CN103088115A

CN103088115A - Method for detecting pathogen and kit

Info

Publication number: CN103088115A
Application number: CN2011103510869A
Authority: CN
Inventors: 黄怡仁; 陈郁璇; 萧耀基; 胡金胜; 陈立祥
Original assignee: TAIWAN SUGAR INDUSTRY Co Ltd
Current assignee: TAIWAN SUGAR INDUSTRY Co Ltd; Taiwan Sugar Corp
Priority date: 2011-11-01
Filing date: 2011-11-01
Publication date: 2013-05-08
Anticipated expiration: 2031-11-01
Also published as: CN103088115B

Abstract

The invention provides a method for detecting pathogen. The method comprises the following steps of: providing a nucleic acid sample; and detecting whether the nucleic acid sample has pathogen by utilizing at least one group of primer sequences, complementary sequences, derivative sequences and degenerate sequences of SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10.

Description

Detect method and the test kit of cause of disease

Technical field

The present invention relates to a kind of method, test kit and primer thereof that detects cause of disease, relate in particular to a kind of while to detect method, test kit and the primer pair combination thereof of multiple cause of disease in a reaction solution.

Background technology

Seedling is the basic of agriculture production, and its quality has close relationship with crop production, and the good seedling of planting, also can not improve the output value although do not need to change cultivation technique, so each developed country all classifies the health seedling management system as important anti-epidemic measure.Taiwan agricultural commission held a meeting in 1997 and selectes 13 kinds of crops is that health seedling promotes object, has wherein comprised sugarcane and oncidiumLuridum.

The Taiwan sugarcane acreage has more than 10,000 1 thousand hectares, refines sugar and eats each approximately 700,000,000 yuan of sugarcane annual value of production raw.Sugarcane also is developed as the biomass energy crop with eating raw purposes except sugaring in recent years, and therefore in Taiwan on limited soil, how effectively plantation acquisition higher output yield sugarcane more can not be ignored.Reach the vegetative period of sugarcane more than 1 year, grow and can reduce sugar yield if the cultivation process suffers the disease invasion and attack will affect sugarcane, and the cause of disease of harm sugarcane has a variety of, the most common and harm is more serious Ustilago scitaminea, Peronosclerospora sacchari, Leifsona xyli subsp, Xanthomonas albilineans etc., can cause respectively smut, reveal that bacterium is sick, ratoon stunting disease and the sick generation of informal voucher.Above-mentioned four kinds of cause of diseases can infect not only a kind of host, and the crop that report can infect smut has sugarcane, corn, Salvia japonica Thunb. and wheat etc.; Report can infect the crop that reveals the bacterium disease corn, vegetables (Cauliflower, celery and wild cabbage etc.) and fruit flowers (fragrant foreign melon, grape and rose etc.).

The disease that above cause of disease causes, can cause plant-growth slow and reduce output more than 5%, causes loss greatly in harvest.Because the time that tradition judgement disease method is required is longer, and need utilize the molecular biosciences detection technique to detect crop pest by special dealer's judgement, start extensively be accepted and pay attention to.

Therefore, the contriver tests and research through concentrated, and, in line with the spirit of working with perseverance, visualizes the present invention and " detect method and the test kit of cause of disease ", is below explanation of the present invention.

Summary of the invention

The present invention is directed to 4 kinds of common cause of disease Ustilago scitaminea, Peronosclerospora sacchari, the quick and sensitive detection technique of Leifsona xyli subsp, Xanthomonas albilineans exploitation, to assist the foundation of judgement disease auxiliary health seedling system.

According to the specific gene sequence design specific primers of cause of disease, successfully set up real-time quantitative polymerase chain reaction (real-timePCR) technology that can in a reaction solution, simultaneously detect multiplex polymerase chain re-action (multiplex PCR) with the high sensitivity of many group cause of diseases in the present invention.For multiplex PCR, detect, when the dNTP concentration of adjusting PCR condition to 400～2000nM (more than being preferably 800nM), and the Mg of 3mM～7mM (more than being preferably 4mM) ²⁺during concentration, can in a reaction solution, detect the arbitrary target cause of disease, limit of detection is 1,000 copy simultaneously.Detect its amplification efficiency of above-mentioned cause of disease with PCR in real time and can reach 90%～110% (30～62 ℃ of annealing temperatures), limit of detection is 10 copies.Except above-mentioned two kinds of PCR, also can carry out Rapid Screening with biochip, it is the gene fragment of utilizing specific primers that the present invention designs to press from both sides out, get wherein approximately 25 continuous nucleotides as probe to reach the purpose of specificity detection.

Main purpose of the present invention is to provide a kind of method that detects cause of disease, comprising: sample of nucleic acid is provided; And utilize at least one group of sequence, its complementary sequence, its derived sequence or its degenerate sequence that is selected from the primer pair of SEQ ID NO.3 and SEQ ID NO.4, SEQID NO.5 and SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8, SEQ IDNO.9 and SEQ ID NO.10 to detect in this sample of nucleic acid whether have cause of disease.

Another aspect of the present invention is to provide a kind of test kit whether existed for detection of cause of disease, comprising: at least one group of sequence, its complementary sequence, its derived sequence or its degenerate sequence that is selected from the primer pair of SEQ ID NO.3 and SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10.

Another aspect of the present invention is to provide a kind of method that detects cause of disease, this cause of disease is to be selected from Ustilago scitaminea, Peronosclerospora sacchari, Leifsona xylisubsp and Xanthomonas albilineans, the method comprises: the chip with nucleic acid probe (a) is provided, wherein utilize at least one group to be selected from SEQ ID NO.3 and SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8, the primer sequence of SEQID NO.9 and SEQ ID NO.10, its complementary sequence, identical or the complementary Nucleotide of the continuous Nucleotide of 20-30 in the nucleic acid fragment that its derived sequence or its degenerate sequence increase is as this nucleic acid probe, (b) provide sample to be tested, (c) make the nucleic acid probe on this sample to be tested and chip carry out hybridization, and (d) the hybridization result of authentication step (c).

Effect of the present invention and purpose, can be by following embodiment explanation, in order to more in depth understand.

The accompanying drawing explanation

Fig. 1: the annealing curve increased with PCR in real time under identical conditions.

Fig. 2: the canonical plotting of real-time PCR reactions.

Fig. 3 (A)～(E): 5 groups of independent PCR limit of detection tests of primer, wherein Fig. 3 (A) is the 2.5S gene; Fig. 3 (B) is the UbE gene; Fig. 3 (C) is the Lxx gene; Fig. 3 (D) is the Xa gene; Fig. 3 (E) is the PS252 gene.

Fig. 4 (A)～(C): PS252-FR primer total amount and Mg ²⁺concentration affects result, the reaction that wherein Fig. 4 (A) is primer summation 200nM to PCR reaction; The reaction that Fig. 4 (B) is primer summation 400nM; The reaction that Fig. 4 (C) is primer summation 800nM.

Fig. 5: different genes is combined in different Mg ²⁺multiplex PCR result under concentration, wherein Fig. 5 (A) is 2 groups of assortment of genes reactions; Fig. 5 (B) is 3 groups of assortment of genes reactions; Fig. 5 (C) is 4 groups of assortment of genes reactions.

Fig. 6 (A)～(B): 5 groups of primer multiplex PCR assay results, wherein Fig. 6 (A) is for adjusting the front 5 groups of primer multiplex PCR assay results of parameter; Fig. 6 (B) is 5 groups of primer multiplex PCR assay results after the adjustment parameter.

Fig. 7: the multiplex PCR concentration difference limit analysis of 5 groups of primers.

Fig. 8: the multiplex PCR assay result of illustration sample.

Embodiment

(1) design of primers

Use altogether 5 groups of primers in embodiments of the invention, be respectively the specific primers for the gene design of sugarcane gene and four kinds of cause of disease Ustilago scitaminea, Peronosclerospora sacchari, Leifsona xyli subsp, Xanthomonas albilineans, as shown in table 1.Ustilago scitaminea compares (Accession No.EF185086, EF185087, U61290, U61291) with bE mating gene, design UbE-FR Auele Specific Primer pair, and 215 base pair products can increase.Leifsona xyli subsp is with ITS sequence (Acession No.AF 034641, AF056603, EU723209, DQ232616, DQ232615, AF034642) design Lxx-FR Auele Specific Primer pair of comparing, and 145 base pair products can increase.Xanthomonas albilineans compares with ITS (AY940633, AF209751, AF251154, AF251156, AY940629, GQ461740, GU223221), and the Auele Specific Primer of design is to being Xa-FR, and 116 base pair products can increase.The sequence that Peronosclerospora sacchari delivers is few, with COX gene order compare (Acession No.DU116052, EU116053, EU116054, AY286225, EU116058, DQ365724, EF406089), design PS252-FR primer pair, 252 base pair products can increase.At internal contrast prescription face, the embodiment of the present invention is to select the 2.5S gene of sugarcane to be designed, and by Acession No.BQ536525 design 2.5S-FR primer pair, 171 base pair products can increase.But those skilled in the art are scrutable, the primer pair of control group can be different according to the sample source, and the sample source can be any plant and the environment that can be infected by above-mentioned pathogeny.Therefore, the primer pair of control group is not limited to the 2.5S-FR primer pair that the embodiment of the present invention is used, and those skilled in the art can select suitable control group primer pair according to the sample source.

In addition, the sequence that primer pair used in the present invention also is not limited to table 1 to be disclosed, as long as the sequence of table 1 still can be for its target fragment separately that increases through a little modification or after changing, those modified or sequences of changing are all in protection scope of the present invention.Above-mentioned modified or sequence that change can be complementary sequence, derived sequence or the degenerate sequence of table 1 sequence.Wherein derived sequence refers in 3 of SEQ ID NO.1 to SEQ ID NO.10 or its complementary sequence ' end or 5 ' end and is modified, make it still with former sequence, there is the nucleotide sequence of 70% above homology, be preferably the nucleotide sequence that there is 90% above homology with former sequence, if only modified at 5 ' end, derived sequence refers to the nucleotide sequence that has 50% above homology with former sequence; Degenerate sequence refers to that in SEQ ID NO.1 to SEQ ID NO.10 or its complementary sequence, the Nucleotide below 30% is replaced by other Nucleotide, and the Nucleotide be preferably below 10% is replaced by other Nucleotide.

The persons of ordinary skill in the technical field of the present invention knows, in the normal PCR reaction, the primer used is wished to get better expanding effect, except binding site, length and the GC content ratio of primer itself, primer concentration, ionic concn, temperature of reaction or time in the time of also must adjusting reaction, so that the fragment of wish amplification is by effective and single-minded amplification.If primer pair quantity and the segments that will increase increase, interaction between primer and primer increases, suitable reaction conditions is also got over harsh, therefore use many group primers in same PCR reaction, and can reach narrow spectrum amplification effect, be not that identical research field is achievable easily.In addition, the persons of ordinary skill in the technical field of the present invention also knows, normal PCR detects the primer used can only be applicable to single detection mode, and is not suitable for Through Several Survey Measure, for the primer/primer pair of dissimilar PCR design, its scope of application is also different.PCR in real time detects necessary identical or approaching, the general practice of amplified production (amplicon) difference in size to each other drawn, is that the difference of gene fragment size is dwindled in 50bp, to obtain preferably detected result.Otherwise various single stage PCR detects the amplified production difference in size to each other drawn and must obviously, so could distinguish on same gel when carrying out follow-up electrophoretic analysis.The primer pair combination that the present invention develops, have avoid forming dipolymer, preferably GC content ratio, the large I of gene are distinguished on same gel, and the PCR annealing temperature needs the characteristics such as close, the primer that has overcome normal PCR detection use can only be applicable to single detection mode, can be applicable to respectively PCR in real time and multiplex PCR, can, in identical conditions, same reaction solution, carry out the specificity pcr amplification reaction simultaneously.

Table 1

The primer title	Primer sequence
		2.5S-F	5′-GTCGGCTCTTCCTATCATTGTG-3′
2.5S-R	5′-GTGAATCAACGGTTCCTCTCG-3′
		UbE-F	5′-CGGCTGGTCCAACATTCTC-3′
UbE-R	5′-TGCTGCTCCATTCTTCTTCG-3′
		Lxx-F	5′-TGGGTGGAACATTGACATTGG-3′
Lxx-R	5′-CTTGAAGACACAGCGATGAGG-3′
		Xa-F	5′-CGCACACGAAGAATTTGAATG-3′
Xa-R	5′-CCGCCTTAGCCACAACG-3′
		PS252-F	5′-GTAGTCAATGGTATTGGAGTTATG-3′
PS252-R	5′-TTAATCTACCTGGACAAGCATC-3′

(2) standard plasmid preparation

Target gene sequence according on GenBank, design corresponding primer sequence, after pcr amplification with Promega sV Gel and PCR Clean-Up System (Promega; Cat#A9281) reclaim target gene, target gene is cloned in to yT& A plasmid (Yeastern; And transform in corresponding intestinal bacteria (Yeastern cat#YC103); Cat#YE607).Get the bacterium liquid of the cultivation overnight that 3mL contains plasmid, remove supernatant liquor after centrifugal, then with the PureYield of Promega company ^tMplasmid prepares test kit (Promega in a small amount; Cat#A1223) extract colibacillary plasmid.The plasmid of purifying cuts at specific site with the DNA restriction enzyme, DNA and sample-loading buffer (loading buffer) are mixed, inject the sample groove of 1% gel, and add DNA molecular amount standard substance (DNA marker), the voltage that imposes 100 volts is separated.After SyBr Green dyeing, observe and the photograph DNA fragmentation separates situation at the UV light source, and can carry out interpretation DNA size by DNA molecular amount standard substance, be confirmed whether as correct band, do sequence after errorless to confirm.Plasmid after purifying can be placed in 4 ℃ of temporary transient preservations, or is stored in-20 ℃ of persistences.Unit conversion method is that the plasmid of confirming the loaded targets gene is surveyed to its A with a minute luminometer ₂₆₀the value obtained is multiplied by extension rate takes advantage of 50 again, to obtain concentration unit amount (μ g/ μ l), want to be converted into copy number (copies number), divided by the base pairs of plasmid, again divided by base pair molecular weight 660 Doltons (Dalton) be converted into mole/μ l, be multiplied by 6.02 * 10 ²³with copied/μ l.

(3) detection mode one: PCR in real time is highly sensitive detection technique, and different from the end point determination of general PCR is that PCR in real time is by amplification procedure monitoring SyBr Green fluorescence volume, carrying out Real-Time Monitoring.Because PCR in real time sensitivity is high, therefore also high to the design requirements of primer.

Please refer to Fig. 1, whether the present invention has high specificity by designed primer judge the present invention with the annealing curve of PCR in real time amplification under identical conditions in.When measuring annealing temperature, because the GC content of each gene is neither same, therefore when continuing to increase the temperature to certain temperature value, there is the double-stranded DNA more than 50% to be opened into strand, show fluorescent value decline figure rapidly in PCR in real time, after differential converts, be presenting of annealing curve figure.As shown in Figure 1, each gene fragment all only has a single crest (being melting peak (melt peak)) to the annealing curve of the amplified production of 5 groups of primers that table one of the present invention discloses, and this result represents that these 5 groups of primers have high specificity.Because primer designed in the present invention has high specificity, and can not form dipolymer, therefore can avoid interference the process of PCR in real time amplification and then increase the accuracy detected.

Accuracy when the primer amplification efficiency also can affect common reaction, best state should be 2 times of amplifications, and therefore best amplification efficiency is 90-110%, and calculation formula is efficiency (η)=[10 ^(1/slope)]-1, the log value that wherein slope is Ct value (Cycle Threshold)/concentration or copy number, so when amplification efficiency is 100%, slope approximately-3.

Want to make a plurality of target gene fragments effectively to increase under same amplification condition, in the present invention, utilize the primer amplification efficiency to seek the common detected temperatures scope of the best of a plurality of primer pairs.5 groups of primers of the present invention's design are respectively 2.5S-FR (being 2.5S-F and 2.5S-R), PS252-FR (being PS252-F and PS252-R), UbE-FR (being UbE-F and UbE-R), Lxx-FR (being Lxx-F and Lxx-R) and Xa-FR (being Xa-F and Xa-R), after getting respectively the plasmid mensuration concentration that comprises target gene, carry out 10 times of serial dilutions, the advanced performing PCR of the gene that wish is analyzed amplifies, carry out real-time PCR reactions and standard curve making, typical curve R ²>0.95 can trust.With iQ SYBR supermix PCR test kit (Bio Rad; Cat#170-8884) allotment PCR reaction solution, carry out the PCR in real time analysis.The PCR in real time condition is, 95 ℃ 10 minutes, " 95 ℃ 15 seconds, 30～62 ℃ 15 seconds, 72 ℃ 15 seconds, 82 ℃ 15 seconds " 30 circulations, 55～95 ℃ are detected annealing temperatures.Understanding according to those skilled in the art to PCR, the above-mentioned condition of part can slightly be done to change and can significantly not affect the PCR result, for example increases and decreases the number of times of circulation.If the resulting primer amplification efficiency of above-mentioned reaction need lower than this scope, mean that design of primers is bad between 90%～110%, the annealing weak effect; If surpass 110% i.e. representative, non-specific amplified production is arranged most probably.As shown in Figure 2, it is that standard substance concentration is contrasted and makes with the Ct value to typical curve.

The amplification efficiency of these 5 groups of primers is compared, arrange as table 2.When these 5 groups of primer annealing temperature are 30～62 ℃, amplification efficiency is between 90～125%.Want to reach better PCR efficiency range 95%～105%, the primer annealing temperature is preferably 30～58 ℃.In addition, through the test of PCR in real time limit of detection, confirm that 5 groups of primers are under suitable detected temperatures scope and condition, its limit of detection is all 10 copies.

Table 2

(4) detection mode two: multiplex PCR

While wanting to make 5 groups of gene fragments in table 1 of the present invention to detect, amplification efficiency is consistent simultaneously, carries out the common limit of detection test under different condition, wherein inquires into 200～800nM primer total amount and 1～5mM MgCl ₂best amplification condition scope under concentration combination.In addition, with the different genes combination, best dNTP concentration and MgCl are discussed by immobilized primer concentration ₂concentration, best multiplex PCR result when more than seeking with this, group genes detect simultaneously.The multiplex PCR condition is 95 ℃ of 1 circulations in 2 minutes, " 95 ℃ 15 seconds, 55 ℃ 15 seconds, 72 ℃ 15 seconds " 30 circulations, 72 ℃ of 1 circulations in 10 minutes, then be held in 4 ℃.Above-mentioned condition can adjust at the situation end that can significantly not affect result, and for example 72 ℃ of last 1 circulations in 10 minutes can make 70 ℃ of 1 circulations in 10 minutes into.

Detection sensitivity is except outside the Pass having with technology category, and the design of reaction conditions, as PCR temperature, primer specificity, primer concentration, Mg ²⁺concentration, K ⁺concentration and Taq kind etc., also can affect its detection sensitivity.Multiple PCR technique is in same reaction solution, carries out the amplification more than one group of gene simultaneously, and required instrument, the consumptive material of this technology is all identical with general PCR with detecting step.But while carrying out multi-PRC reaction, must note whether can interacting between primer, produce not single-minded product, or whether amplification efficiency can be different to organize gene more simultaneously, and causes the portion gene detection sensitivity to descend.Perhaps, when different genes concentration is variant, whether can cause competition to suppress to cause false negative etc., all must further inquire into to get rid of possible impact.

(1) single group of primer PCR condition and limit of detection

Please refer to Fig. 3 (A)～(E), it is 5 groups of independent PCR limit of detection tests of primer.Fig. 3 (A) is the 2.5S gene; Fig. 3 (B) is the UbE gene; Fig. 3 (C) is the Lxx gene; Fig. 3 (D) is the Xa gene; Fig. 3 (E) is the PS252 gene.Swimming lane 1～swimming lane 7 of Fig. 3 (A)～(D) is respectively 1E+07～1E+01 copy, the negative control group NTC of swimming lane 8, and swimming lane 1～swimming lane 5 of Fig. 3 (E) is respectively 1E+06～1E+02 copy.Swimming lane M is that molecular weight indicates swimming lane.5 groups of primer 2 .5S-FR, PS252-FR, UbE-FR, Lxx-FR and Xa-FR of the present invention's design, the product of its amplification is respectively 171bp, 252bp, 215bp, 145bp and 116bp.Work as Mg ²⁺1.5mM, during primer summation 200nM, limit of detection all can reach 1E+03 copy (as shown in Fig. 3 (A)～(D)).And the PS252-FR primer is in this condition, limit of detection is 1E+04 copy (Fig. 3 (E)) only.For making 5 groups of primers all reach the 1E+03 copy detection limit, adjust the primer summation of PS252-FR and change Mg ²⁺concentration, make 5 groups of primers all meet best testing conditions.

Please refer to Fig. 4 (A)～(C), it is PS252-FR primer total amount and Mg ²⁺the affect result of concentration on the PCR reaction.The reaction that Fig. 4 (A) is primer summation 200nM; The reaction that Fig. 4 (B) is primer summation 400nM; The reaction that Fig. 4 (C) is primer summation 800nM.The 1mM-4mM indicated in figure is the Mg2+ concentration of adding in the PCR

process.Swimming lane

1,6,11,16 is the 1E+05 copy, and

swimming lane

2,7,12,17 is the 1E+04 copy, and

swimming lane

3,8,13,18 is the 1E+03 copy, and

swimming lane

4,9,14,19 is the 1E+02 copy, and

swimming lane

5,10,15,20 is the 1E+01 copy.From Fig. 4 (A)～(C), as primer summation 200nM, Mg ²⁺must be 4mM, limit of detection begins to reach 1E+03 copy (Fig. 4 (A)); As primer summation 400nM, Mg ²⁺3mM must reach 1E+03 copy (Fig. 4 (B)) for can make limit of detection; As primer summation 800nM, Mg ²⁺2mM can make limit of detection reach 1E+03 copy (Fig. 4 (C)).From the above results, at fixing dNTP under the condition of q.s, when the primer amount increases, Mg ²⁺required content, under low concentration, can reach limit of detection 1E+3 copy.Otherwise, when primer content is less or efficiency when poor, improve Mg ²⁺concentration also can reach target detect limit 1E+03 copy.Due to Mg ²⁺excessive concentration may have not single-minded product and produce, and the PS252-FR primer of therefore getting 800nM carries out follow-up discussion.

(2) 2 groups, the multiplex PCR of 3 groups, 4 groups arbitrary combination

Please refer to the 5th figure (A)～(C), it is respectively different genes and is combined in different Mg ²⁺multiplex PCR result under concentration.Respectively with 2mM～5mM Mg ²⁺, to 2 groups, 3 groups, with 4 groups of different genes combinations, carry out multiplex PCR assay.Fig. 5 (A) is 2 groups of assortment of genes reactions; Fig. 5 (B) is 3 groups of assortment of genes reactions; Fig. 5 (C) is 4 groups of assortment of genes reactions.Each reactant relative position of organizing gene is shown in the rightmost of figure, from top to bottom is respectively Ps252, UbE, 2.5S, Lxx and Xa.From Fig. 5 (A)～(C), when amplification gene group number improves, required Mg ²⁺concentration also must improve, the 4 groups of genes that therefore will simultaneously increase, Mg ²⁺concentration is preferably more than 4mM.

(3) 5 groups of primer multiplex PCRs

Before adjusting parameter, (5 groups of primer summations are respectively 200nM to Fig. 6 (A), Mg ²⁺1.5mM) the multiplex PCR assay result of 5 groups of primers, swimming lane 1～5 is respectively 5 groups of each 1E+05 of gene, 1E+04,1E+03,1E+02,1E+01 copy.From the result of above-mentioned Fig. 5 and Fig. 6, the increase of primer amount contributes at relatively low Mg ²⁺the situation of content is issued to preferably limit of detection, and when organizing gene more and increasing simultaneously, Mg ²⁺be preferably>=4mM of concentration.Adjust PS252-FR primer summation to 800nM based on the above results, all the other 4 groups of primer summations are respectively 200nM, Mg ²⁺more than 3mM.The multiplex PCR assay result that Fig. 6 (B) is 5 groups of primers after the adjustment parameter, swimming lane 1～4 is respectively 5 groups of each 1E+06 of gene, 1E+05,1E+04,1E+03 copy.From Fig. 6 (A)～(B), 5 groups of common detections by script limit of detection 1E+04 copy of primer are improved as the 1E+03 copy, identical with single primer limit of detection.

(4) 5 groups of gene multiplex PCR concentration difference limit

If gene content difference during multiplex PCR assay, may cause the gene of low levels to increase, this phenomenon is more obvious when different genes group concentration difference is higher.For improving this phenomenon, test dNTP 400nM～2000nM and Mg ²⁺multi-PRC reaction under the condition of concentration 3mM～7mM, result shows that raising dNTP concentration is to 800nM and Mg ²⁺when concentration 4mM is above, originally during 10 times of 5 groups of mrna concentration differences, the phenomenon that the lower concentration gene can't increase (not shown) is improved, and 5 groups of primers can increase effectively simultaneously.Result as shown in Figure 7, the multiplex PCR concentration difference limit analysis that it is 5 groups of primers.Above-mentioned analysis is with dNTP 800nM and 4mM Mg ²⁺10 times, 100 times of mrna concentration differences and the difference of 1000 times are discussed.Swimming lane 1 is the positive controls that 5 groups of genes are respectively the 1E+03 copy; Swimming lane 2～6 is the highest group with 1000 times of minimum quantity differences of gene; Swimming lane 7～11 is the highest group with 100 times of minimum quantity differences of gene; Swimming lane 12～16 is the highest group with 10 times of minimum quantity differences of gene.Wherein the gene of every group of minimum content is respectively PS252, UbE, 2.5S, Lxx and Xa from left to right.For example, in

swimming lane

6,11,16, Xa is 1E+03 copy, but all the other 4 kinds of genes in

swimming lane

6,11,16, be respectively 1000 times, 100 times with 10 times of content.

(5) embodiment-sugarcane sample test

Using sugarcane as sample in this embodiment, but plant, soil or the water etc. of the present invention disclosed four kinds of pathogenic bacteria Ustilago scitaminea, Peronosclerospora sacchari, Leifsona xyli subsp, Xanthomonas albilineans PI all can be used as the detection sample, wherein plant can be sugarcane, corn, Salvia japonica Thunb., tobacco, wheat, vegetables (such as Cauliflower, chilly, celery and wild cabbage etc.), fruit (such as oranges and tangerines, tomato, fragrant foreign melon and grape) or flowers (such as rose etc.).

Carry out the sugarcane sample detection with above-mentioned multiplex PCR and PCR in real time.With the detected result of multiplex PCR assay as described in Figure 8.The negative control group of NC; PC is the positive controls that 5 groups of genes are respectively the 3E+1 copy; Swimming lane 1～27 is respectively sample L2, L3, L4, L5, L11, L12, S13, R14, E15, H16, L17, L18, S18, L19, S19, L20, S20, L21, L22, S22, H24, L24, S24, R24, L28, S29, L31.Wherein code name L represents leaf section sample, and S represents stem's sample, and R represents the root sample, E represents pedotheque, and sample of the present invention is not limited to above-mentioned, also can take from other positions of plant from the sample of plant, such as fruit, petal etc., and also can take from water from the sample of environment.Sample segment is tested root (R), stem (S), leaf (L) and head (H) slightly simultaneously, finds that the different sites sample is influential to detection sensitivity.With the judgement of internal contrast group 2.5S gene, the multiple PCR method that the present invention sets up can effectively detect the pathogen infection situation of sugarcane different sites sample.In the situation that there is no the internal contrast group, also can judge whether according to detected next clip size possibility and the cause of disease kind of pathogen infection.

The PCR in real time detection sensitivity, higher than multiplex PCR, also can detect cause of disease even trace infects PCR in real time.And the cause of disease content difference of same plant different sites, even the low position of cause of disease content also can effectively detect cause of disease (as different sites H24, L24 and the S24 of No. 24 samples) with PCR in real time.Detect more as shown in table 3.

Table 3

* in the table, indicate-negative reaction; Indicate UbE for detecting the smut cause of disease; Indicate PS and reveal the bacterium disease pathogen for detecting; Indicate Lxx for detecting the ratoon stunting disease cause of disease; Indicate Xa for detecting the informal voucher disease pathogen.

(6) applying biochip is identified the method for cause of disease

The present invention utilizes SEQ ID NO.1 and SEQ ID NO.2, SEQ ID NO.3 and SEQID NO.4, SEQ ID NO.5 and SEQ ID NO.6, SEQ ID NO.7 and SEQ IDNO.8, SEQ ID NO.9 and SEQ ID NO.10, its complementary sequence, its derived sequence or its degenerate sequence are respectively with pcr amplification sugarcane and four kinds of pathogenic bacteria Ustilago scitaminea, Peronosclerospora sacchari, Leifsona xyli subsp, Xanthomonas albilineans's is nucleic acid-templated, the PCR condition is 95 ℃ of 1 circulations in 2 minutes, " 95 ℃ 15 seconds, 55 ℃ 15 seconds, 72 ℃ 15 seconds " 30 circulations, 72 ℃ of 1 circulations in 10 minutes, then be held in 4 ℃.Get gene fragment after amplification wherein approximately 25 (for example 20-30) continuous Nucleotide or its complementary sequence as the probe of biochip.Because the sequence of above-mentioned probe can be learnt by order-checking, therefore also direct synthesising probing needle fragment on chip.

Below provide an embodiment of biochip making method, but the present invention is not limited to this method.At first, one blank chip is toasted approximately 5 minutes under 45 ℃, respectively (μ M) oligonucleotide probe of 40 micro-molar concentrations is added probe solution to mix, the position of setting with spot film machine point again, be placed in 45 ℃ of baking 1-2 minute, finally again with ultraviolet-crosslinkable device (UVcrosslinker) in 0.8 Jiao Er, under the condition of 6 minutes, oligonucleotide probe is fixed on chip.

Utilize the tagged molecule labeled primer, described tagged molecule includes but not limited to the tagged molecule such as vitamin H (Biotin), fluorescein, foxglove.Carry out polymerase chain reaction with the primer of vitamin H (Biotin) mark and the DNA of sample to be tested in the present embodiment, its each reaction solution cumulative volume is 25 μ l, include 2ng sample to be tested template DNA, 250 μ M dNTPs, 1XPCR damping fluid, 0.2 μ M primer, and 0.5U Taq Plus heat-resisting polymerase (Bio BasicInc., Canada).Reaction conditions is 95 ℃ of 1 circulations in 2 minutes, " 95 ℃ 15 seconds, 55 ℃ 15 seconds; 72 ℃ 15 seconds " 30 circulations, 72 ℃ of 1 circulations in 10 minutes complete reaction, and obtain having the amplified production of biotin labeled sample to be tested, are the target (target) of heterozygosis reaction.

Oligonucleotide probe on biotin labeled target dna fragment and biochip of the present invention is hybridized.According to the preferred embodiments of the present invention, the method of carrying out hybridization comprise by the PCR reactant be heated to 95 ℃ open two strands after, first add hybridization reaction solution at each reactive tank of chip, add again appropriate biotin labeled target dna fragment to mix with it, be placed in 45～60 ℃ of environment lower 1 hour, to carry out hybridization.Wash away afterwards the not target fragment of hybridization, add again developer (4 μ l NBT/BCIP+196 μ l Detection buffer) to be placed in darkroom reaction 10 minutes, after reaction, with clear water, clean, and dry under 45 ℃, judge in sample to be tested having or not of color spot to occur whether existing of cause of disease is arranged.

In sum, can illustrate with the disclosed primer of the present invention and can effectively detect at the same time or separately 4 kinds of cause of diseases by above-described embodiment.Those skilled in the art can understand easily the designed any one group of above primer pair of the present invention and can use with primer in order to detect other cause of diseases or for the primer collocation of the host's of those cause of diseases gene design, to reach the broader applications of the designed primer of the present invention.In addition, in the scope of also protecting in institute of the present invention wish for detection of the test kit that comprises the designed primer of the present invention or the wrapped product of four kinds of cause of diseases of the present invention.According to the current content of many these type of commercialization commodity, mentioned reagent box or wrapped product can further comprise as required and extract the required reagent of sample nucleic acid required reagent, PCR process, carry out electrophoresis (such as discontinuous agarose coagulation electrophoresis) and analyze needed reagent etc.

Embodiment

1. a method that detects cause of disease comprises:

Sample of nucleic acid is provided; And

Utilize at least one group of sequence, its complementary sequence, its derived sequence or its degenerate sequence that is selected from SEQ ID NO.3 and SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10 to detect in this sample of nucleic acid whether have cause of disease.

2. according to the described method of embodiment 1, wherein this cause of disease comprises at least one in Ustilago scitaminea, Peronosclerospora sacchari, Leifsona xyli subsp and Xanthomonas albilineans.

3. according to the described method of embodiment 1-2 any one, wherein this sample of nucleic acid is from plant or environmental samples.

4. according to the described method of embodiment 3, wherein this environmental samples comprise soil and water at least one of them.

5. according to the described method of embodiment 1-4 any one, also comprise:

Use the primer pair of control group, wherein this control group primer pair is SEQ ID NO.1 and SEQ ID NO.2, its complementary sequence, its derived sequence or its degenerate sequence.

6. according to the described method of embodiment 5, wherein this derived sequence refers in 3 of SEQ ID NO.1 to SEQ ID NO.10 or its complementary sequence ' end or 5 ' end and is modified, and makes it still with former sequence, have the nucleotide sequence of 70% above homology.

7. according to the described method of embodiment 5, wherein this derived sequence refers in 5 of SEQ ID NO.1 to SEQ ID NO.10 or its complementary sequence ' end and is modified, and makes it still with former sequence, have the nucleotide sequence of 50% above homology.

8. according to the described method of embodiment 5-7 any one, wherein this derived sequence refers in 3 of SEQ ID NO.1 to SEQ ID NO.10 or its complementary sequence ' end or 5 ' end and is modified, and makes it still with former sequence, have the nucleotide sequence of 90% above homology.

9. according to the described method of embodiment 5-8 any one, wherein this degenerate sequence refers to that in SEQID NO.1 to SEQ ID NO.10 or its complementary sequence, the Nucleotide below 30% is replaced by other Nucleotide.

10. according to the described method of embodiment 5-9 any one, wherein this degenerate sequence refers to that in SEQID NO.1 to SEQ ID NO.10 or its complementary sequence, the Nucleotide below 10% is replaced by other Nucleotide.

11., according to the described method of embodiment 5-10 any one, wherein this sample of nucleic acid is from plant.

12. according to the described method of embodiment 11, the wherein gene order complementation of the primer pair of this control group and this plant, wherein this plant comprises sugarcane, tobacco, Salvia japonica Thunb., wheat, corn, Cauliflower, fragrant foreign melon, grape, oranges and tangerines, tomato, chilly, rose and leaf vegetables.

13., according to the described method of embodiment 1-12 any one, wherein said detection comprises:

Carry out multiplex polymerase chain re-action (multiplex PCR) and real-time quantitative polymerase chain reaction (real-time PCR) one of them.

14., according to the described method of embodiment 1-13 any one, also comprise and be adjusted to identical by the ultimate value of each primer pair.

15. according to the described method of embodiment 13, wherein:

When carrying out multiplex polymerase chain re-action, use between the dNTP of 400～2000nM concentration, be preferably 800nM, reach the Mg between 3～7mM ²⁺concentration, more than being preferably 4mM; When carrying out the real-time quantitative Polymerase Chain Reaction, use the annealing temperature (annealing temperature) between 30～62 ℃, be preferably below 58 ℃.

16. the test kit whether existed for detection of cause of disease comprises:

At least one group of primer sequence, its complementary sequence, its derived sequence or its degenerate sequence that is selected from SEQ ID NO.3 and SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8, SEQ ID NO.9 and SEQID NO.10.

17., according to the described test kit of embodiment 16, also comprise:

Extract the required reagent of sample nucleic acid.

18., according to the described test kit of embodiment 16-17 any one, also comprise:

The polymerase chain reaction solution formula.

19., according to the described test kit of embodiment 16-18 any one, also comprise:

Carry out the needed reagent of electrophoretic analysis, the needed reagent of described electrophoretic analysis includes but not limited to the needed reagent of discontinuous agarose coagulation electrophoresis.

20. a method of utilizing multiplex polymerase chain re-action (multiplex PCR) to detect cause of disease comprises:

Sample of nucleic acid is provided, and wherein this sample of nucleic acid is one of them from plant and environmental samples, and this environmental samples comprises soil and water;

Utilize at least three groups to be selected from SEQ ID NO.3 and SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8, the primer sequence of SEQ ID NO.9 and SEQ ID NO.10, its complementary sequence, its derived sequence or its degenerate sequence and control group sequence are carried out multiplex polymerase chain re-action with the nucleic acid in this sample that increases, wherein this control group primer pair is SEQ ID NO.1 and SEQ ID NO.2, its complementary sequence, its derived sequence or its degenerate sequence, wherein this derived sequence refers in 3 of SEQ ID NO.1 to SEQID NO.10 or its complementary sequence ' end or 5 ' terminal modified other nucleotide sequences, make it still with former sequence, there is the nucleotide sequence of 90% above homology, this degenerate sequence refers to that in SEQ IDNO.1 to SEQ ID NO.10 or its complementary sequence, the Nucleotide below 10% is replaced by other Nucleotide, wherein dNTP concentration is adjusted in 400～2000nM, Mg ²⁺concentration is adjusted between 3～7mM, and is adjusted to identical by the ultimate value of each primer pair, and

Utilize the gene order increased to detect this sample of nucleic acid and whether have cause of disease, wherein this cause of disease comprises at least one in Ustilago scitaminea, Peronosclerospora sacchari, Leifsona xyli subsp and Xanthomonas albilineans.

21. a method of utilizing real-time quantitative polymerase chain reaction (real-time PCR) to detect cause of disease comprises:

Utilize at least one group to be selected from SEQ ID NO.3 and SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8, the primer sequence of SEQ ID NO.9 and SEQ ID NO.10, its complementary sequence, its derived sequence or its degenerate sequence and control group sequence are carried out the real-time quantitative Polymerase Chain Reaction with the nucleic acid in this sample that increases, wherein this control group primer pair is SEQ ID NO.1 and SEQ ID NO.2, its complementary sequence, its derived sequence or its degenerate sequence, wherein this derived sequence refers in 3 of SEQ ID NO.1 to SEQ ID NO.10 or its complementary sequence ' end or 5 ' terminal modified other nucleotide sequences, make it still with former sequence, there is the nucleotide sequence of 90% above homology, this degenerate sequence refers to that in SEQID NO.1 to SEQ ID NO.10 or its complementary sequence, the Nucleotide below 10% is replaced by other Nucleotide, wherein annealing temperature (annealing temperature) is adjusted between 30～62 ℃, and be adjusted to identical by the ultimate value of each primer pair, and

22. a method that detects cause of disease, this cause of disease is selected from Ustilago scitaminea, Peronosclerospora sacchari, Leifsona xyli subsp and Xanthomonas albilineans, and the method comprises:

(a) providing the chip with nucleic acid probe, is wherein that the identical or complementary Nucleotide of 20-30 continuous Nucleotide in the nucleic acid fragment that utilizes at least one group of primer pair sequence, its complementary sequence, its derived sequence or its degenerate sequence that is selected from SEQ IDNO.3 and SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10 to increase is as this nucleic acid probe;

(b) provide sample to be tested;

(c) make the nucleic acid probe on this sample to be tested and chip carry out hybridization; And

(d) the hybridization result of authentication step (c).

23., according to the described method of embodiment 22, wherein step (a) also comprises: the identical or complementary Nucleotide of 20-30 continuous Nucleotide in the nucleic acid fragment that uses the control group primer pair to increase is nucleic acid probe as a control group.

24., according to the described method of embodiment 22-23 any one, wherein the primer pair of this control group is SEQ ID NO.1 and SEQ ID NO.2, its complementary sequence, its derived sequence or its degenerate sequence, and this host is sugarcane.

25., according to the described method of embodiment 22-24 any one, wherein step (c) also comprises before:

This sample to be tested increases; And

Product after this sample to be tested amplification of applying marking molecule marker.

26., according to the described method of embodiment 22-25 any one, wherein this tagged molecule is vitamin H (Biotin).

27., according to the described method of embodiment 22-26 any one, wherein in step (d), the hybridization result of authentication step (c) is to utilize enzyme to carry out color reaction.

28., according to the described method of embodiment 22-27 any one, wherein this enzyme is combined with this vitamin H.

The present invention can carry out multiple modification by those skilled in the art, but does not depart from the scope of the present invention.

Claims

1. a method that detects cause of disease comprises:

Sample of nucleic acid is provided; And

Utilize at least one group of primer, its complementary sequence, its derived sequence or its degenerate sequence that is selected from SEQ ID NO.3 and SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10 to detect in this sample of nucleic acid whether have cause of disease.

2. method as claimed in claim 1, wherein this cause of disease comprises at least one in Ustilago scitaminea, Peronosclerospora sacchari, Leifsona xyli subsp and Xanthomonas albilineans.

3. method as claimed in claim 1, wherein this sample of nucleic acid is from plant or environmental samples, and this environmental samples comprise soil and water at least one of them.

4. method as claimed in claim 1 also comprises:

5. method as claimed in claim 4, wherein this derived sequence refers in 3 of SEQ ID NO.1 to SEQ ID NO.10 or its complementary sequence ' end or 5 ' end and is modified, and makes it still with former sequence, have the nucleotide sequence of 70% above homology; And this degenerate sequence refers to that in SEQ IDNO.1 to SEQ ID NO.10 or its complementary sequence, the Nucleotide below 30% is replaced by other Nucleotide.

6. method as claimed in claim 4, wherein this derived sequence refers in 3 of SEQ ID NO.1 to SEQ ID NO.10 or its complementary sequence ' end or 5 ' end and is modified, and makes it still with former sequence, have the nucleotide sequence of 90% above homology; And this degenerate sequence refers to that in SEQ IDNO.1 to SEQ ID NO.10 or its complementary sequence, the Nucleotide below 10% is replaced by other Nucleotide.

7. method as claimed in claim 4, wherein this sample of nucleic acid is from plant, and the gene order complementation of this control group primer pair and this plant, wherein this plant comprise sugarcane, tobacco, Salvia japonica Thunb., wheat, corn, Cauliflower, fragrant foreign melon, grape, oranges and tangerines, tomato, chilly, rose and leaf vegetables at least one of them.

8. method as claimed in claim 2, wherein said detection comprises

Carry out multiplex polymerase chain re-action and real-time quantitative polymerase chain reaction one of them.

9. method as claimed in claim 8, also comprise and be adjusted to identical by the ultimate value of each primer pair.

10. method as claimed in claim 8, wherein:

When carrying out multiple Polymerase Chain Reaction, use between the dNTP of 400～2000nM concentration, reach the Mg between 3～7mM ²⁺concentration; And

When carrying out the real-time quantitative Polymerase Chain Reaction, use the annealing temperature (annealing temperature) between 30～62 ℃.

11. the test kit whether existed for detection of cause of disease comprises:

12. a method that detects cause of disease, this cause of disease is selected from Ustilago scitaminea, Peronosclerospora sacchari, Leifsona xyli subsp and Xanthomonasalbilineans, and the method comprises:

(b) providing the chip with nucleic acid probe, is wherein that the identical or complementary Nucleotide of 20-30 continuous Nucleotide in the nucleic acid fragment that utilizes at least one group of primer sequence, its complementary sequence, its derived sequence or its degenerate sequence that is selected from SEQ IDNO.3 and SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10 to increase is as this nucleic acid probe;

(b) provide sample to be tested;

(d) the hybridization result of authentication step (c).

13., as the method for claim 12, wherein this step (a) also comprises: the identical or complementary Nucleotide of 20-30 continuous Nucleotide in the nucleic acid fragment that uses the primer pair of control group to increase is nucleic acid probe as a control group.

14., as the method for claim 13, wherein this control group primer pair is SEQ ID NO.1 and SEQ ID NO.2, its complementary sequence, its derived sequence or its degenerate sequence, and this host is sugarcane.

15., as the method for claim 12, wherein step (c) also comprises before:

This sample to be tested increases; And