CN101906465B - Cladosporium cucumerinum assay kit and detection method thereof - Google Patents
Cladosporium cucumerinum assay kit and detection method thereof Download PDFInfo
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- CN101906465B CN101906465B CN2009100523681A CN200910052368A CN101906465B CN 101906465 B CN101906465 B CN 101906465B CN 2009100523681 A CN2009100523681 A CN 2009100523681A CN 200910052368 A CN200910052368 A CN 200910052368A CN 101906465 B CN101906465 B CN 101906465B
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Abstract
The invention provides primer composition used for specifically detecting cladosporium cucumerinum and/or fusarium oxysporum and mycosphaerella melonis in sample and a PCR assay kit containing the primer composition. The invention also provide a method for detecting cladosporium cucumerinum and/or fusarium oxysporum and mycosphaerella melonis in sample. The method of the invention not only can rapidly, simply and accurately detect disease, and detection cost can be greatly saved.
Description
Technical field
The application relates to biological technical field, especially field of biological detection.Particularly, the application relates to the detection kit and the detection method thereof of melon black star germ.
Background technology
Melon scab cladosporium sp (Cladosporium cucumerinum is called the melon black star germ again) can cause melon diseases such as cucumber, watermelon, summer squash, pumpkin, muskmelon, wax gourd, with the morbidity of the black spot on the cucumber the most common [1].The melon black spot is a kind of destructive disease on the melon, because its harm is serious, is difficult to control, and China classifies it as national Plant Quarantine property harmful organism.Cucurbits fusarium wilt bacterium Fusarium oxysporum, melon didymella bryoniae (Didymellabryoniae; Mostly domestic report is Mycosphaerella meloni) also be melon important pathogenic bacteria, these three kinds of pathogenic bacterias be melon in three kinds of very common pathogenic bacterias.
Scab of cucumber in the seedling phase, become the strain phase can both be susceptible, pathogenic bacteria can be infected tender leaf, tender stem and young melon, be melon in common disease, in case the seriously underproduction, even have no harvest takes place in disease.Cucurbits fusarium wilt, melon climing rot can take place simultaneously, are the main diseases that causes melon loss.Cucurbit wilt, the melon grower is " dead rattan ", generally begin to show symptom successively in the field at the plant blossom fruiting period, from seedling to becoming the strain phase all can fall ill, in the phase of yielding positive results the heaviest [5].The climing rot cause of disease of melon belongs to the Ascomycotina fungi, and morbidity is mainly in the melon growth middle and later periods, and plant is climing, leaf, fruit all can be fallen ill main harm blade and melon climing [6].Three kinds of diseases are difficult to identify from symptom at initial phase, therefore, give in time, effectively prevent and treat and bring difficulty.
Because these three kinds of disease spread approach, disease symptom and onset conditions are similar, can carry disease germs through soil and infect, soil, invalid body, flowing water, wind and rain all can make disease spread.Under, the improper ventilation situation big in soil continuous cropping, field humidity, the disease ratio is easier to take place.These three kinds of diseases are on protection ground or morbidity is all more common in outdoor cropping.
Therefore; Press in this area and can develop detection method and the test kit that makes new advances; So that at the initial stage of a disease or in field soil, identify multiple melon main disease simultaneously, thereby a situation arises, in time take effectively preventing method control pathogenic bacteria to propagate for the prediction disease, reduces melon grower's loss.
Summary of the invention
For realizing above-mentioned purpose, the present inventor is through measuring the ITS sequence of melon black star germ (Cladosporium cucumerinum) rDNA, and the ITS sequence homology of comparison sibling species and melon different infective pathogen bacterium has been designed Auele Specific Primer HX-1/HX-2.
Therefore; First aspect present invention provides a kind of compsn that is used for specific detection sample melon black star germ; Said combination comprises a pair of Auele Specific Primer, and the right sequence of said Auele Specific Primer is respectively the sequence shown in SEQ ID NO:1 and the SEQID NO:2.
The present invention provides a kind of PCR detection kit that is used for specific detection sample melon black star germ on the other hand; Said test kit comprises the PCR reaction system; Said reaction system comprises a pair of Auele Specific Primer, and the right sequence of said Auele Specific Primer is respectively the sequence shown in SEQ ID NO:1 and the SEQ ID NO:2.
The present invention provides the method for melon black star germ in a kind of test sample on the other hand, and this method may further comprise the steps: a) with the above-mentioned PCR detection kit of the present invention said sample is carried out pcr amplification, obtain pcr amplification product; B) size of the pcr amplification product that a) obtains of determination step if there is the dna fragmentation of 190bp in the product, then shows melon black star germ in the said sample.In a preferable embodiment, the amplification of said step a) is included in 94 ℃ of preparatory sex change 5 minutes; 94 ℃ of sex change 45 seconds, 60 ℃ of annealing 1 minute, 72 ℃ were extended 1 minute, totally 32 circulations, last 72 ℃ were extended 7 minutes.In another preferable embodiment, said melon is selected from watermelon, muskmelon, cucumber, wax gourd, flesh melon, balsam pear, pumpkin, summer squash and sponge gourd.
The present invention also provides a kind of compsn of specific detection sample cucurbit wilt bacterium, melon didymella bryoniae and melon black star germ simultaneously that is used on the other hand; Said compsn comprises three pairs of Auele Specific Primers; Wherein, The right sequence of first primer is the sequence shown in SEQ ID NO:1 and the SEQ ID NO:2, and the right sequence of second primer is the sequence shown in SEQ ID NO:3 and the SEQ ID NO:4, and the right sequence of three-primer is the sequence shown in SEQ ID NO:5 and the SEQ ID NO:6.
The present invention provides a kind of PCR detection kit of specific detection sample cucurbit wilt bacterium, melon didymella bryoniae and melon black star germ simultaneously that is used on the other hand; Said test kit comprises the PCR reaction system; Said reaction system comprises three pairs of Auele Specific Primers; Wherein, The right sequence of first primer is the sequence shown in SEQ ID NO:1 and the SEQ ID NO:2, and the right sequence of second primer is the sequence shown in SEQ ID NO:3 and the SEQ ID NO:4, and the right sequence of three-primer is the sequence shown in SEQ ID NO:5 and the SEQ ID NO:6.
On the other hand; The invention provides the method for cucurbit wilt bacterium, melon didymella bryoniae and melon black star germ in a kind of while test sample; This method may further comprise the steps; A) with the above-mentioned PCR detection kit that is used for while specific detection sample cucurbit wilt bacterium, melon didymella bryoniae and melon black star germ said sample is carried out pcr amplification, obtain pcr amplification product; B) size of the pcr amplification product that a) obtains of determination step if there is the dna fragmentation of 190bp in the product, then shows melon black star germ in the said sample; If have the dna fragmentation of 327bp in the product, then show cucurbit wilt bacterium in the said sample; If have the dna fragmentation of 442bp in the product, then show melon didymella bryoniae in the said sample.In a preferable embodiment aspect this, the amplification of said step a) is included in 94 ℃ of preparatory sex change 6 minutes, gets into circulation, 94 ℃ of sex change 30 seconds, and 46 ℃ of annealing 1 minute, 72 ℃ were extended 2 minutes, totally 40 circulations, last 72 ℃ were extended 10 minutes.In another the preferable embodiment aspect this, said sample is melon tissue or its DNA extraction thing, and said melon is selected from watermelon, muskmelon, cucumber, wax gourd, flesh melon, balsam pear, pumpkin, summer squash and sponge gourd.
The present invention relates to adopt round pcr to Rapid identification and detection that melon black star germ carries out, overcome long, shortcoming such as program is loaded down with trivial details of traditional time that from disease plant and melon body, exists in the method for isolation identification pathogenic bacteria.Can be exactly latent period of disease and their early stage to disease diagnose, monitoring and pathogen identification so that in time take the effectively preventing method, the generation of control disease reduces financial loss,
Description of drawings
Fig. 1 has shown with primer HX1/HX2 has been carried out the result of provicial commander's pcr amplification, among the figure: M:100bp dna ladder mark; Swimming lane 1-12: represent 58.0 ℃, 58.6 ℃, 59.7 ℃, 61.2 ℃, 63.0 ℃, 64.4 ℃, 65.5 ℃, 66 ℃ annealing temperature respectively.
Fig. 2 has shown with the product of primer to the HX1/HX2 specific amplification.Among the figure, the M:DL2000 mark; Swimming lane 1 and 12: negative control; Swimming lane 2-3 and swimming lane 13-14:Cladosporium cucumerinum isolate; Swimming lane 4-11 and 15-22: the isolate of other different fungies.
Fig. 3 has shown the specific amplification products with primer HX-1/HX-2, Fn-1/Fn-2 and Mn-1/Mn-2.Among the figure, the M:DL2000 mark; Swimming lane 1: negative control; The isolate of swimming lane 2-3:Fusarium oxysporum, Cladosporium cucumerinum and Didymella bryoniae; Swimming lane 4-11: the isolate of other different fungies.
Fig. 4 has shown the sensitivity of carrying out Molecular Detection with primer HX-1/HX-2, M:DL2000 mark among the figure; Swimming lane 1-9: the product that uses concentration in 25 μ l PCR reaction systems, to increase as the DNA of 10ng, 1ng, 100pg, 10pg, 1pg, 100fg, 10fg, 1fg.
Fig. 5 has shown the sensitivity of carrying out the triple PCR detection with different DNA concentration respectively with primer HX-1/HX-2, Fn-1/Fn-2, Mn-1/Mn-2, wherein M:DL2000 mark; Swimming lane 1: negative control; Swimming lane 2-9: use DNA the amplified production in 25 μ l PCR reaction systems of concentration as 10ng, 1ng, 100pg, 10pg, 1pg, 100fg, 10fg, 1fg.
Fig. 6 has shown that the DNA with different concns carries out the sensitivity that triple PCR detects, M:DL2000 mark among the figure with primer HX-1/HX-2, Fn-1/Fn-2, Mn-1/Mn-2 respectively; Swimming lane 1: negative control; Swimming lane 2-9: concentration is the amplified production of DNA in 25 μ l PCR reaction systems of 30ng, 3ng, 300pg, 30pg, 3pg, 300fg, 30fg, 3fg.
Fig. 7 has shown with primer the PCR detected result of HX-1/HX-2 on infected watermelon sample, M:DL2000 mark among the figure; Swimming lane 1: negative control; Swimming lane 2-5: infected fruit; Swimming lane 6-7: the fruit of no pathogenic bacteria; Swimming lane 8-9: the leaf of no pathogenic bacteria.
Fig. 8 has shown with the product of triple PCR from infected melon tissue specificity amplification, M:DL2000 mark among the figure; Swimming lane 1: negative control; Swimming lane 2-5: infected fruit; Swimming lane 6-7: the fruit of no pathogenic bacteria; Swimming lane 8-9: the leaf of no pathogenic bacteria.
Embodiment
The present invention is through measuring the ITS sequence of dosporium cucumerinumand its rDNA, and the ITS sequence homology of comparison sibling species and melon other main infective pathogen bacterium is designed Auele Specific Primer; And with other melons on main pathogenic bacterium detect primer and carry out primer screening, combination; The multiplex PCR system is optimized, has realized the triple PCR of main disease on three kinds of melons is detected, can disposablely detect three kinds of diseases; Not only disease can be detected fast, succinctly, exactly, and the detection cost can be practiced thrift greatly.
Particularly, the present invention at first provides a kind of compsn that is used for specific detection sample melon black star germ, and said combination comprises a pair of Auele Specific Primer, and the right sequence of said Auele Specific Primer is distinguished as follows:
HX-1：5′-TGTTGTCCGACTCTGTTGC-3′(SEQ?ID?NO：1)
HX-2：5′-CATTTCGCTGCGTTCTTC-3′?(SEQ?ID?NO：2)
Primer of the present invention can use any known method to produce; People's [Proc.Natl.Acad.Sci.USA (1983) 80:7461] such as people's [J.Am.Chem.Soc. (1981) 103:3185] such as Matteucci three ester compound methods or Urdea method for example, or synthetic with commercially available automatic oligonucleotide synthesizer.In addition, can also select the chemical feature of primer according to preference.Use for some, DNA or RNA are suitable.For other application, can also add modification, for example backbone modification like thiophosphatephosphorothioate or methyl phosphorodithioate, changes RNA avidity, and increase ribozyme resistances etc. are [for example referring to Agrawal and Iyer (1995) Curr Opin Biotechnol 6:12-19; Agrawal (1996) TIBTECH 14:376-387]; Also can adopt analogue such as PNAG3 [for example referring to Corey (1997) TIBTECH15:224-229; People such as Buchardt (1993) TIBTECH 11:384-386].
In a preferred embodiment, the compsn that is used for specific detection sample melon black star germ of the present invention is made up of the sequence shown in SEQ ID NO:1 and the SEQ ID NO:2.
Second aspect present invention provides a kind of PCR detection kit that is used for specific detection sample melon black star germ; Said test kit comprises the PCR reaction system; Said reaction system comprises a pair of Auele Specific Primer, and the right sequence of said Auele Specific Primer is respectively the sequence shown in SEQ ID NO:1 and the SEQ ID NO:2.
Term " PCR " or " polymerase chain reaction " used among the present invention refer to a kind of process or technology (like United States Patent(USP) No. 4,683, that kind described in 195 (mandates on July 28th, 1987)).Usually, need to obtain the sequence information of area-of-interest two ends or its extension, thus can the design oligonucleotides primer; These primers should be same or similar with the sequence of template opposite strand to be amplified.5 ' terminal nucleotide of two primers can overlap with the two ends of amplification material just.Round pcr can be used to increase among the full gene group DNA, transcribe specific RNA sequence, specific dna sequence the cDNA of acquisition from whole-cell rna, phage or plasmid sequence etc.See Mullis etc., Cold Spring Harbor Symp.Quant.Biol.51:263 (1987); Erlich edits, PCR Technology (Stockton Press, NY, 1989).
Polymerase chain reaction technique is a kind of means that detect a small amount of target nucleic acid well known to those skilled in the art.Similar test is at people such as Mullis [Meth.Enzymol. (1987) 155:335-350]; USP 4,683 is described in 195 and 4,683,202 to some extent.With two " primer " Nucleotide and target nucleic acid hybridization, and be used for guiding reaction.Primer can comprise not the sequence with amplified target sequence (or its complementary sequence) hybridization, helping the stability of duplex, or for example can insert an easy restriction site.In order to detect the existence of target nucleic acid, the selection of Auele Specific Primer is vital.
Those skilled in the art can be according to the PCR reaction system in the technological suitable selection test kit of knowing of the present invention.In a preferable embodiment of the present invention, the reaction mixture of PCR comprises, and is 25 μ l in TV, ExTaq enzyme 12.5 μ l, and each 0.5 μ l of the primer of 10 μ M, template DNA is some.In addition, also can attach process specifications in the PCR detection kit of the present invention.
The present invention has also designed the method for melon black star germ in a kind of test sample on the other hand, and this method may further comprise the steps: a) with the above-mentioned PCR detection kit of the present invention said sample is carried out pcr amplification, obtain pcr amplification product; B) size of the pcr amplification product that a) obtains of determination step if there is the dna fragmentation of 190bp in the product, then shows melon black star germ in the said sample.
In said method, sample for example can be, but is not limited to, the cotyledon of melon plant, young stem, true leaf, stem, pod and seed, preferably the DNA extraction thing of above-mentioned plant tissue.Said DNA extraction thing can use commercially available DNA extraction test kit or other conventional DNA extraction methods to obtain.Though the embodiment of the invention describes with watermelon and cucumber as an example, be to be understood that the melon that the inventive method is suitable for is not limited to watermelon and cucumber, it also can comprise muskmelon, wax gourd, flesh melon, balsam pear, pumpkin, summer squash and sponge gourd.In another preferable embodiment, said sample also can contain any sample of melon black star germ from soil or other suspection.
Pcr amplification reaction can carry out on the commercially available pcr amplification instrument of any routine (for example PTC-200PCR appearance).In a preferable embodiment, the amplification of said step a) is included in 94 ℃ of preparatory sex change 5 minutes; 94 ℃ of sex change 45 seconds, 60 ℃ of annealing 1 minute, 72 ℃ were extended 1 minute, totally 32 circulations, last 72 ℃ were extended 7 minutes.Yet those skilled in the art also can be according to concrete test conditions, augmentation apparatus etc. to the said process change.These are all within those skilled in the art's routine test limit of power.
After having obtained amplified production with the pcr amplification method, can adopt ordinary method, measure the size of pcr amplification product like gel electrophoresis (like agarose gel electrophoresis).If of course, can also adopt more accurate measuring method, as product being checked order etc.In a specific embodiments of the present invention, employing be that method is to get 5 μ l samples at sepharose electrophoresis in 0.5 * TAE of 3.0%, and take a picture with the Bio-rad gel imaging system.If the result shows above-mentioned two pairs of primers and has successfully amplified the band with expectation length (190bp) that then showing has the melon black star germ in the said sample.Should be understood that various variations (like disappearance, increase or replacement) may take place the dna sequence dna of melon black star germ, therefore the length of the actual amplified production that records may have certain difference with above-mentioned length.So; The implication of specification sheets of the present invention used length " XXX bp " is in the whole text not merely represented these length itself; But comprised near the length that said length, (for example differs 20 bp or more, be preferably and differ 10 bp, be more preferred from and differ 5 bp's).
In addition, the present inventor also considers and has designed the multiplex PCR system, so that while adding in a PCR reaction system is many to Auele Specific Primer, and a plurality of goal gene that increase simultaneously, thus can save time, reduce cost, raise the efficiency.The multi-PRC reaction system is difficult to be set up, and can not interact between the primer in requiring to guarantee to react, and the amplified production size is close but can separate through electrophoresis.The multiplex PCR amplification requires very high to test operation, any experiment condition control is improper, all will be easy to cause the failure [7] of amplification condition.The foundation of the multiplex PCR system of domestic report; Great majority all are a plurality of fragments that increase of the different zones to same dna profiling; But seldom to the foundation of the multiplex PCR system of different templates; The foundation that particularly detects different phytopathogen multiplex PCR systems is very rare; ZhengguangZhang [4] places two pairs of Auele Specific Primers of withered germ of water-melon and watermelon didymella bryoniae same reaction tubes to carry out pcr amplification, through optimizing reaction conditions, in primary first-order equation, can two kinds of pathogens successfully be detected.But the system of triple PCR is set up difficult more a lot than the foundation of double PCR; An ideal multi-PRC reaction system is not the simple mixing of single PCR, needs to title product; Carry out multianalysis, TE, set up suitable reaction system and reaction conditions [8].Xie Jianyun etc. [9] adopt the reaction conditions identical with single PCR; Carry out double and the triple PCR detection to inbred mouse; The double PCR of result obtains expected results, and interference appears in triple PCR, therefore thinks when not changing the PCR reaction conditions; Carry out double PCR ratio and be easier to obtain expected results, and relatively more difficult to triple or above pcr amplification.
For overcoming the problems referred to above; The present inventor is through discovering that designed primer of the present invention not only can detect the melon black spot to HX-1/HX-2 from melon pathogenic bacteria; And can also make up with primers F n-1/Fn-2, Mn-1/Mn-2; Set up the triple PCR system, thereby disposablely can detect three kinds of melon important diseases, practice thrift greatly and detect cost.Tangible efficient is high because the relative regular-PCR of multiplex PCR has, low cost and other advantages.
Particularly, the present invention also provides a kind of compsn of specific detection sample cucurbit wilt bacterium, melon didymella bryoniae and melon black star germ simultaneously that is used on the other hand, and said compsn comprises three pairs of Auele Specific Primers,
First primer is to (HX-1/HX-2)
HX-1：5′-TGTTGTCCGACTCTGTTGC-3′(SEQ?ID?NO：1)
HX-2：5′-CATTTCGCTGCGTTCTTC-3′?(SEQ?ID?NO：2)
Second primer is to (Fn-1/Fn-2)
Fn-1：5′-TACCACTTGTTGCCTCGGC-3′(SEQ?ID?NO：3)
Fn-2：5′-TTGAGGAACGCGAATTAAC-3′(SEQ?ID?NO：4)
Three-primer is to (Mn-1/Mn-2)
Mn-1：5′-GGATCATTACCTAGAGTTG-3′(SEQ?ID?NO：5)
Mn-2：5′-ACGTCGTCGTTGTGAGTG-3′?(SEQ?ID?NO：6)
The present invention provides a kind of PCR detection kit of specific detection sample cucurbit wilt bacterium, melon didymella bryoniae and melon black star germ simultaneously that is used on the other hand; Said test kit comprises the PCR reaction system; Said reaction system comprises three pairs of Auele Specific Primers; Wherein, The right sequence of first primer is the sequence shown in SEQ ID NO:1 and the SEQ ID NO:2, and the right sequence of second primer is the sequence shown in SEQ ID NO:3 and the SEQ ID NO:4, and the right sequence of three-primer is the sequence shown in SEQ ID NO:5 and the SEQ ID NO:6.
On the other hand; The invention provides the method for cucurbit wilt bacterium, melon didymella bryoniae and melon black star germ in a kind of while test sample; This method may further comprise the steps; A) with the above-mentioned PCR detection kit that is used for while specific detection sample cucurbit wilt bacterium, melon didymella bryoniae and melon black star germ said sample is carried out pcr amplification, obtain pcr amplification product; B) size of the pcr amplification product that a) obtains of determination step if there is the dna fragmentation of 190bp in the product, then shows melon black star germ in the said sample; If have the dna fragmentation of 327bp in the product, then show cucurbit wilt bacterium in the said sample; If have the dna fragmentation of 442bp in the product, then show melon didymella bryoniae in the said sample.In a preferable embodiment aspect this; The reaction mixture of triple PCR comprises; With TV is that 25 μ l calculate; ExTaq enzyme 12.5 μ l, primer HX-1, HX-2, Fn-1, Fn-2, Mn-1, the Mn-2 of 10 μ M are respectively 0.5 μ l, 0.5 μ l, 0.7 μ l, 0.7 μ l, 0.7 μ l, 0.7 μ l, each 1 μ l of template.The amplification of said step a) is included in 94 ℃ of preparatory sex change 6 minutes, gets into circulation, 94 ℃ of sex change 30 seconds, and 46 ℃ of annealing 1 minute, 72 ℃ were extended 2 minutes, totally 40 circulations, last 72 ℃ were extended 10 minutes.
Concrete numerical point in the above-mentioned embodiment only has been the effect of enumerating, and unqualified effect.It will be appreciated by those skilled in the art that at the numerical value in the scope of said concrete numerical value 5% and also should realize identical/similar purpose that produce identical/similar effect, these technical schemes are also all in full scope of equivalents of the present invention.
In said method, sample for example can be, but is not limited to, the cotyledon of melon plant, young stem, true leaf, stem, pod and seed, preferably the DNA extraction thing of above-mentioned plant tissue.Said DNA extraction thing can use commercially available DNA extraction test kit or other conventional DNA extraction methods to obtain.Though the embodiment of the invention describes with watermelon and cucumber as an example, be to be understood that the melon that the inventive method is suitable for is not limited to watermelon and cucumber, it also can comprise muskmelon, wax gourd, flesh melon, balsam pear, pumpkin, summer squash and sponge gourd.In another preferable embodiment, said sample also can contain any sample of melon black star germ from soil or other suspection.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in the restriction scope of the present invention.Only if description is arranged in addition, enforcement of the present invention will be adopted molecular biology, microbiology, recombinant DNA and immunologic routine techniques, and these all are that those skilled in the art know.These technology have complete description in following document: for example, and Sambrook " molecular cloning experiment guide " the 2nd edition (1989); " dna clone " I and II volume (D.N.Glover edits 1985); " oligonucleotide is synthetic " (M.J.Gait edits, 1984); " nucleic acid hybridization " (B.D.Hames and S.J.Higgins edit .1984); " protein purification: principle and put into practice " the 2nd edition (Springer-Verlag, N.Y.).Perhaps, can carry out according to the specification sheets that the reagent manufacturer is provided.
Embodiment
1 material method:
1.1 strains tested:
The strains tested kind name, source and the quantity that are used for the Auele Specific Primer screening of the employing of present embodiment see the following form.
Table 1
Annotate :+for amplified production is arranged;-be no amplified production.
1.2 mycelium is cultivated and is collected
With the dosporium cucumerinumand its that supplies examination on the PDA flat board; Cultivate after 5-7 days for 21 ℃, cut the mycelia piece from colony edge and go to (dress 100ml nutrient solution in every 250ml Erlenmeyer flask) the PDB liquid nutrient medium (potato glucose liquid nutrient medium), 21 ℃; 100rpm shaking culture 7 days; Filter and collect mycelia, grind to form hypha powder through freezing draining ,-20 ℃ of preservations are subsequent use.
With the withered germ of water-melon that supplies examination, watermelon didymella bryoniae on PDA (potato dextrose agar) flat board; Cultivate after 7 days for 26 ℃, cut the mycelia piece from colony edge and go to (dress 100ml nutrient solution in every 250ml Erlenmeyer flask) the PDB liquid nutrient medium, 28 ℃; 100rpm shaking culture 7 days; Filter and collect mycelia, grind to form hypha powder through freezing draining ,-20 ℃ of preservations are subsequent use.
1.3 the extraction of genomic dna
1.3.1 the extraction of mycelia genomic dna
According to Sun etc.
[2]Method improvement is optimized:
A, 0.008g hypha powder sample, the silica sand mortar grinds, and moves to 1.5 milliliters of pipes, adds 25 microlitre 20%SDS, adds 700 microlitre extracting solutions, 50 microlitre N,O-Diacetylmuramidases, vortex mixing;
B, 37 ℃ of water-baths 30 minutes add 700 microlitre chloroforms: primary isoamyl alcohol (24: 1), and careful the mixing, 12000rpm (rev/min) centrifugal 5 minutes;
C, get supernatant, add isopyknic chloroform: primary isoamyl alcohol (24: 1), carefully mix centrifugal 5 minutes of 12000rpm;
D, get supernatant, add isopyknic primary isoamyl alcohol, the NaAc of 10% volume (pH5.2) solution ,-20 ℃ of depositions 20 minutes;
Centrifugal 5 minutes of E, 12000rpm abandon supernatant, wash deposition 3 times with 70% ethanol, add fully dissolving of 100 microlitre TE (Nucleotide is preserved damping fluid, and staple is Tris-HCl and EDTA) after the drying ,-20 ℃ of preservations.
1.3.2 the rapid extraction of the DNA of incidence tissue:
According to Hong etc.
[3]Method improvement optimization: the plant tissue (blade or melon meat) of getting one section neopathy; Every milligram of tissue adds 20 μ l 0.5M NaOH; Be transferred to after in mortar, fully grinding in the Eppendorf pipe of 1.5ml; Centrifugal 5 minutes of 12000rpm gets 5 μ l supernatants and adds 495 μ l 0.1mMTris damping fluids (PH 8.0), gets 1 μ l behind the mixing and directly is used for the PCR reaction.
1.4 pcr amplification and the sequencing of ribosomal gene transcribed spacer ITS
Adopt Eukaryotic universal primer ITS4/ITS5 amplification black spot genomic dna, it is synthetic that primer is given birth to the worker by Shanghai:
ITS4(5-TCC?TCC?GCT?TAT?TGA?TAT?GC-3’)
ITS?5(5-GGAAGTAAAAGTCGTAACAAGG-3)
The pcr amplification reaction program: 95 ℃ of preparatory sex change 10 minutes, get into circulation, 94 ℃ of sex change 30 seconds, 42 ℃ of annealing 2 minutes, 72 ℃ were extended 2 minutes, totally 30 circulations, last 72 ℃ were extended 10 minutes.After reaction finishes, get 5 μ l samples, and take a picture with the Bio-rad gel imaging system at sepharose electrophoresis in 0.5 * TAE of 3.0%.Amplified production is given birth to worker's order-checking by Shanghai.
1.5. the design of primer HX1/HX2
ITS sequence according to dosporium cucumerinumand its Cladosporium cucumerinum rDNA; The ITS sequence homology of comparison sibling species and melon different infective pathogen bacterium; In order to detect the combination of primers multiplex PCR with melon disease such as cucurbit wilt, the climing rot of melon, melon anthrax, melon sclerotium disease, amplified production is set about 200bp best simultaneously.Design Auele Specific Primer HX-1/HX-2 with primer-design software Primer Premier 5, sequence is following:
HX-15′-TGTTGTCCGACTCTGTTGC-3′
HX-25′-CATTTCGCTGCGTTCTTC-3′
And primer analyzed and estimate, the size of pcr amplification product should be 190bp in theory.
1.6 the selection of primer HX1/HX2 annealing temperature
Base sequence preresearch estimates annealing temperature according to HX1/HX2 is 58 ℃; Design grads PCR program between 58 ℃ to 66 ℃; Totally 8 gradients; Be followed successively by: 58.0 ℃, 58.6 ℃, 59.7 ℃, 61.2 ℃, 63.0 ℃, 64.4 ℃, 65.5 ℃, 66 ℃, find out the optimum annealing temperature of primer amplification.
1.7 the pcr amplification of primer HX1/HX2
The PCR reaction mixture TV that primer HX-1/HX-2 detects black spot is 25 μ l:ExTaq enzymes, 12.5 μ l, each 0.5 μ l of the primer of 10 μ M, and template DNA is some. on the PTC-200PCR appearance, increases.Response procedures is: 94 ℃ of preparatory sex change 5 minutes; 94 ℃ of sex change 45 seconds, 60 ℃ of annealing 1 minute, 72 ℃ were extended 1 minute, totally 32 circulations, last 72 ℃ were extended 7 minutes.After reaction finishes, get 5 μ l samples, and take a picture with the Bio-rad gel imaging system at sepharose electrophoresis in 0.5 * TAE of 3.0%.
1.8 the foundation of triple PCR system
1.8.1 the screening of triple PCR primer
The amplified production size that at first will consider primer when selecting suitable primer to carry out Multiple Combination can not be too approaching, is convenient to the electrophoretogram analysis, also is convenient to practical application and promotes.Comparatively suitable when the amplified production scope generally differs 40-100bp, differ less and bigger combination for amplified fragments, all be unsuitable for the statistics and analysis that carries out data.
Primer to existing bibliographical information detects melon disease is searched for, according to selected two couples of primer: Fn-1/Fn-2 of amplified production clip size and Mn-1/Mn-2.Combination of primers used herein is as shown in table 2 below:
Table 2
| Fungi | Primer sequence | Product (bp) |
| Cladosporium cucumerinum | HX-1：5′-TGTTGTCCGACTCTGTTGC-3′ HX-2：5′-CATTTCGCTGCGTTCTTC-3′ | 190 |
| Fusarium oxysporum | Fn-1：5′-TACCACTTGTTGCCTCGGC-3′ Fn-2：5′-TTGAGGAACGCGAATTAAC-3′ | ?327 |
| Didymella?bryoniae | Mn-1：5′-GGATCATTACCTAGAGTTG-3′ Mn-2：5′-ACGTCGTCGTTGTGAGTG-3′ | ?442 |
1.8.2 the optimization of the triple PCR reaction system that primer HX-1/HX-2, Fn-1/Fn-2, Mn-1/Mn-2 form
Amplified production band brightness according to gained is optimized the primer amount, a little less than the band brightness; Suitably increase the primer amount: three gradients setting the amount of 10 μ M primers F n-1, Fn-2: 0.6 μ l, 0.7 μ l, 0.8 μ l, set three gradients of the amount of 10 μ M primer Mn-1, Mn-2: 0.6 μ l, 0.7 μ l, 0.8 μ l, do orthogonal experiment; During at 46 ℃, other conditions are constant, to triple PCR system primer amount optimized choice in annealing temperature; The result shows as 10 μ M primers F n-1, Fn-2, Mn-1, when the Mn-2 amount all is 0.7 μ l; Reaction result is best, and 327bp, 190bp and three bands of 442bp are all brighter, and brightness is more or less the same.
The reaction mixture TV that final optimization pass goes out the triple PCR of primer HX-1/HX-2, Fn-1/Fn-2, Mn-1/Mn-2 composition is 25 μ l:ExTaq enzymes, 12.5 μ l; Primer HX-1, HX-2, Fn-1, Fn-2, Mn-1, the Mn-2 of 10 μ M is respectively 0.5 μ l, 0.5 μ l, 0.7 μ l, 0.7 μ l, 0.7 μ l, 0.7 μ l, each 1 μ l of template.Response procedures: 94 ℃ of preparatory sex change 6 minutes, get into circulation, 94 ℃ of sex change 30S, 46 ℃ of annealing 1 minute, 72 ℃ were extended 2 minutes, totally 40 circulations, last 72 ℃ were extended 10 minutes.After reaction finishes, get 5 μ l samples, and take a picture with the Bio-rad gel imaging system at sepharose electrophoresis in 0.5 * TAE of 3.0%.
1.8.3 the sensitivity of primer HX-1/HX-2 detection architecture and triple PCR detection architecture:
The blight of being cultivated, didymella bryoniae, the liquid cooling of black star germ bacterium frozen drain; Utilize and optimize good Wyler's process extraction genomic dna; Measure the nucleotide concentration of genomic dna according to adjusting quantitative instrument, the genomic dna template concentrations is transferred to 10ng, 1ng, 100pg, 10pg, 1pg, 100fg, 10fg, 1fg, increase optimizing under good primer HX-1/HX-2 detection architecture and the triple PCR detection architecture condition; According to the electrophoresis situation of amplified production, confirm reaction sensitivity.
1.9 the rapid detection of watermelon incidence tissue
Get blight, climing rot, the black spot axis tissue of 0.5g morbidity respectively, after the liquid nitrogen grinding, extract the diseased tissue genomic dna with the CTAB method after improving.Utilize then and optimize good primer HX-1/HX-2 detection architecture and triple PCR system augmentation detection.
2 results and analysis
2.1 the selection of primer HX1/HX2 annealing temperature
Its annealing temperature of base sequence preresearch estimates according to primer HX1/HX2 is 58 ℃, in order to improve the specificity that primer detects, improve annealing temperature as far as possible; The gradient of design from 58 ℃ to 66 ℃ comes dosporium cucumerinumand its is increased, and the result shows that obviously very bright band is arranged between 58 ℃ to 61.2 ℃; More weak at 63.0 ℃ of banding patterns; And banding pattern almost cannot see between 64 ℃ to 66 ℃, and for the stability that keeps detecting, setting annealing temperature is 60 ℃ (Fig. 1).
2.2 the specificity of primer HX-1/HX-2 check
ITS sequence according to dosporium cucumerinumand its Cladosporium cucumerinum rDNA; The ITS sequence homology of comparison sibling species and melon different infective pathogen bacterium; Utilize Primer Premier5.0 software design to go out a pair of primer HX1/HX2; Can from 9 Cladosporium cucumerinum bacterial strain DNA that supply examination, amplify the product of 190bp, and Cladosporium fulvum all there is not amplified band (Fig. 2) with other 65 relevant and approximate bacterial strains and blank.
2.3 the triple PCR reaction system specific detection that primer HX-1/HX-2, Fn-1/Fn-2, Mn-1/Mn-2 form
According to the triple PCR reaction system among the 1.8.2; The genomic dna of the melon black star germ of the examination of amplification confession simultaneously, wilt, didymella bryoniae; Can amplify the band of 190bp, 327bp, 442bp simultaneously, and relevant or approximate bacterial strain and blank all there is not amplified band (Fig. 3).
2.4 primer HX1/HX2 sensitivity detects
The melon black star germ liquid cooling of being cultivated frozen drain; Utilize and optimize good Wyler's process extraction genomic dna; Measure the nucleotide concentration of genomic dna according to adjusting quantitative instrument, the genomic dna template concentrations is transferred to 10ng, 1ng, 100pg, 10pg, 1pg, 100fg, 10fg, 1fg, under the good PCR condition of optimization, increase; According to the electrophoresis situation of amplified production, confirm reaction sensitivity (Fig. 4).
2.5 the triple PCR system detection sensitivity that primer HX-1/HX-2, Fn-1/Fn-2, Mn-1/Mn-2 form
The cucurbit wilt of being cultivated, climing rot, black star germ liquid cooling frozen drain; Utilize and optimize good Wyler's process extraction genomic dna; Measure the nucleotide concentration of genomic dna according to adjusting quantitative instrument; Genomic dna is begun successively 10 times from 10ng/ μ l down be diluted to 1fg/ μ l, it is template that every concentration is got 1 μ l, carries out pcr amplification with the triple PCR system.The result is illustrated in the reaction system of 25 μ l; When primer HX-1/HX-2, Fn-1/Fn-2, Mn-1/Mn-2 increase single template; Can clearly detect the genomic dna (Fig. 5) of 10pg/ μ l; When primer HX-1/HX-2, Fn-1/Fn-2, Mn-1/Mn-2 increase three templates simultaneously, can detect the genomic dna (Fig. 6) of 300pg/ μ l.
2.6 solid tissue detects
Extract DNA according to the method in 1.7 from the incidence tissue and the health tissues of scab of cucumber, watermelon blight and the climing rot of watermelon of morbidity, detect with primer HX-1/HX-2PCR system and triple PCR system.The result shows, can amplify the band of 190bp with primer HX-1/HX-2 at the DNA of the black spot tissue extraction of morbidity, and not produce band (Fig. 7) on other the health tissues.According to incidence tissue and the health tissues extraction DNA of the method among the 1.8.2, detect with the triple PCR system from scab of cucumber, watermelon blight and the climing rot of watermelon of morbidity.The result shows that the DNA that in incidence tissue, extracts can amplify band, and does not produce band (Fig. 8) on other the health tissues.
Although the invention describes concrete example, having a bit is significantly to those skilled in the art, promptly can do various variations and change to the present invention under the premise without departing from the spirit and scope of the present invention.Therefore, accompanying claims has covered all these changes within the scope of the present invention.It is for referencial use that this paper is all included in all publications, patent and the patented claim that this paper quotes in.
Reference:
[1] Wang Yushui, Liu Yongying, Xu Huicai, Ji Shengdong. environmental factors is to the influence of dosporium cucumerinumand its spore germination. northwest 17 4 phases of volume of agricultural journal .2008.
[2]Sun?Y，Zhang?W，Li?F?L，et?al.Identification?and?genetic?mapping?of?novel?genesthat?regulate?leaf?development?in?Arabidopsis.Cell?Research，2000，10(4)：325-335.
[3]Hong?Wang，Meiqing?Qi，Adrian?J.Cutle?r.A?simple?method?of?preparing?plantsamples?for?PCR.Nucleic?Acids?Research，1993，21(17)：4153～4154.
[4]Zhenggang?Zhang，Jingyu?Zhang，Yuchao?Wang，Xiaobo?Zheng.Moleculardetection?of?Fusarium?oxysporum?f.sp.niveum?and?Mycosphaerella?melonis?in?infectedplant?tissues?and?soil.FEMS?Microbiology?Letters，249(2005)39?47
[5]Zhou，X.G.and?Everts，K.L.Suppression?of?Fusarium?wilt?of?watermelon?bywoil?amendment?with?Hairy?Vetch.Plant?Dis.2004，88，13571365.
[6]Keinath，A.P.，Farnham，M.W.and?Zitter，T.A.Morphological，pathological?andgenetic?differentiation?of?Didymella?bryoniae?and?Phoma?spp.isolated?from?cucurbits.Phytopathology，1995，85，364-369.
[7] yellow honeysuckle flower, Hu Xiaoxiang accounts for profit. influence the factor of multiplex PCR expanding effect. and heredity, 2003,25 (1): 65～68.
[8] Chen Mingjie, square unconventional, Ke Tao; Xiong Zhiyong, what light source. multiplex PCR-a kind of is Protocols in Molecular Biology fast efficiently. Wuhan University of Technology's journal, 2005; 27 (10): 33-36.Chen M J, Fang T, Ke T; Xiong Z Y, HeG Y.Multiplex PCR-A molecular biotechnique of high eficiency and speed.Journal ofWuhan University ofTechnology.2005,27 (10): 33-36.
[9] Xie J.Y.; Shao W.J.; Gao C., Genetic Monitoring in Ten Inbred Strains of Miceby Multiplex PCR.Zhongguo Shiyan DongwuXuebao (AetaLabortorium Animalis SeientiaSinica), ll (2): 92.95. thanks and builds cloud; Shao Weijuan is really high. the application pre-test of multiplex PCR in several inbred mouse Genetic Detection. 11 2 phases of volume of Chinese experimental animal journal .2003
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Claims (8)
1. a compsn that is used for specific detection sample melon black star germ is characterized in that it, and said compsn comprises a pair of Auele Specific Primer, and the right sequence of said Auele Specific Primer is respectively the sequence shown in SEQ ID NO:1 and the SEQ ID NO:2.
2. PCR detection kit that is used for specific detection sample melon black star germ; It is characterized in that it; Said test kit comprises the PCR reaction system; Said reaction system comprises a pair of Auele Specific Primer, and the right sequence of said Auele Specific Primer is respectively the sequence shown in SEQ ID NO:1 and the SEQ ID NO:2.
3. the method for melon black star germ in the test sample is characterized in that it, and this method may further comprise the steps,
A) with the described PCR detection kit of claim 2 said sample is carried out pcr amplification, obtain pcr amplification product, said amplification is included in 94 ℃ of preparatory sex change 5 minutes; 94 ℃ of sex change 45 seconds, 60 ℃ of annealing 1 minute, 72 ℃ were extended 1 minute, totally 32 circulations, last 72 ℃ were extended 7 minutes;
B) size of the pcr amplification product that a) obtains of determination step, if there is the dna fragmentation of 190bp in the product, then showing has the melon black star germ in the said sample.
4. method as claimed in claim 3 is characterized in that it, and said sample is melon tissue or its DNA extraction thing, and said melon is selected from watermelon, muskmelon, cucumber, wax gourd, flesh melon, balsam pear, pumpkin, summer squash and sponge gourd.
5. one kind is used for the compsn of specific detection sample cucurbit wilt bacterium, melon didymella bryoniae and melon black star germ simultaneously; It is characterized in that it; Said compsn comprises three pairs of Auele Specific Primers, and wherein, the right sequence of first primer is the sequence shown in SEQ ID NO:1 and the SEQ ID NO:2; The right sequence of second primer is the sequence shown in SEQ ID NO:3 and the SEQ ID NO:4, and the right sequence of three-primer is the sequence shown in SEQ ID NO:5 and the SEQ ID NO:6.
6. one kind is used for the PCR detection kit of specific detection sample cucurbit wilt bacterium, melon didymella bryoniae and melon black star germ simultaneously; It is characterized in that it; Said test kit comprises the PCR reaction system; Said reaction system comprises three pairs of Auele Specific Primers, and wherein, the right sequence of first primer is the sequence shown in SEQ ID NO:1 and the SEQ ID NO:2; The right sequence of second primer is the sequence shown in SEQ ID NO:3 and the SEQ ID NO:4, and the right sequence of three-primer is the sequence shown in SEQ ID NO:5 and the SEQ ID NO:6.
7. the method for cucurbit wilt bacterium, melon didymella bryoniae and melon black star germ in the test sample simultaneously is characterized in that it, and this method may further comprise the steps,
A) with the described PCR detection kit of claim 6 said sample is carried out pcr amplification, obtain pcr amplification product, said amplification is included in 94 ℃ of preparatory sex change 6 minutes; Get into circulation; 94 ℃ of sex change 30 seconds, 46 ℃ of annealing 1 minute, 72 ℃ were extended 2 minutes; Totally 40 circulations, last 72 ℃ were extended 10 minutes;
B) size of the pcr amplification product that a) obtains of determination step, if there is the dna fragmentation of 190bp in the product, then showing has the melon black star germ in the said sample; If there is the dna fragmentation of 327bp in the product, then showing has the cucurbit wilt bacterium in the said sample; If there is the dna fragmentation of 442bp in the product, then showing has the melon didymella bryoniae in the said sample.
8. method as claimed in claim 7 is characterized in that it, and said sample is melon tissue or its DNA extraction thing, and said melon is selected from watermelon, muskmelon, cucumber, wax gourd, flesh melon, balsam pear, pumpkin, summer squash and sponge gourd.
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Non-Patent Citations (4)
| Title |
|---|
| "A Polymerase Chain Reaction (PCR) Assay for the Detection of Inoculum ofSclerotinia sclerotiorum";Jacqueline Freeman;《BIOM EDICAL AND LIFE SCIENCES》;20021231;第108卷(第9期);第877-886页 * |
| "Molecular detection of Fusarium oxysporum f. sp. niveum and Mycosphaerella melonis in infected plant tissues and soil";Zhang Z;《FEMS Microbiol Lett.》;20050801;第249卷(第1期);第39-47页 * |
| Jacqueline Freeman."A Polymerase Chain Reaction (PCR) Assay for the Detection of Inoculum ofSclerotinia sclerotiorum".《BIOM EDICAL AND LIFE SCIENCES》.2002,第108卷(第9期),第877-886页. |
| Zhang Z."Molecular detection of Fusarium oxysporum f. sp. niveum and Mycosphaerella melonis in infected plant tissues and soil".《FEMS Microbiol Lett.》.2005,第249卷(第1期),第39-47页. |
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