CN104498473A - Method for rapid separation of pathogen DNA from rice blast single scab - Google Patents

Abstract

The invention provides a method for direct extraction of rice blast pathogen DNA from rice leaf infected by rice blast or neck blast single scab. The method of the invention can directly extract rice blast pathogen DNA from rice leaf or neck infected by rice blast, the extraction method is simple, fast and low-cost, and the extracted DNA is applicable to PCR based analysis on rice blast pathogen nontoxic gene detection and colony genetic diversity.

Description

A kind of method from rice blast list scab sharp separation pathogenic bacteria DNA

Technical field

The invention belongs to microbial molecules detection field, especially, belong to a kind of method from rice blast list scab sharp separation pathogenic bacteria DNA.

Background technology

By Pyricularia oryzae (Magnaporthe oryzae; Invisible element: Pyricularia oryzae) (Couch and Hohn, 2002) rice blast of causing is one of main limiting factor of Rice Production, grain-production (Ou, 1985 in the world in serious threat; Zeigler et al., 1994).The interaction of paddy rice and Pyricularia oryzae meets classical " gene-for-gene " theory (Flor, 1971; Jia et al., 2000).Research the pathogenic of rice blast fungus population is configured with the popularization and layout that are beneficial to and instruct paddy disease-resistant new variety.Along with rice blast bacterium pathogenicity research is constantly goed deep into, the nontoxic genes such as avirulence gene of rice blast Avr1Pi-CO39, ACE1, AvrPiz-t, AvrPita, AvrPik/Pikm/kp, AvrPia and the AvrPii corresponding with rice varieties disease-resistant gene are constantly by successful clone (Orbach et al., 2000; Farman et al., 2002; Bohnert et al., 2004; Li etal., 2009; Yoshida et al., 2009).The successful clone of nontoxic gene, for based on the nontoxic gene DNA sequence dna of having cloned, the molecule marker that design nontoxic gene is special, fast monitored field Pyricularia oryzae population composing, for the prediction of rice blast and the rational deployment of disease-resistant variety provide foundation.Carry out Molecular Detection and the monitoring of pathogenic bacteria population, the successful extraction of DNA is important step and the basis of carrying out associated molecule detect delay.The method of DNA extraction has many, as adopted classical CTAB (Doyle and Doyle, 1987); , fungal DNA isolation kit method, microwave boiling method (Tendulkaret al., 2003), extraction of spores method (Xu and Hamer, 1995), DNA in a small amount extraction process (Sweigard et al., 1990; Saitoh et al., 2006) extraction and the Related Experimental Study of Pyricularia oryzae DNA etc. is applied to.

Traditional CTAB extraction method, fungal DNA isolation kit method, microwave boiling method (Tendulkar et al., 2003), extraction of spores method (Xu and Hamer, 1995) and DNA in a small amount extraction process (Sweigard et al., 1990; Saitoh et al., 2006) though meet the relevant molecular biology experiment of Pyricularia oryzae to a certain extent.But adopt these methods extract DNA all need early stage carry out pathogenic bacteria separation, preservation, cultivation etc. early stage a couple of days preparation, cause the experimental period of early-stage preparations long; And it is also comparatively loaded down with trivial details to there is process when utilizing these methods to carry out the extraction of DNA, as needed grinding or the process such as cell wall breaking cracking and extracting repeatedly of liquid nitrogen, Part Methods, as the more high weakness of experimental cost utilizing test kit to extract DNA, is difficult to meet be based upon on round pcr basis and carries out the object that field pathogenic bacterium colony is extensive, rapid detection is analyzed.

It is necessary to provide a kind of quicker, simple method and reagent extracting Pyricularia oryzae DNA, meet the needs that field pathogenic bacterium colony is extensive, rapid detection is analyzed.

Summary of the invention

Rice blast can be divided into leaf pest by the position of morbidity, fringe pest, joint pest, grain pest, branch stalk pest, field the most commonly leaf Wen with Miho neck pest, research and develop a kind of method directly extracting DNA from the single scab of incidence of leaf or rice blast Miho neck pest, for Molecular Detection and the monitoring of rice blast fungus population nontoxic gene, to greatly increase work efficiency, save the separation of Pathogen of Rice Blast Fungus, preserve, cultivate the set-up procedure waiting a couple of days in early stage, and the extraction of DNA only needs a step to complete, the template that the DNA extracted directly can be used as the amplification of PCR effectively increases, compared with existing DNA extraction method, greatly can shorten the time of DNA extraction, the triviality of minimizing work, the rapid detection that the field Pyricularia oryzae population nontoxic gene realizing PCR-based technology is formed and monitoring.

On the one hand, the invention provides a kind of method from rice leaf or fringe portion extracting directly DNA, the method comprises: 1), in advance prepare following four kinds of reagent respectively, for subsequent use after sterilizing: (1), 100mM TrisHCl (pH8.7), (2), 10mMEDTA (pH 8.0), (3), 1M KCl and (4), 10%Tween 20;

2), gather the rice leaf Huo Miho neck of infection rice blast to be detected, cut the incidence of leaf of about 2mm × 2mm size from the single scab of rice leaf of morbidity or be positioned in PCR-96 orifice plate respectively from the Fa Bing Miho neck that morbidity Miho neck gets 1-2mm long;

3), quantity per sample, after 100mM TrisHCl (pH8.7) in step (1), 10mM EDTA (pH8.0) and 1M KCl respectively being got the mixing of appropriate equal-volume, and then add the Tween 20 of 10% by the amount of 1000:1 according to the cumulative volume of 3 kinds of mixed solutions; After finally mixing, add 100 μ L extracting solutions in the every hole of the PCR-96 orifice plate putting into sample in advance;

4), by the above-mentioned sample handled well put into PCR instrument, 95 DEG C of heat treated 10min, or sample put into moist heat sterilization pot, at 105 DEG C, process 10min, take out sample be positioned over-20 DEG C for subsequent use.

In some preferred modes, PCR-96 orifice plate can replace by any container, as long as can be carried out the process of step 4 by heating just passable for this container.

In some preferred modes, but the area of the size any appropriate of the single scab of rice leaf, be generally the size of single scab, such as 2mm × 2mm, 1mm × 2mm, 0.5mm × 2mm etc.Except the rice blast scab on rice leaf or Miho neck, the single scab of rice blast of its hetero-organization also can adopt method of the present invention to carry out.The susceptible scab length of rice blast varies, field is generally more than 1-5mm (Miho neck), long exceeded 1cm square (blade), and the present invention is only extracting directly and rapid detection that a very little part of getting single scab just can realize DNA.In fact, the 2 × 2mm (blade) in the present invention or person are little parts of the 1-2mm just scab in fact of Miho neck, approximately only have 1/5th or less of whole single scab.This is conducive to the detection of avirulence gene of rice blast, if when needing to be separated Pyricularia oryzae, can complete the separation of pathogenic bacteria from remaining scab; And too many requirement is not had for the size of sampling, the single scab of the part of small area like this can realize extraction and the detection of Pyricularia oryzae gene DNA, achieve the object that trace samplings detects.

In some preferred modes, by our experiment, also there is concrete requirement to handling the Heating temperature after sample well, generally needing more than 90 DEG C, preferably more than 95 DEG C and 105 DEG C, so only need the leaching process that just can complete the DNA of whole Pyricularia oryzae for general 5-10 minute.

In other one side, the invention provides a kind of from the infection rice leaf of rice blast or the method for fringe neck extracting directly Pyricularia oryzae DNA, the method is made up of following steps: 1), in advance prepare following four kinds of reagent respectively, for subsequent use after sterilizing: (1), 100mM TrisHCl (pH8.7), (2), 10mM EDTA (pH 8.0), (3), 1M KCl and (4), 10%Tween 20; 2), gather the rice leaf Huo Miho neck of infection rice blast to be detected, cut the incidence of leaf of 2mm × 2mm size from the single scab of rice leaf of morbidity or be positioned in PCR-96 orifice plate respectively from the Fa Bing Miho neck that morbidity Miho neck gets 1-2mm long; 3), quantity per sample, after 100mM TrisHCl (pH8.7), 10mM EDTA (pH 8.0) in step (1) and 1M KCl respectively being got the mixing of appropriate equal-volume, and then add the Tween 20 of 10% by the amount of 1000:1 according to the cumulative volume of 3 kinds of mixed solutions; After finally mixing, add 100 μ L extracting solutions in the every hole of the PCR-96 orifice plate putting into sample in advance; 4), by the above-mentioned sample handled well put into PCR instrument, 95 DEG C of heat treated 10min, or sample put into moist heat sterilization pot, at 105 DEG C, process 10min, take out sample be positioned over-20 DEG C for subsequent use.

Beneficial effect

By this technological invention, (1). the process that the separation of pathogenic bacteria in traditional ordinary method, cultivation and mycelia are collected can be saved, simplify program; (2). realize single stage method is directly separated Pyricularia oryzae DNA from the single scab of rice tissue, eliminate the tedious steps of repeatedly extracting in routine; (3). the DNA of acquisition can be used for detection and the population diversity analysis of avirulence gene of rice blast.

Accompanying drawing explanation

Fig. 1 is from infected leaves with Miho neck extracts the pcr amplification result of Pyricularia oryzae DNA.Wherein, M, molecular weight marker, DL2000; Fig. 1 a refers to extract result with ITS1/ITS4 primer pair amplifies after DNA from infected leaves, and expection clip size 544bp, is wherein with 1-15 to be the DNA cloning result that infected leaves extracts, and band 16 makes blank for healthy leaves; Fig. 1 b refers to extract result with AvrPizt-1Fw/AvrPizt-1Rv primer pair amplifies after DNA from infected leaves, and expection clip size 1186bp, is wherein with 1-15 to be the DNA cloning result that infected leaves extracts, and band 16 makes blank for healthy leaves; By the result of ITS1/ITS4 primer pair amplifies after Fig. 1 c refers to extract DNA from Gan Bing Miho neck, be wherein with 1-15 to be the DNA cloning result that Gan Bing Miho neck extracts, band 16 makes blank for Jian Kang Miho neck; By the result of AvrPizt-1Fw/AvrPizt-1Rv primer pair amplifies after Fig. 1 d refers to extract DNA from Gan Bing Miho neck, expection clip size 1186bp, be wherein with 1-15 to be the DNA cloning result that Gan Bing Miho neck extracts, band 16 makes blank for Jian Kang Miho neck.

Embodiment

Be exactly illustrate how the present invention carries out by way of example, these citings are only used to illustrate how the present invention realizes, or preferred forms, these examples of implementation can not do any restriction to the present invention, and scope of the present invention is limited by claim.

Examples of implementation 1: rice leaf is with the extraction of DNA of Miho neck Pyricularia oryzae and checking.

The method of concrete enforcement:

1) reagent: prepare following reagent in advance, for subsequent use after sterilizing: (1), 100mM TrisHCl (pH8.7), (2), 10mM EDTA (pH 8.0), (3) 1M KCl, (4), 10%Tween 20.

2) preparation of samples: choose drying at room temperature after gathering from field at random respectively and be stored in 15 blades of typical case's morbidity in laboratory with Miho neck, cut the incidence of leaf of about 2mm × 2mm size from the single scab of rice leaf of morbidity, get the Fa Bing Miho neck of 1-2mm length from morbidity Miho neck, be positioned in PCR-96 orifice plate respectively, a sample is placed in every hole, and blade is with Miho neck arranges a healthy rice material in contrast.

3) according to the quantity extracting sample, 100mM TrisHCl (pH8.7), 10mM EDTA (pH 8.0) and 1M KCl are respectively got the mixing of 1.2mL equal-volume, then the Tween 20 of 3.6 μ L 10% is added, 100 μ L extracting solutions are added in the every hole of the PCR-96 orifice plate putting into sample in advance after mixing, and cover lid.If there is sample floating, the toothpick of available disposable tip or sterilizing is pressed in liquid, after waiting completing steps 3, can not needed wait time, and directly carry out step 4.Certainly, if in the operation carrying out step 4 after waiting for some time a little, so also passable.

4) DNA preparation: the above-mentioned sample handled well, if sample only has within 96, is put into PCR instrument by (1), 95 DEG C of heat treated 10min, be stored in afterwards-20 degrees Celsius for subsequent use; (2) if sample is many, sample can be put into moist heat sterilization pot, process 10min at 105 DEG C after, take out sample and be positioned over-20 DEG C (the method is applicable to the preparation of a large amount of sample) for subsequent use.

5) PCR reaction: the PCR of sample detects and undertaken by 2 pairs of PCR primer, it is for a pair the universal primer of the ITS sequence detecting Pyricularia oryzae rDNA, amplification size is 544bps, forward primer and reverse primer sequences are respectively, ITS1:TCC GTA GGT GAA CCT GCG G, ITS4:TCC TCC GCT TAT TGA TAT GC; Other is for a pair primer for detecting avirulence gene of rice blast AvrPizt, amplification size is 1186bps, forward primer and reverse primer sequences are respectively, AvrPizt-1Fw:AAG CGT CAC AAA ATA TCA TCA AGT, AvrPizt-1Rv:CGG CTA CCA TCG AGA AAA GT.PCR reaction system 20 μ L, PCR reaction mixture comprises: 2 × Taq MasterMix 10 μ L, forward primer and each 1 μ L of reverse primer (primer concentration 10 μMs), the DNA profiling 1 μ L prepared, aseptic double-distilled water 6 μ L.Pcr amplification condition is: 95 DEG C of denaturation 3min, then enters the amplified reaction of 35 circulations, i.e. 95 DEG C of sex change 30min, 55 DEG C of annealing 30min, and 72 DEG C extend 1.5min, extend 7min again, then PCR primer is positioned over 4 DEG C after loop ends; PCR reaction is carried out (BioRad) on C1000.

6) PCR primer detects: 5 μ L electrophoresis staining examine on 1.2% sepharose got by the every sample of PCR primer.

Experimental result:

As shown in Figure 1, the DNA utilizing the scab on infected leaves to extract increases, and successfully goes out to expect the band of clip size 544bp with ITS1/ITS4 primer pair amplifies; Equally, utilize AvrPizt-1Fw/AvrPizt-1Rv primer pair, Successful amplification goes out to expect the object tape of clip size 1186bp; Similar to the DNA extracted from infected leaves, utilize 2 pairs of primers Successful amplification Cong the Pyricularia oryzae DNA that Miho neck extracts to go out the target fragment of 544bp and 1186bp respectively.

As can be seen from Figure 1, M, molecular weight marker, DL2000; Fig. 1 a refers to extract result with ITS1/ITS4 primer pair amplifies after DNA from infected leaves, and expection clip size 544bp, is wherein with 1-15 to be the DNA cloning result that infected leaves extracts, and band 16 makes blank, without any the amplification of band for healthy leaves; By the result of AvrPizt-1Fw/AvrPizt-1Rv primer pair amplifies after Fig. 1 b refers to extract DNA from infected leaves, expection clip size 1186bp, wherein be with 1-15 to be the DNA cloning result that infected leaves extracts, band 16 makes blank, without any the amplification of band for healthy leaves; By the result of ITS1/ITS4 primer pair amplifies after Fig. 1 c refers to extract DNA from Gan Bing Miho neck, be wherein with 1-15 to be the DNA cloning result that Gan Bing Miho neck extracts, band 16 makes blank for Jian Kang Miho neck, without any the amplification of band; By the result of AvrPizt-1Fw/AvrPizt-1Rv primer pair amplifies after Fig. 1 d refers to extract DNA from Gan Bing Miho neck, expection clip size 1186bp, wherein be with 1-15 to be the DNA cloning result that Gan Bing Miho neck extracts, band 16 makes blank for Jian Kang Miho neck, without any the amplification of band.

We are by comparative experiments, and adopt traditional extracting method, although also can obtain above-mentioned result, traditional method is time-consuming, efficiency is low and cost is high, is also unfavorable for large batch of operation.

Found out by above experimental result, utilize the method for this technological invention, can successfully from morbidity rice leaf He Miho neck extracting directly Pyricularia oryzae DNA be used for Molecular Detection analysis, the DNA of extraction effectively can amplify the DNA fragmentation more than 1kb, can meet the needs of general test experience.By this technological invention, achieve the summary of (1) DNA extraction: the tissue of minute quantity direct onestep extraction germ DNA can be utilized, without the need to traditional multi step strategy, more summary from the scab of rice leaf and susceptible Miho neck; (2) high efficiency of DNA extraction: because only a step process can complete the extraction of DNA, the pathogenic bacteria DNA extraction of 500-1000 part disease standard specimen can be completed for each person every day, for fast monitored and detection provide guarantee, and use conventional methods, the pathogenic bacteria DNA extraction of 20-50 part disease standard specimen can only be completed for each person every day; (3) reagent cost is low, intensive: the 4 kinds of reagent adopting present method to use are the conventional reagent in laboratory, low price, and it is convenient to buy, than the price of the test kit cheap more than 90% that business is bought; (4) equipment requirements is simplified: without special plant and instrument demand, the PCR instrument of Routine Test Lab or high-pressure sterilizing pot can meet experiment.Adopt present method can obtain a large amount of DNA detecting sample fast, support for the monitoring and detection analysis of carrying out field avirulence gene of rice blast formation and population diversity fast provide important technology.

Claims (4)

1., from the infection rice leaf of rice blast or a method of fringe neck extracting directly Pyricularia oryzae DNA, the method comprises:

(1), in advance following four kinds of reagent are prepared respectively, for subsequent use after sterilizing: (1) 100mM TrisHCl (pH8.7), (2) 10mM EDTA (pH 8.0), (3) 1M KCl and (4) 10%Tween 20;

(2), gather the rice leaf Huo Miho neck of infection rice blast to be detected as sample, cut the incidence of leaf of about 2mm × 2mm size from the single scab of rice leaf of morbidity or be positioned in PCR-96 orifice plate respectively from the Fa Bing Miho neck that the single scab morbidity Miho neck gets 1-2mm long;

(3), quantity per sample, after 100mM TrisHCl (pH8.7) in step (1), 10mM EDTA (pH8.0) and 1M KCl respectively being got the mixing of appropriate equal-volume, and then add the Tween 20 of 10% by the amount of 1000:1 according to the cumulative volume of 3 kinds of mixed solutions; After finally mixing, add 100 μ L extracting solutions in the every hole of the PCR-96 orifice plate putting into sample in advance;

(4), by the sample that step (3) is handled well put into PCR instrument, 95 DEG C of heat treated 10min, or sample put into moist heat sterilization pot, at 105 DEG C, process 10min, take out sample be positioned over-20 DEG C for subsequent use.

2. method according to claim 1, wherein, PCR-96 orifice plate can by plastic test tube or glass test tube replace.

3., from the infection rice leaf of rice blast or a method of fringe neck extracting directly Pyricularia oryzae DNA, the method is made up of following steps:

(1), in advance following four kinds of reagent are prepared respectively, for subsequent use after sterilizing: (1), 100mM TrisHCl (pH8.7), (2), 10mM EDTA (pH 8.0), (3), 1M KCl and (4), 10%Tween 20;

(2), gather the rice leaf Huo Miho neck of infection rice blast to be detected, cut the incidence of leaf of 2mm × 2mm size from the single scab of rice leaf of morbidity or be positioned in PCR-96 orifice plate respectively from the Fa Bing Miho neck that the single scab morbidity Miho neck gets 1-2mm long;

(3), quantity per sample, after 100mM TrisHCl (pH8.7), 10mM EDTA (pH 8.0) in step (1) and 1M KCl respectively being got the mixing of appropriate equal-volume, and then add the Tween 20 of 10% by the amount of 1000:1 according to the cumulative volume of 3 kinds of mixed solutions; After finally mixing, add 100 μ L extracting solutions in the every hole of the PCR-96 orifice plate putting into sample in advance;

(4), the sample that above-mentioned steps (3) is handled well is put into PCR instrument immediately, 95 DEG C of heat treated 10min, or moist heat sterilization pot put into immediately by the sample that just above-mentioned steps (3) is handled well, processes 10min at 105 DEG C, take out sample be positioned over-20 DEG C for subsequent use.

4. the method according to claim 1 or 4, the method also comprises the Molecular Detection whether sample being existed to Pyricularia oryzae further, the method detected is as follows: the DNA of the sample after extraction is carried out PCR detection by 2 pairs of PCR primer, it is for a pair the universal primer of the ITS sequence detecting Pyricularia oryzae rDNA, amplification size is 544bps, forward primer and reverse primer sequences are respectively, ITS1:TCC GTA GGT GAA CCT GCG G, ITS4:TCC TCC GCT TAT TGA TAT GC; Other is for a pair primer for detecting avirulence gene of rice blast AvrPizt, amplification size is 1186bps, forward primer and reverse primer sequences are respectively, AvrPizt-1Fw:AAG CGT CAC AAA ATA TCA TCA AGT, AvrPizt-1Rv:CGG CTA CCA TCG AGA AAA GT; PCR reaction system 20 μ L, comprises: 2 × Taq MasterMix 10 μ L, forward primer and each 1 μ L of reverse primer (primer concentration 10 μMs), the DNA profiling 1 μ L prepared, aseptic double-distilled water 6 μ L; Pcr amplification condition is: 95 DEG C of denaturation 3min, then enters the amplified reaction of 35 circulations, i.e. 95 DEG C of sex change 30min, and 55 DEG C of annealing 30min, 72 DEG C extend 1.5min, extend 7min again, then PCR primer is positioned over 4 DEG C after loop ends.