CN105039535B - A kind of primer for detecting Pear black spot bacterium and the detection method using its detection Pear black spot bacterium - Google Patents
A kind of primer for detecting Pear black spot bacterium and the detection method using its detection Pear black spot bacterium Download PDFInfo
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Abstract
The invention discloses a kind of for detecting the primer of Pear black spot bacterium, including pair of primers, and upstream primer sequence is as described in HB1, downstream primer sequence is as described in HB2, wherein, HB1 are as follows: TCACCCTTGTCTTTTGCGTA, HB2 are as follows: ACCTTTGCTGATAGAGAGTG.And the method using the primer detection Pear black spot bacterium.The Pear black spot bacterium in plant tissue can be effectively detected in conjunction with the method for PCR amplification by designing a pair of of specific primer in the present invention.It is accurate, quick, simple to operation that the method for the present invention has many advantages, such as, pathogen can be identified at disease infestation initial stage, it can refer to the inspection applied to field investigation and plant product, it is of great significance to the large area outburst and trans-regional propagation of control Pear black spot, meanwhile the foundation of system of the present invention also provides technological guidance and theoretical foundation for the detection of other pathogens.
Description
Technical field
The present invention relates to a kind of for detecting the primer of Pear black spot bacterium, and the utilization primer detection Pear black spot bacterium
Molecular detecting method.
Background technique
Pear black spot germ (Alternaria alternata) main harm leaf, fruit and young sprout, cause tree body weak,
And storage period fruit is caused to be fallen ill, cause heavy economic losses.The disease is in Japan, and France, South Korea etc. has been reported that, in China
There is generation in multiple provinces, seriously affect the development of China's pears industry.But it is directly seen using traditional plant disease detection method
It examines, the case where early stage does not show symptom can be omitted, and rely on morphologic detection method vulnerable to human factor and ring
The interference of border condition, time-consuming for traditional classification method in addition, and program is cumbersome, is not suitable for the requirement quickly detected, is difficult to realize
Propagation and the plant disease epidemic of the timely monitoring and effectively control pathogens that occur to disease.With the hair of Protocols in Molecular Biology
Exhibition, uses molecular detecting method to provide good detection means for the diagnosis of the disease.
Summary of the invention
Technical problem to be solved by the invention is to provide the detection methods and its primer of a kind of Pear black spot bacterium, utilize this
Primer and detection method detect Pear black spot bacterium, and speed is fast, and accurately, high sensitivity is reliable and stable.
Present invention provide the technical scheme that
It is a kind of for detecting the primer of Pear black spot bacterium, including pair of primers, it is characterised in that: its upstream primer sequence
As described in HB1, downstream primer sequence is as described in HB2, wherein
HB1 are as follows: TCACCCTTGTCTTTTGCGTA
HB2 are as follows: ACCTTTGCTGATAGAGAGTG.
A method of using above-mentioned primer detection Pear black spot bacterium, process is: extracting the DNA of sample to be tested as mould
Plate carries out PCR reaction using the primer, takes PCR reaction amplified production to be detected, is about if there is molecular weight
The DNA band of 400bp then proves to contain Pear black spot bacterium in institute's test sample.
The method of above-mentioned detection Pear black spot bacterium, the amplification system of the PCR reaction are as follows: 10 × Taq buffer 2.
5µL、25mmol/L MgCl22.0 μ L, 2.0 μ L of 2.5mmol/L dNTP, each 1. 0 μ L of 10 μm of forward and reverse primers of ol/L, 5U/L
0. 2 μ L of Tagase, 50 ng/ μ LDNA template, 2 μ L, moisturizing to 25 μ L of total volume.
The method of the detection Pear black spot bacterium, the response procedures of the PCR reaction are as follows: 94 DEG C of 4min; 94℃30
S, 55.8 DEG C of 30 s, 72 DEG C of 30 s, 35 circulations; 72℃10min.4 DEG C of preservations.
The present invention also provides the methods of another detection Pear black spot bacterium, and process is: extracting the DNA conduct of sample to be tested
Template carries out sleeve type PCR reaction using two pairs of primers, and the template of first round PCR reaction is to extract the DNA of sample to be tested, primer
Template for fungi ITS sequence universal primer ITS1 and ITS4, the second wheel PCR reaction is first round PCR reaction product, and primer is
The primer HB1 and HB2 takes the second wheel PCR reaction amplified production to carry out detected through gel electrophoresis, is about if there is molecular weight
The DNA band of 400bp then proves to contain Pear black spot bacterium in institute's sample product.
The invention has the following advantages:
Plant tissue can be effectively detected in conjunction with the method for PCR amplification by designing a pair of of specific primer in the present invention
In Pear black spot bacterium.It is accurate, quick, simple to operation that the method for the present invention has many advantages, such as, can reflect at disease infestation initial stage
Make pathogen, can refer to the inspection applied to field investigation and plant product, to control Pear black spot large area outburst and
Trans-regional propagation is of great significance, meanwhile, the foundation of system of the present invention also provides technology for the detection of other pathogens and refers to
It leads and theoretical foundation.
The present invention analyzes rDNA-ITS sequences that Pear black spot bacterium and other categories are obtained from GeneBank, devises
A pair of of specific primer carries out specific amplification using other pathogens of the primer pair, and discovery can only be expanded from the category and be obtained
The band of one 400bp, other bacterial strains occur without amplified band, show that this has specificity among genus to primer.With fungi
RDNA-ITS sequence design specific primer has obtained extensively to carry out diagnosing for disease with the Molecular Detection of plant pathogenic fungi
Using.
High sensitivity is an important indicator of detection of pathogens, it be related to can Fast Detection Technique be directly applied to
In the inspection and quarantine of plant product.In the reaction system of 25 μ L, common PCR method can detect the genomic DNA of 1ng,
100 times have been increased to using the sensitivity that sleeve type PCR technology will test genomic DNA in the system that the present invention establishes, has only been needed
The DNA of 10pg is wanted to can be detected the presence of germ.
Detailed description of the invention
Fig. 1 is to carry out analysis design according to Pear black spot bacterium all in GeneBank and other rDNA-ITS sequences belonged to
A pair of of specific primer；
Fig. 2 is the verifying of specific primer, M:2000bp marker；Swimming lane 1: Pear black spot bacterium；Swimming lane 2-23: other
Bacterial strain；Swimming lane 24: negative control；
Fig. 3 is sensitivity technique of the Standard PCR to Pear black spot bacterium, M:2000bp marker；Swimming lane 1-9: template is dense
Degree is respectively 100ng, 10ng, 1ng, 100pg, 10pg, 1pg, 100fg, 10fg, 1fg；Swimming lane 10: negative control；
Fig. 4 is sensitivity technique of the sleeve type PCR to Pear black spot bacterium, M:2000bp marker；Swimming lane 1-9: template is dense
Degree is respectively 100ng, 10ng, 1ng, 100pg, 10pg, 1pg, 100fg, 10fg, 1fg；Swimming lane 10: negative control；
Fig. 5 is the PCR detection of Pear black spot bacterium in disease plant, M:2000bp marker；Swimming lane 1: it is mentioned with separating germ
Taking DNA is the PCR product of template；Swimming lane 2-3: using disease plant coarse extraction DNA as the pcr amplification product of template；Swimming lane 4: strong
Health plant pair is shone；Swimming lane 5: negative control.
Specific embodiment
Come that the present invention is furture elucidated below by the detailed description of specific embodiment, but is not to limit of the invention
System, only illustrates.
Embodiment 1: design, synthetic primer and the PCR reaction system for establishing Pear black spot bacterium
One, design of primers and synthesis
The design of specific primer: Pear black spot bacterium in GeneBank and other rDNA-ITS sequences belonged to are divided
Analysis, devises a pair of of specific primer, sequence is as follows:
HB1:TCACCCTTGTCTTTTGCGTA
HB2:ACCTTTGCTGATAGAGAGTG.
Again its specificity is verified in BLAST analysis to the primer of design in GeneBank.
Outer primer of a pair of of plant pathogenic fungi ITS universal primer ITS1 and ITS4 as sleeve type PCR is also used simultaneously,
Sequence is as follows:
ITS1:TCCGTAGGTGAACCTGCGG
ITS4:TCCTCCGCTTATTGATATGC
All primers all entrust the raw work combining unit synthesis in Shanghai.
Two, routine PCR reaction system is established
Standard PCR amplification system are as follows: 10 × Taq buffer, 2. 5 μ L, 25mmol/L MgCl2 2.0µL、2.5mmol/L
2.0 μ L of dNTP, each 1.0 μ L of 10 μm of forward and reverse primers of ol/L, 0.2 μ L of 5U/L Tagase, 20 ng/ μ LDNA template, 2 μ L are mended
Water carries out amplification reaction, response procedures to 25 μ L of total volume in PCR amplification instrument are as follows: 94 DEG C of initial denaturation 4min；94 DEG C of denaturation
30s, 55.8 DEG C of annealing 30s, 72 DEG C of extension 30s, totally 35 recycle；72 DEG C of extension 10min, 4 DEG C of preservations.
Three, sleeve type PCR reaction system is established
To improve detection sensitivity, sleeve type PCR reaction system further established.With plant pathogenic fungi universal primer
ITS1/ITS4 is the first round to react primer, for example above-mentioned Standard PCR system of reaction system.Annealing temperature is 58 DEG C in response procedures,
Other parameters are the same as above-mentioned routine PCR reaction program.Then with specific primer, second is carried out by template of first round PCR product
PCR amplification is taken turns, the primer is specific primer HB1 and HB2 described in claim 1.System is same as above, annealing temperature 55.8
DEG C, other parameters are constant.All reagents are purchased from the macro biological Co., Ltd of Hefei will.Take 5 μ L amplified productions in 1.0% agarose
Electrophoresis in gel, detects and is taken pictures on gel imaging system, and the DNA band for being about 400bp if there is molecular weight then proves
Contain Pear black spot bacterium in institute's test sample.
Embodiment 2: DNA profiling is prepared
The template that the DNA of all kinds of samples is reacted as PCR is extracted, detailed process is as follows:
1. mycelial culture, collection and DNA are extracted
PDA culture medium: agar powder 20g, potato 200g, glucose 20g add water to be settled to 1L.
Will for examination germ go on PDA culture medium plate, after 20 DEG C of dark culturing 5d from colony edge cut 10 pieces of 2cm ×
2cm bacterium colony block, goes to PDA liquid medium, 28 DEG C shaken cultivation 7 days, mycelia is collected by filtration, pulverizes through liquid nitrogen frozen ,-
20 DEG C save backup.
The CTAB method that the extraction of genomic DNA is provided according to molecular cloning, concrete operations are as follows:
A small amount of hypha powder is taken, 900 μ L, 2% CTAB extracting solution and 90 μ L10%SDS are added, is vortexed and mixes, in 60 DEG C of water-baths
During which 1h turns upside down several times every 10min, 12000rpm is centrifuged 10min under normal temperature condition；Supernatant is taken, is added isometric
Phenol/chloroform/isoamyl alcohol (25:24:1), is mixed by inversion, and 12000rpm is centrifuged 5min under normal temperature condition；Supernatant is transferred to new
In EP pipe, isometric chloroform is added, is gently mixed by inversion, 12000rpm is centrifuged 5min under normal temperature condition；Supernatant is transferred to new EP
Guan Zhong, is added the isopropanol of 1 times of supernatant volume, and -20 DEG C of conditions precipitate 15min；12000rpm is centrifuged under normal temperature condition
5min, incline supernatant, and precipitating is done under the conditions of 70% ethanol washing 2 times, 37 DEG C to ethyl alcohol volatilization；Add appropriate sterilizing ultrapure water or
Person TE(pH8.0) dissolution precipitating (containing 20 μ g/ml RNase), 37 DEG C of resolution 30min rear electrophoresis detections, -20 DEG C save backup.
2. the extraction for plant tissue DNA of falling ill
It will be gone on PDA culture medium plate for examination germ, and cut 2cm × 2cm from colony edge after 28 DEG C of dark culturing 5d
Bacterium colony block, wound inoculation is on pomegranate plant.Incidence tissue is cut after a period of time and extracts genomic DNA, concrete operations: takes one
The plant tissue fallen ill a bit, every milligram of tissue are added 10 μ L, 0.5mol/LNaOH, are transferred to after being fully ground in mortar
In the centrifuge tube of 1.5ml, 12,000g centrifugation 5min take 5 μ L supernatants that 495 μ L, 0.1mmol/L Tris (PH8.0) are added, mix
1 μ L is taken to be directly used in PCR reaction after even.
Embodiment 3: the specificity and sensitivity of detection primer
One, specific detection
Bacterial strain uses therefor of the present invention and relevant information are shown in Table 1.Owned using specific primer designed by the present invention in table 1
Strains tested genomic DNA carry out PCR amplification, be only capable of specifically amplifying a 400bp's from Pear black spot bacteria strain
Band, and other strains testeds and blank control are without amplified band.Show that the primer has species specificity, it can be black by pears
Pinta bacterium distinguishes with other kinds。
For screening the bacterial strain of primer specificity in 1 the present embodiment of table
| Bacterial strain | Number of strains | With HB1/HB2 amplification |
| Alternaria alternata | 1 | + |
| Botrytis cinerea | 1 | - |
| Botryosphaeria dothidea | 1 | - |
| Coniella granati | 1 | - |
| Coniothyrium diplodiella | 1 | - |
| Colletotrichum gloeosporioides | 1 | - |
| Cercospora circumscissa | 1 | - |
| Gymnosporangium yamadae | 1 | - |
| Gymnosporangium haraeanum | 1 | - |
| Glomerella acutata | 1 | - |
| Elsinoe fawcetti | 1 | - |
| Phyllosticta pirina | 1 | - |
| Pestalotiopsis theae | 1 | - |
| Fusicladium virescens | 1 | - |
| Marssonina coronaria | 1 | - |
| Podosphaera leucotricha | 1 | - |
| Podosphaera tridactyla | 1 | - |
| Penicillium italicum | 1 | - |
| Uncinula necator | 1 | - |
| Phytophthora capsici | 1 | - |
| Plasmopara viticola | 1 | - |
| Rhizopus stolonifer | 1 | - |
| Septoria piricola | 1 | - |
In upper table :+indicate the specific amplification band with primer HB1/HB2, length 400bp；It indicates to produce without amplification
Object.
Two, sensitivity technique
The sensitivity of above-mentioned established detection architecture is determined in the reaction system of 25 μ L, primer HB1 and HB2 can be steady
Surely from at least genomic templates of 1ng, amplification obtains the specific band of 400bp.And drawn with the general of plant pathogenic fungi
The product that object ITS1 and ITS4 carries out PCR amplification to the genomic DNA of Pear black spot bacteria strain various concentration is template, is reused
Specific primer HB1 and HB2 carry out the second wheel shell type PCR amplification, can steadily detect the genomic DNA of 10pg, make sensitive
Degree improves 100 times.
Embodiment 4: the cause of disease analyte detection in Diseased Plant Tissues
From clip incidence tissue on the plant of artificial infection, extracts DNA and carry out PCR amplification, morbidity sample amplifies
The specific band of 400bp, and healthy plant expands not shaping band, illustrates that the set technology can be used for pears blackspot in Diseased Plant Tissues
The rapid molecular of germ detects.
Claims (5)
1. a kind of for detecting the primer of Pear black spot bacterium, including pair of primers, it is characterised in that: its upstream primer sequence is such as
Described in HB1, downstream primer sequence is as described in HB2, wherein HB1 are as follows: TCACCCTTGTCTTTTGCGTA, HB2 are as follows:
ACCTTTGCTGATAGAGAGTG。
2. a kind of method using primer detection Pear black spot bacterium described in claim 1, it is characterised in that: extract sample to be tested
DNA as template, carry out PCR reaction using the primer of above-mentioned design, PCR reaction amplified production taken to be detected, if
There are the DNA band that molecular weight is 400bp, then prove to contain Pear black spot bacterium in institute's test sample.
3. the method for detection Pear black spot bacterium according to claim 2, it is characterised in that: the amplification body of the PCR reaction
System are as follows: 10 × Taq buffer, 2.5 μ L, 25mmol/L MgCl2 2.0µL、2.5mmol/L dNTP 2.0µL、10µmol/L
Each 1. 0 μ L of forward and reverse primer, 0.2 μ L of 5U/L Tagase, 20 ng/ μ LDNA template, 2 μ L, moisturizing to 25 μ L of total volume.
4. the method for detection Pear black spot bacterium according to claim 2, it is characterised in that: the reaction interval of the PCR reaction
Sequence are as follows: 94 DEG C, 4min；94 DEG C, 30 s, 55.8 DEG C, 30 s, 72 DEG C, 30 s, 35 circulations；72 DEG C, 10min, 4 DEG C of guarantors
It deposits.
5. a kind of method using primer detection Pear black spot bacterium described in claim 1, which is characterized in that concrete operations are such as
Under: the DNA of sample to be tested is extracted as template, carries out sleeve type PCR reaction, the template of first round PCR reaction using two pairs of primers
For the DNA for extracting sample to be tested, primer is fungi ITS sequence universal primer ITS1 and ITS4, and the template of the second wheel PCR reaction is
First round PCR reaction product, primer are the primer HB1 and HB2, and the second wheel PCR reaction amplified production is taken to carry out gel electricity
Swimming detection, the DNA band for being about 400bp if there is molecular weight then prove to contain Pear black spot bacterium in institute's sample product.
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| CN105724138A (en) * | 2016-03-14 | 2016-07-06 | 阜阳市颍州区金湖丰种植农民专业合作社 | Method for reducing high-incidence season disease incidence of black spot of late-autumn yellow pears |
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| 出口果园中库尔勒香梨黑斑病快速检测及疫情监测技术研究;马海波;《中国优秀硕士学位论文全文数据库（农业科技辑）》;20150515;170-173 * |
| 实时荧光PCR检测香梨黑斑病菌;王凤军等;《食品科学》;20131231;第34卷(第20期);第9页第1段，第25页第1段，第27页第3.1.4.1节 * |
| 砀山梨黑斑病分子检测技术研究;李云飞等;《中国农学通报》;20160205;第32卷(第4期);150-154 * |
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