection of cyst roundworm pattern nematode beet cyst roundworm, Amiri etc. pass through H.betae, H.ciceri, H.glycines and H.medicaginis, H.schachtii, the ITS sequence of H.trifolii is analyzed, filter out Auele Specific Primer SHF6, with this primer and AB28(or rDNA2) primer is combined, made up detection method (the Amiri S. of beet cyst roundworm one one-step dual PCR, Subbotin S.A., Moens M., An efficient meth od for identification of the Heterodera schachtii sensu stricto group using PCR with specific primers.Nematol.medit.2001,29:241-246).
Subbotin S A, Peng D L, Moens M.(2001) use double PCR to detect the method for soy bean cyst roundworm, in a PCR reaction system, use simultaneously universal primer D3A and D3B and Auele Specific Primer GlyF1 and rDNA2, from 52 soy bean cyst roundworm colonies, all amplify two dna segments (181bp and345bp), the susceptibility that detects is up to single sporangiocyst, the minim DNA of single head second instar larvae (Subboton S.A., Peng D L, Moens M., A rapid method forthe identification of the soybean cyst nematode Heterodera glycines using duplex PCR.Nematology 2001, vol.3 (4): 365-371).Ou, S.Q. and Peng D.L adopt the RAPD technology, have obtained the specific SCAR label based on the soy bean cyst roundworm genomic dna, and while and D2A and D3B combine, and have invented an one-step dual PCR method and have detected soy bean cyst roundworm.Under the pcr amplification condition, soy bean cyst roundworm colony has all obtained specific SCAR label fragment and 800bp fragment (the Ou S.Q. of 500bp, Peng D.L., Li Y., Moens M., Identification of Heterodera glycines using PCR with sequence characterised amplified region (SCAR) primers.Nematology2008,10 (3): 397-403).
QiXiao Li etc. adopt the method for RAPD, work out cereal cyst nematode specific SCAR molecule marker, the method can be special cereal cyst nematode and Philips's cyst roundworm, barley cyst roundworm, upland rice cyst roundworm and pea cyst roundworm are distinguished, detection sensitivity is up to 1/80 2 instar larvae (QiXiao Li, Peng Deliang, Peng Huan, Longhai City's ripple, Huang Wenkun, He Wenting. based on wheat cearal cyst nematode (Heterodera avenae) the rapid molecular detection technique of SCAR mark. Scientia Agricultura Sinica, 2012,45 (21): 4388-4395).Simultaneously, Ophel-Keller etc. develop the real time PCR detection technique of direct-detection cereal cyst nematode in the worm soil, sensitivity reaches 1 ovum/1g (Ophel-Keller Kathy, McKay Alan, Hartley Di, Herdina and Curran, John.Development of a routine DNA-based testing servicefor soilborne diseases in Australia.Australian Plant Pathology, 2008,37:243-253).Molecular assay method take PCR as the basis has remedied the defective in the traditional form evaluation to a certain extent.But PCR detects professional instrument and the molecular biology reagent that needs PCR instrument, gel electrophoresis and imaging system (ultraviolet device) etc. expensive, and need molecular biology Specialty Experiment personnel operation, above detection can only just can detect under laboratory condition, need the long time, limited PCR detection method applying aborning, in the investigation of cereal cyst nematode occurrence and distribution, in the urgent need to a kind of simple and efficient detection means.
Circulation constant temperature amplification technique (loop-mediated isothermal amplification of DNA, LAMP) is a kind of New Cycle constant temperature nucleic acid amplification technology of the people such as the Japanese Rong Yan Notomi of Co., Ltd. exploitation in 2000.(Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N and Hase T.Loop-mediated isothermal amplification ofDNA ［ J ］ .Nucleic Acids Res, 2000,28 (12): e63.), this technology by 6 special zones on the identification target sequence primer and utilize the Bst archaeal dna polymerase with strand displacement function, can be special under constant temperature, Gao Min, amplified target sequence rapidly.This technology technology has following characteristics: 1) high specificity, highly sensitive.The LAMP reaction is just designed 4-6 bar primer for 6-8 specificity site of target-gene sequence, compares common other molecular detecting methods and has stronger specificity.2) the amplified reaction time is short, equipment requirements is simple.The LAMP reaction has high amplification efficiency, target gene can be increased 10 in 30-90 minute
9-10
10, amplification procedure need not the equipment such as PCR instrument of specialty simultaneously, and simple water-bath can be finished reaction.3) detected result can the naked eyes direct viewing, and is simple and efficient.Because the characteristics such as this detection method has high specificity, and is highly sensitive, fast and convenient extensively are incorporated in people and animals' pathogen, food safety and the sanitary detection at present.But the utilization in plant pathogeny line insect detects is at the early-stage, Japanese scientist utilized the LAMP technology to set up method (the Kikuchi T that direct-detection pine wood nematode LAMP detects from diseased wood first in 2008, Aikawa T, OedaY, Karim N, Kanzaki N.A Rapid and Precise Diagnostic Methodfor Detecting the Pinewood Nematode Bursaphelenchus xylophilus by Loop-Mediated Isothermal Amplification.Nematology, 2009,12:1365-1369).Niu et al in 2011 etc. have set up Meloidogyne incognita LAMP detection technique, can from soil and root knot, detect Meloidogyne incognita (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) and Meloidogyne enterolobii (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 on5S rDNA-IGS2regionsfor detecting Meloidogyne enterolobii.Plant Pathology, 2011,02562.x).Peng De in 2012 has set up Meloidogyne enterolobii LAMP Fast Detection Technique very first take Meloidogyne enterolobii ITS as target, and detection sensitivity reaches 1/200000 larva (patent No.: 201110034960).(the Peng H such as Peng Huan in 2012, 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.) work out from the plant tissue of falling ill with LAMP fast and the method for specific detection radopholus similes thorne, limit of detection reaches 1/20000 larva, and the conventional PCR of remolding sensitivity detects high 100 times.In addition, the LAMP detection technique there is no relevant report at cereal cyst nematode, and the present invention has set up cereal cyst nematode LAMP method for quick first.
Summary of the invention
The objective of the invention is to set up the LAMP method for quick that circulation constant temperature amplification technique (LAMP) detects cereal cyst nematode, the characteristics such as that the method has is highly sensitive, high specificity, be applicable to the use of testing under the various experiment conditions, also be adapted at the outdoor detection of experiment condition deficiency.
A kind of cereal cyst nematode LAMP method for quick is characterized in that: wherein the employed primer of LAMP reaction system is:
①HA5-F3：5`-TGATAGGGAAAAAATTCTCCAA-3`；
②HA5-B3：5`-GTCGGCAATGGTCAACAA-3`;
③HA5-BIP：5`-TTGTGGTGCCGTAGTGTTAGAAATGCTTCATTTGAGAACAATG-3`;
④HA5-FIP：5`-ACACAACGCTTAGTCCGGTCCCAATGGCATGGCAAAGA-3`;
⑤HA5-LB：5`-CGGGGATGAAAGAAGGCAAAGTG-3`;
LAMP reaction system wherein comprises:
1) primer mixed solution: each 0.2 μ mol/L of outer primer HA5-F3 and HA5-B3, each 1.4 μ mol/L of inner primer HA5-FIP and HA5-BIP, ring primer HA5-LB is 0.4 μ mol/L;
2) reaction mixture: 3.2mmol/L dNTP, 20mmol/L Tris-HCl (pH8.8), 10mmol/L KCl, 3mmol/LMgS0
4, 10mmol/L (NH4)
2S0
4, 0.1%Triton x-100,8U Bst archaeal dna polymerase large fragment;
3) 1 μ lDNA template;
Add the completion of sterilization bi-distilled water to 25 μ l.
Wherein the LAMP reaction conditions is as follows: primer mixed solution and reaction mixture are mixed rear adding 1 μ lDNA template, 61～65 ℃ of insulation 30～90min, 85 ℃ of insulation 10min.
Add developer in the final product of LAMP reaction, observe after gently throwing away core barrel.
Described developer is the mixture of SYBRgreen I and PCR level DMSO, and its volume ratio is 1:9.
The extracting method of described dna profiling is: the single cereal sporangiocyst of picking is put into 10 μ lddH is housed
2In the 0.2mL centrifuge tube of O, liquid nitrogen freezing, taking-up is placed on ice, glass stick with sterilization turns to ice-out in centrifuge tube, sporangiocyst is broken, and discharges ovum, the LB solution that adds 8 μ l, the 600 μ g/ml Proteinase K solution of 2 μ l are then at-80 ℃ of lower freezing 30min.Centrifuge tube is taken out, and at 65 ℃ of lower incubation 90min, supernatant liquor was directly used in LAMP and PCR reaction as the nematode dna profiling after 95 ℃ of reaction 10min processed, and described LB solution is 500mmol/L KCl, 10mmol/L Tris-HCl, 15mmol/L MgCl
2, 1.0mmol/L DTT, 4.5%Tween20, equal-volume mixing, filtration sterilization.
Above-mentioned arbitrary detection method is in the cereal cyst nematode infection conditions of diagnosis of plant, soil or the application in the discriminating cereal cyst nematode.
The present invention utilizes circulation constant temperature amplification technique (Loop-mediated isothermal amplifcation, LAMP) foundation for the detection method of cereal cyst nematode.Present method has the amplification of many primers, and formed ring texture with the primer function at two ends, this many primers combination and the principle that can certainly produce primer make it have the characteristics such as highly sensitive, high specificity, because LAMP operation step simply reaches the precipitation that comprises a large amount of nucleic acid and magnesium pyrophosphate in the reaction product, the judgement reaction result can detect by an unaided eye after the fluorescent agent colour developing, be applicable to the use of testing under the various experiment conditions, also be adapted at the outdoor detection of experiment condition deficiency.
The solution of the present invention implementation step is as follows:
1. nematode DNA extraction reagent preparation
1) LB (Lysis Buffer) solution: 500mmol/L KCl, 10mmol/L Tris-HCl, 15mmol/L MgCl
2, 1.0mmol/L DTT, 4.5%Tween20, equal-volume mixing, filtration sterilization.
2) Proteinase K: 600 μ g/ml Proteinase Ks.
2.DNA extraction
The single cereal sporangiocyst of picking is put into 10 μ l ddH is housed
2In the 0.2mL centrifuge tube of O, liquid nitrogen freezing, taking-up is placed on ice, glass stick with sterilization turns to ice-out in centrifuge tube, sporangiocyst is broken, and discharges ovum, the LB solution that adds 8 μ l, the 600 μ g/ml Proteinase K solution of 2 μ l are then at-80 ℃ of lower freezing 30min.Centrifuge tube is taken out, and at 65 ℃ of lower incubation 90min, supernatant liquor was directly used in LAMP and PCR reaction as the nematode dna profiling after 95 ℃ of reaction 10min processed.
3. cereal cyst nematode RAPD amplification and sequential analysis
Use SCAR labeled primer OPD13-HaF1 (5`-TGACGAGAACATATGATGGGGATGAT-3`) and OPD13-HaR1(5`-GAGGGGGTGGGAATGAAATGGAT--3`) amplification cereal cyst nematode SCAR fragment.The pcr amplification reaction system is 50 μ l, and the PCR reaction system is 10 * Buffer (containing Mg2+), 5 μ l, 10mM dNTP4 μ l, each 1 μ l of primer OPD13-HaF1 and OPD13-HaR1 (10 μ mol/L), Taq enzyme (5U/ μ l, Takara) 0.5 μ l, template DNA 5 μ l, sterilization ddH
2O complements to 50 μ l.The pcr amplification condition is: 95 ℃ of denaturation 5min, and 59 ℃ of annealing 30sec, 72 ℃ are extended 1.5min; 35 circulations; 72 ℃ are extended 10min again, 4 ℃ of preservations.Behind the pcr amplification, get 5 μ l amplified productions and add 1 μ l sample loading buffer electrophoresis on 1.5% sepharose, EB dyeing, observation and take a picture recovery, Cloning and sequencing under ultraviolet lamp, sequencing is finished by the large Gene Tech. Company Limited of Beijing six directions China.
4. cereal cyst nematode LAMP design of primers
Be template according to cereal cyst nematode RAPD sequencing result, design following 5 primers, sequence is as follows:
①HA5-F3：5`-TGATAGGGAAAAAATTCTCCAA-3`；
②HA5-B3：5`-GTCGGCAATGGTCAACAA-3`;
③HA5-BIP：5`-TTGTGGTGCCGTAGTGTTAGAAATGC?TTCATTTGAGAACAATG-3`;
④HA5-FIP：5`-ACACAACGCTTAGTCCGGTCCCAATGGCA?TGGCAAAGA-3`;
⑤HA5-LB：5`-CGGGGATGAAAGAAGGCAAAGTG-3`;
5.LAMP reaction system configuration: each 0.2 μ mol/L of outer primer HA5-F3 and HA5-B3, each 1.4 μ mol/L of inner primer HA5-FIP and HA5-BIP, ring primer HA5-LB is 0.4 μ mol/L, 3.2mmol/L dNTP, 20mmol/L Tris-HCl (pH8.8), 10mmol/L KCl, 3mmol/L MgS0
4, 10mmol/L (NH4)
2S0
4, 0.1%Triton x-100 and 8U Bst archaeal dna polymerase large fragment, 1 μ lDNA template is supplied 25 μ l with the sterilization bi-distilled water.
6.LAMP reaction amplification condition: above mixed solution is placed 65 ℃ of water bath with thermostatic control isothermal duplication 75min, and 85 ℃ are incubated 10min again, observations behind the developer mixing that adding 1 μ l prepared after reaction finished.
7.LAMP the result detects: the result can adopt following two kinds of detection methods:
1) developer of adding 1 μ l in the system that above-mentioned reaction is complete.Light rolling mixing, i.e. observable;
2) get 3 μ l amplified productions electrophoresis in 2% agarose gel electrophoresis and can be observed trapezoid belt;
Cereal cyst nematode LAMP method for quick provided by the present invention has the following advantages:
One, highly sensitive.Limit of detection to cereal cyst nematode can reach 1/200000 larva level, and the detection sensitivity that detects cereal cyst nematode than conventional PCR is high 100 times, illustrates that also the primer specificity that the present invention designs is very good.
Two, high specificity.Used special primer is designed 5 primers according to six different zones of RAPD fragment of cereal cyst nematode, and specificity is more eager to excel in whatever one does than conventional PCR.
Three, detection time is short.Can obtain detected result about 1 hour, detect than conventional PCR and shorten 2～4h.
Four, do not need the PCR instrument.Low to the plant and instrument requirement, without any need for PCR instrument, gel electrophoresis and imaging system, only need a water-bath or vacuum flask just can finish detection.
Five, simple to operate, the result is obvious.Whole testing process does not relate to complex instrument and equipment, and those skilled in the art can finish detection, and the result easily distinguishes, by different colours, naked eyes get final product observations, do not need loaded down with trivial details electrophoresis and ultraviolet visualization.
Six, friendly to human and environment.Testing process does not need to use the toxic reagents such as EB, and is very safe to human and environment.
In sum, the present invention has the method that detects cereal cyst nematode than existing round pcr and has higher specificity, sensitivity and portability.Can in actual production, rig-site utilization detect.This technology can be applicable to the generation early stage rapid molecular of cereal cyst nematode field soil sample and Wheat cyst nematode is detected, and has actual using value.
Description of drawings
Fig. 1 is the LAMP design of primers schematic diagram of cereal cyst nematode RAPD sequence,
Fig. 2 is that cereal cyst nematode LAMP method detects,
A is for adding the detection of fluorescent dyes result, 1: positive findings has green fluorescence, 2: negative control is sorrel.
The B detected result is electrophorogram, M:D2000DNA standard molecular weight (Takara) 1: positive findings is LAMP amplification trapezoid-shaped strips, 2: negative control, and without amplified production.
Fig. 3 is as a result figure of cereal cyst nematode LAMP detection method specific detection
A is that LAMP adds as a result figure of detection of fluorescent dyes, and 1 is positive, and 2 ~ 11 is negative,
B is LAMP detected result electrophorogram,
Fig. 4 is cereal cyst nematode sensitivity detected result
A is that LAMP adds as a result figure of detection of fluorescent dyes, and 1～7 is respectively: single head nematode, 10
-1, 10
-2, 10
-3, 10
-4, 10
-5, 10
-6And negative control.
B is LAMP detected result electrophorogram, M:D2000DNA standard molecular weight (Takara), and 1～7 is respectively: single head nematode, 10
-1, 10
-2, 10
-3, 10
-4, 10
-5, 10
-6And negative control.
C is regular-PCR method detected result electrophorogram, M:D2000DNA standard molecular weight (Takara), and 1～7 is respectively: single head nematode, 10
-1, 10
-2, 10
-3, 10
-4, 10
-5, 10
-6, negative control.
Embodiment
The present invention is described in further detail below by embodiment.
Described reagent is commercially available, and all there is preservation in used cereal cyst nematode the applicant laboratory, can freely provide to the public.
Main agents: the Taq archaeal dna polymerase is available from sky root company; DNA marker is available from TaKaRa company; Primer is given birth to worker Bioisystech Co., Ltd by Shanghai and is synthesized; PGEM-T EasyVector is purchased from U.S. promega Pro K(Proteinase K) available from Roche company; Bst archaeal dna polymerase large fragment is available from New England Biolabs company; SYBR green I is available from Invitrogen company.
The extraction of embodiment 1 cereal cyst nematode DNA, RAPD amplification and sequential analysis
1.1 the extraction of cereal cyst nematode DNA
The single cereal sporangiocyst of picking is put into 10 μ lddH is housed
2In the 0.2mL centrifuge tube of O, liquid nitrogen freezing, taking-up is placed on ice, in centrifuge tube, turn to ice-out with aseptic glass stick, sporangiocyst is broken, discharge ovum, the LB solution that adds 8 μ l, the 600 μ g/ml Proteinase K solution of 2 μ l are then at-80 ℃ of lower freezing 30min.Centrifuge tube is taken out, and at 65 ℃ of lower incubation 90min, supernatant liquor was directly used in LAMP and PCR reaction as the nematode dna profiling after 95 ℃ of reaction 10min processed.
1.2 cereal cyst nematode RAPD amplification and sequential analysis
Use SCAR labeled primer OPD13-HaF1 (5`-TGACGAGAACATATGATGGGGAT-GAT-3`) and OPD13-HaR1(5`-GAGGGGGTGGGAATGAAATGGAT-3`) amplification cereal cyst nematode specific SCAR fragment.The pcr amplification reaction system is 50 μ l, and the PCR reaction system is that 10 * Buffer (contains Mg
2+) 5 μ l, 10mMdNTP4 μ l, each 1 μ l of primer OPD13-HaF1 and OPD13-HaR1 (10 μ mol/L), Taq enzyme (5U/ μ l, Takara) 0.5 μ l, template DNA 5 μ l, sterilization ddH
2O complements to 50 μ l.The pcr amplification condition is: 95 ℃ of denaturation 5min, and 59 ℃ of annealing 30sec, 72 ℃ are extended 1.5min; 35 circulations; 72 ℃ are extended 10min again, 4 ℃ of preservations.Behind the pcr amplification, get 5 μ l amplified productions and add 1 μ l sample loading buffer electrophoresis on 1.5% sepharose, EB dyeing, observation and take a picture recovery, Cloning and sequencing under ultraviolet lamp, sequencing is finished by the large Gene Tech. Company Limited of Beijing six directions China.
The foundation of embodiment 2LAMP technology for detection cereal cyst nematode method
2.1LAMP design of primers
According to cereal cyst nematode RAPD extension increasing sequence sequencing result, design and screen following LAMP primer (see figure 1), it is synthetic that primer is handed in marine life engineering Services Co., Ltd.Primer sequence is as follows:
①HA5-F3：5`-TGATAGGGAAAAAATTCTCCAA-3`；
②HA5-B3：5`-GTCGGCAATGGTCAACAA-3`;
③HA5-BIP：5`-TTGTGGTGCCGTAGTGTTAGAAATGC?TTCATTTGAGAACAATG--3`;
④HA5-FIP：5`-ACACAACGCTTAGTCCGGTCCCAATGGCA?TGGCAAAGA--3`;
⑤HA5-LB：5`-CGGGGATGAAAGAAGGCAAAGTG--3`;
2.2LAMP reaction system configuration:
The primer mixed solution: each 0.2 μ mol/L of outer primer HA5-F3 and HA5-B3, each 1.4 μ mol/L of inner primer HA5-FIP and HA5BIP, ring primer HA5-LB is 0.4 μ mol/L;
Reaction mixture: 3.2mmol/L dNTP, 20mmol/L Tris-HCl (pH8.8), 10mmol/L KCl, 3mmol/L MgS0
4, 10mmol/L (NH4)
2S0
4, 0.1%Triton x-100,8U Bst archaeal dna polymerase large fragment, 1 μ lDNA template adds the completion of sterilization bi-distilled water to 25 μ l.
2.3LAMP reaction amplification condition: above mixed solution is placed 65 ℃ of water bath with thermostatic control isothermal duplication 75min, 85 ℃ of insulation 10min, observations (A among Fig. 2) behind the developer mixing that adding 1 μ l prepared after reaction finished.Get 2 μ l products electrophoresis on 2% sepharose, EB dyeing, observation and photograph can see that the characteristic of LAMP trapezoid belt (B among Fig. 2) is arranged under ultraviolet lamp.
Collect cereal cyst nematode, Philips's cyst roundworm, soy bean cyst roundworm, upland rice cyst roundworm, pea cyst roundworm, barley cyst roundworm, Meloidogyne incognita, javanese root knot nematode, peanut root-knot nematode, radopholus similes thorne (seeing Table 1), extract respectively its DNA and carry out the LAMP detection as template with the cereal cyst nematode dna profiling, to detect the specificity of cereal cyst nematode LAMP detection method.
Table 1 is for other Plant nematode colony sample code and source of examination
After above-mentioned primer mixed solution and reaction buffer mixture mix, add 1 μ l template DNA, reaction conditions by 2.3 carries out, behind the developer mixing that adding 1 μ l prepared after reaction finished, observe colour-change, the 1st pipe can be observed green fluorescence for cereal sporangiocyst DNA, and the nematode of other pipes and negative control are sorrel (shown in A among Fig. 3).Get 3 μ l products electrophoresis on 2% sepharose, EB dyeing, observation and photograph (shown in B among Fig. 3) can see that the 1st swimming lane has the characteristic trapezoid belt of LAMP under ultraviolet lamp, other swimming lanes all find no amplified production.The result shows that above-mentioned LAMP primer and reaction system have very high specificity when detecting cereal cyst nematode.
Single head cereal cyst nematode dna profiling is diluted to 1～1.0 * 10 by 10 times
-56 concentration, respectively get 1 μ lDNA and do template, after above-mentioned primer mixed solution and reaction mixture mixed, undertaken by 2.3 reaction conditionss, behind the developer mixing that adding 1 μ l prepared after reaction finished, observe colour-change (shown in Fig. 4 A), 1～5 pipe all can be seen green fluorescence.Get 3 μ l products electrophoresis on 2% sepharose, EB dyeing, observation and photograph (shown in B among Fig. 4) can find out that 1～5 swimming lane all has the characteristic trapezoid belt of LAMP under ultraviolet lamp, sensitivity can reach 1/200000 nematode.Simultaneously take above-mentioned dilution DNA as template, take SCAR mark Auele Specific Primer Aa289 and Aa740 as primer,, to carry out conventional PCR and detect, system is as follows: 2.5 μ l10 * PCRBuffer (contain Mg
2+), 2.5 μ l10mM dNTPs, 1 μ l primer pair Aa289/Aa740 (10uM), 0.3 μ lTaq archaeal dna polymerase (5U/ul), 1 μ l template DNA, sterilization ddH
2O complements to 25 μ l, adopts without the negative contrast of nematode dna profiling.Gel electrophoresis observations (shown in C among Fig. 4).The result shows at DNA and is diluted to 10
-2Times the time, can observe amplified band, conventional PCR then can not detect during redilution.
Above presentation of results: above-mentioned LAMP detection system can detect 1/200000 cereal cyst nematode, has high sensitivity, detects sensitive 100 times than conventional PCR.
Claims (7)
1. cereal cyst nematode LAMP method for quick, it is characterized in that: wherein the employed primer of LAMP reaction system is:
①HA5-F3：5`-TGATAGGGAAAAAATTCTCCAA-3`；
②HA5-B3：5`-GTCGGCAATGGTCAACAA-3`;
③HA5-BIP：5`-TTGTGGTGCCGTAGTGTTAGAAATGC?TTCATTTGAGAACAATG-3`;
④HA5-FIP：5`-ACACAACGCTTAGTCCGGTCCCAATGGCATGGCAAAGA-3`;
⑤HA5-LB：5`-CGGGGATGAAAGAAGGCAAAGTG-3`。
2. detection method according to claim 1, LAMP reaction system wherein comprises:
1) primer mixed solution: each 0.2 μ mol/L of outer primer HA5-F3 and HA5-B3, each 1.4 μ mol/L of inner primer HA5-FIP and HA5-BIP, ring primer HA5-LB is 0.4 μ mol/L;
2) reaction mixture: 3.2mmol/L dNTP, 20mmol/L Tris-HCl (pH8.8), 10mmol/L KCl, 3mmol/L MgS0
4, 10mmol/L (NH
4)
2S0
4, 0.1%Triton x-100,8U Bst archaeal dna polymerase large fragment;
3) 1 μ l dna profiling;
Add the completion of sterilization bi-distilled water to 25 μ l.
3. detection method according to claim 2, wherein the LAMP reaction conditions is as follows: primer mixed solution and reaction mixture are mixed rear adding 1 μ l dna profiling, 61～65 ℃ of insulation 30～90min, 85 ℃ of insulation 10min.
4. arbitrary described detection method according to claim 1-3 adds developer in the final product of LAMP reaction, observes after gently throwing away core barrel.
5. detection method according to claim 1, described developer is the mixture of SYBR green I and PCR level DMSO, its volume ratio is 1:9.
6. detection method according to claim 1, the extracting method of described dna profiling is: the single cereal sporangiocyst of picking is put into the 0.2mL centrifuge tube that 10 μ l ddH2O are housed, liquid nitrogen freezing, taking-up is placed on ice, turns to ice-out with the glass stick of sterilizing in centrifuge tube, and sporangiocyst is broken, discharge ovum, the LB solution that adds 8 μ l, the 600 μ g/ml Proteinase K solution of 2 μ l are then at-80 ℃ of lower freezing 30min.Centrifuge tube is taken out, and at 65 ℃ of lower incubation 90min, supernatant liquor was directly used in LAMP and PCR reaction as the nematode dna profiling after 95 ℃ of reaction 10min processed, and described LB solution is 500mmol/L KCl, 10mmol/L Tris-HCl, 15mmol/L MgCl
2, 1.0mmol/L DTT, 4.5%Tween20, equal-volume mixing, filtration sterilization.
7. such as the application of the described arbitrary detection method of claim 1-6 in the cereal cyst nematode infection conditions of diagnosis of plant, soil or discriminating cereal cyst nematode.
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| CN114410802A (en) * | 2022-01-28 | 2022-04-29 | 中国农业科学院植物保护研究所 | RPA primer, probe, kit and application for detecting heterodera avenae wollenweber |
| CN114410802B (en) * | 2022-01-28 | 2023-02-03 | 中国农业科学院植物保护研究所 | RPA primer, probe, kit and application for detecting heterodera avenae wollenweber |
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