CN109652584A - For detecting the LAMP primer and detection kit of the black star bacterium of peach - Google Patents
For detecting the LAMP primer and detection kit of the black star bacterium of peach Download PDFInfo
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Abstract
The present invention is provided to detect the LAMP primer and detection kit of the black star bacterium of peach.The sequence of the LAMP primer is as shown in Seq ID No.1-4.Loop-mediated isothermal amplification technology is used for the quick detection of the black star bacterium of peach by the present invention, can accurately detect the black star bacterium of peach from complicated pathogen environment such as morbidity plant tissues.The specificity of this method and sensitivity are higher compared with conventional PCR method, can detect to the brood body such as mycelia, spore of various forms etc. of the black star bacterium of peach, are of great significance to the cause of disease monitoring etc. of the early warning of peach scab epidemic situation, epidemic-stricken area；High instrument investment can be exempted simultaneously, promoted the use of convenient for base.
Description
Technical field
The invention belongs to phytopathogen detection technique fields, specifically, being related to the LAMP for detecting the black star bacterium of peach
Primer and detection kit.
Background technique
By the black star bacterium of peach, (Venturia carpophila also known as the black star bacterium of thermophilic fruit belong to the black star of Ascomycotina Sphaerials
Pseudomonas fungi) caused by shot hole be important one of the disease in peach producing region, peach scab has seriously affected the quality of peach
And value.The disease generates crineous to black dot in fruit face, symptom easily with peach shot hole, black spot, canker and early stage charcoal
Subcutaneous ulcer disease is obscured, and basic level Agro-technology extension personnel and orchard worker are difficult to differentiate between, the case of Chang Fasheng disease screening mistake；The black star bacterium of peach is being posted
Your Majesty's incubation period is long, difficulty separation when the pure culture of laboratory, grow it is slow, easy to pollute.Shot hole is accurately identified to the scientific prevention and cure disease
Evil is most important.
If being capable of precise Identification Species of Pathogens before spray, so that it may which accurate fungicide of formulating sprays plan, with raising
Control efficiency reduces Pesticide use.Identification method more commonly used at present includes two kinds, i.e., the tradition based on disease symptom
Identification method and molecular methods based on target gene.Traditional symptom identification method is identified according to the disease symptom of disease.
This method need practitioner have disease recognition ability and disease recognition experience abundant really up to the mark, but due to different cultivars,
Different geographical, different stages of growth Disease symptoms are different, and shot hole disease symptom difficulty and shothole disease, black spot, canker, morning
Phase anthrax disease symptoms are distinguished, therefore symptom identification method is difficult to promote.Simultaneously as the separation of peach black star bacterium difficulty, grow it is slow, easy to pollute etc.
Attribute, molecular methods usually require several weeks, work difficulty and heavy workload, measure limited sample size, it is desirable that stringent
Sterile working.And common PCR detection technique, required instrument price are expensive, and it is higher to the technical requirements of operator, it is uncomfortable
It closes and is promoted in field or basic agriculture engineering department.
Summary of the invention
The present invention is directed to overcome existing method detection cycle long, heavy workload needs aseptic condition and round pcr pair
Instrument and the high defect of personnel's technical requirements, provide the LAMP primer and detection kit for detecting the black star bacterium of peach, Yi Ji
Establish that a kind of convenience based on molecular level, sensitive, result judgement are intuitive and the peach promoted the use of convenient for base is black on the basis of this
Star bacterium detection method.
Present inventive concept is as follows: carrying out the ITS1/ITS4 primer amplification segment the study found that the black star bacterium of peach to the black star bacterium of peach
147~163 bit bases and close kind of venturia inaequalis (Venturia inaequalis), Pear scab (Venturia
) and the confusing Peach black spot bacterium of symptom (Alternaria tenuissima), peach ulcer bacteria nashicola
(Phomopsis amygdali), there are notable differences for anthracnose of peach bacterium (Colletotrichum acutatum).It is verified,
Black 147~163 bit base section of star bacterium of peach is the specific site for being different from other pathogens.Design is accordingly drawn on this basis
Object realizes the detection to the black star bacterium of peach according to amplification.
In order to achieve the object of the present invention, in a first aspect, the present invention is provided to detect the black star bacterium (Venturia of peach
Carpophila LAMP primer), including (SEQ ID NO:1-4):
Outside forward primer VC-F3:5 '-AGCAAGCCCTGCCTAGA-3 '；
Outside reverse primer VC-B3:5 '-TGCCCCCCGGAATACCA-3 '；And
Inside forward primer VC-FIP (F1c+F2): 5 '-TGCCAGAACCAAGAGATCCGTTGTTGAAGTCTGAGGAG
AAAGCC-3'；
Inside reverse primer VC-BIP (B1c+B2): 5 '-GATGAAGAACGCAGCGAAATGCGCAATGTGCGTTCAAA
GATTCGA-3’。
Second aspect, the present invention provide the reagent and kit for being used to detect the black star bacterium of peach containing the LAMP primer.
Optionally, the kit further includes dNTPs, Bst archaeal dna polymerase, Thermopol Buffer, MgSO4Or
MgCl2, at least one of glycine betaine.
Optionally, the kit further includes standard positive template.
Optionally, the kit further includes TE lysate.
The third aspect, the present invention provide following any application of the LAMP primer or the detection reagent/kit:
I) application in the detection black star bacterium of peach；
Ii) in detection by the application in the microbial plant disease of the black star of peach.
The plant disease includes but is not limited to peach scab.
It is above-mentioned application the following steps are included:
1) DNA in sample is extracted；
2) it using the DNA extracted in step 1) as template, is carried out amplification reaction using the LAMP primer；
3) amplified production is analyzed.
Preferably, amplification reaction system is calculated as with 25 μ l: 4U Bst archaeal dna polymerase, 2.5 μ L10 × ThermoPol
Buffer, 5mM MgSO4, 0.8mM dNTPs, 1.2mM VC-FIP, 1.2mM VC-BIP, 0.4mM VC-F3,0.4mM VC-
B3,0.8M glycine betaine, 1 μ L DNA profiling.
Amplification reaction condition: 75min is reacted in 61 DEG C of water-baths.Step 3) specifically: into the amplified production of step 2)
It is added SYBR Green I dyestuff (1 μ L1000 × SYBR Green I dyestuff is added in each reaction tube), according to colour developing situation
Determine result: display green is the positive, orange for feminine gender.
Fourth aspect, the present invention provide based on LAMP technology (loop-mediated isothermal amplification technology) black star bacterium of detection peach and by
The method of the microbial plant disease of the black star of peach.The method includes utilizing the LAMP primer pair as shown in SEQ ID NO:1-4
Sample to be tested is detected.
By above-mentioned technical proposal, the present invention at least have following advantages and the utility model has the advantages that
(1) loop-mediated isothermal amplification technology is used for the quick detection of the black star bacterium germ of peach by the present invention, can be from morbidity plant
Complicated pathogen environment accurately detects the black star bacterium of peach in tissue and soil.The specificity of this method and sensitivity are relatively often
It is higher to advise PCR method, the brood body such as mycelia, spore of various forms etc. of the black star bacterium of peach can be detected, to the black star bacterium of peach
The early warning of epidemic situation, the cause of disease monitoring in epidemic-stricken area etc. are of great significance；High instrument investment can be exempted simultaneously, just
It is promoted the use of in base.
(2) using kit provided by the invention can in 2h from the sample of field quickly, it is easy, accurate, delicately examine
The black star bacterium of peach is measured, it is simpler, more more efficient than traditional symptom identification method and common molecular detection method.
(3) LAMP primer specificity provided by the invention is good, high sensitivity, and the detection of the black star bacterium DNA of peach is limited to
5.66fg/μl。
Detailed description of the invention
Fig. 1 is LAMP specific primer in the embodiment of the present invention 2 to the black star bacterium of peach and 7 kinds of pathogen DNA cloning results.
Fig. 2 is the Mg in the embodiment of the present invention 3 in reaction system2+Concentration optimization result.
Fig. 3 is the dNTPs concentration optimization result in the embodiment of the present invention 3 in reaction system.
Fig. 4 is LAMP reaction temperature optimum results in the embodiment of the present invention 3.
Fig. 5 is LAMP reaction time optimum results in the embodiment of the present invention 3.
Fig. 6 is the sensitivity analysis result that LAMP primer detects the black star bacterium of peach in the embodiment of the present invention 6.
Fig. 7 is LAMP specific primer in the embodiment of the present invention 4 to the expansion to the black star bacterium of 23 plants of peaches for picking up from 14 provinces and regions
Increase result.
Fig. 8 is LAMP specific primer in the embodiment of the present invention 5 to the amplification of susceptible and healthy pericarp DNA.
Fig. 9 is the ITS1/ of the black star bacterium of peach edge species (venturia inaequalis, Pear scab) close with its in the embodiment of the present invention 1
ITS4 sequence alignment result.
Figure 10 is the design site of the black star bacterium LAMP primer of peach in the embodiment of the present invention 1.
Figure 11 is the amplification of the 10 groups of LAMP primers designed in the embodiment of the present invention 1.Wherein, M:DNA Maker, 1
~10 correspond respectively to 1-10 group LAMP primer.
Specific embodiment
The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..Unless otherwise specified, embodiment
According to conventional laboratory conditions, such as Sambrook molecular cloning experiment handbook (Sambrook J&Russell DW,
Molecular Cloning:a Laboratory Manual, 2001), or according to the condition of manufacturer's specification suggestion.
Embodiment 1 is used to detect the design and synthesis of the LAMP primer of the black star bacterium of peach
The ITS1/ITS4 sequence of the black star bacterium of peach edge species (venturia inaequalis, Pear scab) close with its is compared and (is used
In amplification the black star bacterium ITS1/ITS4 sequence of peach primer be ITS1:5 '-TCCGTAGGTGAACCTGCGG-3 ' and ITS4:5 '-
TCCTCCGCTTATTGATATGC-3 '), the results showed that black 147~163 site of star bacterium ITS1/ITS4 amplified fragments of peach is difference
In the specific recognition sites (Fig. 9) of other pathogens.10 groups of LAMP primers (table 1) are devised on this basis, the results showed that only
When scalariform band (Figure 11) can be generated to the black star bacterium DNA cloning of peach using the 8th group of primer (SEQ ID NO:1-4), with reality
Now to the detection of the black star bacterium of peach.Figure 10 is seen in the design site of 8th group of LAMP primer.
For detecting the LAMP primer of the black star bacterium of peach, including (SEQ ID NO:1-4):
Outside forward primer VC-F3:5 '-AGCAAGCCCTGCCTAGA-3 '；
Outside reverse primer VC-B3:5 '-TGCCCCCCGGAATACCA-3 '；And
Inside forward primer VC-FIP (F1c+F2): 5 '-TGCCAGAACCAAGAGATCCGTTGTTGAAGTCTGAGGAG
AAAGCC-3'；
Inside reverse primer VC-BIP (B1c+B2): 5 '-GATGAAGAACGCAGCGAAATGCGCAATGTGCGTTCAAA
GATTCGA-3’。
Wherein, VC-FIP is made of the complementary series F1c and F2 of F1, and VC-BIP is made of the complementary series B1c and B2 of B1.
Primer synthesis is completed by one Hui Yuan Biotechnology Co., Ltd of Wuhan Tian.
Table 1
Embodiment 2 is analyzed using the specificity of the LAMP primer detection black star bacterium of peach
1, reagent and equipment
Water-bath, pipettor etc..
2, sample
The phytopathogen that the present embodiment uses includes peach black star germ (Venturia carpophila), the black star of apple
Germ (Venturia inaequalis), pear cucumerinum (Venturia nashicola), peach ulcer bacteria (Phomopsis
Amygdali), peach gummosis germ (Botryosphaeria dothidea), Peach black spot bacterium (Alternaria
Tenuissima), anthracnose of peach bacterium (Colletotrichum acutatum), peach shot hole bacterium (Xanthomonas
campestris)。
3, DNA is extracted
The a small amount of mycelia of picking is in 50 μ 10 × TE of L lysate (100mM Tris-HCL, 10mM EDTA, pH from scab
8.0) in, boiling water boiling 2 minutes.
4, LAMP reaction and detection
LAMP reaction system (25 μ l): 4U Bst archaeal dna polymerase, 2.5 μ L10 × ThermoPol Buffer, 5mM
MgSO4, 0.8mM dNTPs, 1.2mM VC-FIP, 1.2mM VC-BIP, 0.4mM VC-F3,0.4mM VC-B3,0.8M beet
Alkali, 1 μ L DNA profiling (lysate of above-mentioned processed mycelia).
Reaction condition: 75min is reacted in 61 DEG C of water-baths.
1 μ L 1000 × SYBR Green I dyestuff is added into gained amplified production, visually observes colour developing situation, obtains
As a result: display green is the positive, orange for feminine gender.
5, result
Amplification of the LAMP specific primer VC-F3/VC-B3/VC-FIP/VC-BIP to the black star bacterium of peach and 7 kinds of pathogen DNA
As a result SYBR Green I staining conditions are shown in Fig. 1.Wherein, 1: peach black star germ (Venturia carpophila)；2: apple
Black star germ (Venturia inaequalis)；3: pear cucumerinum (Venturia nashicola)；4: peach ulcer bacteria
(Phomopsis amygdali)；5: peach gummosis germ (Botryosphaeria dothidea)；6: Peach black spot bacterium
(Alternaria tenuissima)；7: anthracnose of peach bacterium (Colletotrichum acutatum)；8: peach shot hole bacterium
(Xanthomonas campestris)。
Pipe 1 is the black star bacterium germ of peach, and green chromogenic reaction occurs in reaction system, and display reaction is the positive.And other kind right
For the reaction system answered in orange, display reaction is feminine gender.This result shows that primer of the present invention high specificity.
The optimization of embodiment 3LAMP reaction system and reaction condition
1、Mg2+Concentration optimization
Using the reaction system and reaction condition of embodiment 2, only change Mg2+Concentration.
Colour developing situation after amplified production addition SYBR Green I dyestuff is shown in Fig. 2.Wherein, the corresponding Mg of 1-82+Final concentration
Respectively 2mM, 3mM, 4mM, 5mM, 6mM, 7mM, 8mM, 9mM.The result shows that 5mM Mg2+Corresponding color developing effect is best.
2, dNTPs concentration optimization
Using the reaction system and reaction condition of embodiment 2, only change dNTPs concentration.
Colour developing situation after amplified production addition SYBR Green I dyestuff is shown in Fig. 3.Wherein, the corresponding dNTPs of 1-8 is dense eventually
Degree is respectively 0.4mM, 0.8mM, 1.0mM, 1.2mM, 1.4mM, 1.6mM, 2.0mM, 2.4mM.The result shows that 0.8mM dNTPs
Corresponding color developing effect is best.
3, LAMP reaction temperature optimizes
Using the reaction system of embodiment 2,75min is reacted under different reaction temperatures.
Colour developing situation after amplified production addition SYBR Green I dyestuff is shown in Fig. 4.Wherein, the corresponding reaction temperature of 1-12
Respectively 57 DEG C, 58 DEG C, 59 DEG C, 60 DEG C, 61 DEG C, 62 DEG C, 63 DEG C, 64 DEG C, 65 DEG C, 66 DEG C, 67 DEG C, 68 DEG C.The result shows that 61
The amplified production reacted at DEG C, corresponding color developing effect are best.
4, the LAMP reaction time optimizes
Using the reaction system of embodiment 2, reaction temperature is 61 DEG C, sets the different reaction time.
Colour developing situation after amplified production addition SYBR Green I dyestuff is shown in Fig. 5.Wherein, the 1-4 corresponding reaction time
Respectively 45min, 60min, 75min, 90min.The result shows that reaction time 75min, corresponding color developing effect is best.
The sensitivity analysis of the black star bacterium of embodiment 4LAMP primer detection peach
The black star bacterium DNA of peach extracted to embodiment 2 is diluted, and is obtained the DNA solution of various concentration as template, is used
2 method of embodiment carries out LAMP reaction and detection.
Colour developing situation after amplified production addition SYBR Green I dyestuff is shown in Fig. 6.Wherein, the corresponding DNA concentration point of 1-8
It Wei not 5.66ng/ μ l, 5.66 × 105fg/μl、5.66×104fg/μl、5.66×103fg/μl、5.66×102fg/μl、5.66
×101fg/μl、5.66fg/μl、5.66×10-1fg/μl、5.66×10-2fg/μl.The result shows that the detection of the black star bacterium DNA of peach
It is limited to 5.66fg/ μ l.
Embodiment 5 detects the black star bacterium of peach from different regions using LAMP primer
Experimental method is the same as embodiment 2.
Fig. 7 is shown in the amplified production SYBR Green I staining conditions for the black star bacterium of 23 plants of peaches for picking up from 14 provinces and regions.Wherein,
1: Huangshi bacterial strain；2: Suizhou, hubei bacterial strain；3: Hubei Xiaogan bacterial strain；4: Beibei, chongqing bacterial strain；5: Chongqing Ba Nan bacterial strain；Weight
Celebrate Hechuan bacterial strain；6: Shandong Laixi bacterial strain；7: Shandong Linyi bacterial strain；8: Qingdao bacterial strain；9: Shandong Weifang bacterial strain；10: Zhejiang
Jiang Hangzhou bacterial strain 11: Henan Jiaozuo bacterial strain；12: Jiangsu Suqian bacterial strain；13: Nanjing bacterial strain；14: Jianyang of Sichuan's bacterial strain；15:
Sichuan Chengdu bacterial strain；16: Beijing bacterial strain；17: Hebei bacterial strain；18: Guilin bacterial strain；19: Yangling Shaanxi bacterial strain；20: Shaanxi
Xi'an bacterial strain；21: Kunming, Yunnan bacterial strain；22: Tongren district Guizhou Province bacterial strain；23: Lechang of Guangdong's bacterial strain；CK is distilled water control.
There is green chromogenic reaction in the reaction system of pipe 1-23, and display reaction is the positive.It should be the result shows that primer of the present invention
Applicability it is good, be suitable for the detection of the black star bacterium of different regions peach.
Embodiment 6 detects the infection conditions of the black star bacterium of incidence tissue peach using LAMP primer
1, DNA is extracted from the susceptible fruit for picking up from peach black star bacterium epidemic-stricken area and healthy pericarp
DNA extraction method is the same as embodiment 2.
2, LAMP reaction and detection
Reaction system, reaction condition, reaction result detection method are the same as embodiment 2.
3, result
LAMP specific primer is shown in figure to the SYBR Green I staining conditions of the amplified production of susceptible and healthy pericarp DNA
8.Wherein, 1~10: peach scab sample, 11~20: healthy fruit sample.
There is green chromogenic reaction in 1~10 reaction system of pipe, and display reaction is the positive.The reaction system of pipe 11~20 is in orange
Color, display reaction are feminine gender.This result shows that primer of the present invention high specificity, and can be directly used for the detection of field diseases.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, fall within the scope of the claimed invention without departing from theon the basis of the spirit of the present invention.
Sequence table
<110>Hua Zhong Agriculture University
<120>for detecting the LAMP primer and detection kit of the black star bacterium of peach
<130> KHP191110222.9
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tgccccccgg aatacca 17
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tgccagaacc aagagatccg ttgttgaagt ctgaggagaa agcc 44
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Claims (10)
1. the LAMP primer for detecting the black star bacterium of peach (Venturia carpophila) characterized by comprising
Outside forward primer VC-F3:5 '-AGCAAGCCCTGCCTAGA-3 '；
Outside reverse primer VC-B3:5 '-TGCCCCCCGGAATACCA-3 '；And
Inside forward primer VC-FIP:5 '-TGCCAGAACCAAGAGATCCGTTGTTGAAGTCTGAGGAGAAAGCC-3；'
Inside reverse primer VC-BIP:5 '-GATGAAGAACGCAGCGAAATGCGCAATGTGCGTTCAAAGATTCGA-3 '.
2. the kit for being used to detect the black star bacterium of peach containing LAMP primer described in claim 1.
3. kit according to claim 2, which is characterized in that the kit further includes dNTPs, Bst DNA polymerization
Enzyme, Thermopol Buffer, MgSO4Or MgCl2, at least one of glycine betaine.
4. kit according to claim 2 or 3, which is characterized in that the kit further includes standard positive template.
5. following any application of any one of LAMP primer described in claim 1 or claim the 2-4 kit:
I) application in the detection black star bacterium of peach；
Ii) in detection by the application in the microbial plant disease of the black star of peach.
6. application according to claim 5, which is characterized in that the plant disease includes peach scab.
7. application according to claim 5 or 6, which comprises the following steps:
1) DNA in sample is extracted；
2) it using the DNA extracted in step 1) as template, is carried out amplification reaction using the LAMP primer；
3) amplified production is analyzed.
8. application according to claim 7, which is characterized in that amplification reaction system is calculated as with 25 μ l: 4U Bst DNA is poly-
Synthase, 2.5 μ 10 × ThermoPol of L Buffer, 5mM MgSO4, 0.8mM dNTPs, 1.2mM VC-FIP, 1.2mM VC-
BIP, 0.4mM VC-F3,0.4mM VC-B3,0.8M glycine betaine, 1 μ L DNA profiling.
9. application according to claim 7, which is characterized in that amplification reaction condition: reacting 75min in 61 DEG C of water-baths.
10. according to the described in any item applications of claim 7-9, which is characterized in that step 3) specifically: the amplification to step 2)
SYBR Green I dyestuff is added in product, determines result according to colour developing situation: display green is the positive, orange for feminine gender.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111197105A (en) * | 2020-03-18 | 2020-05-26 | 福建省农业科学院植物保护研究所 | Application of LAMP primer in detection of alternaria alternata |
| CN111500765A (en) * | 2020-06-11 | 2020-08-07 | 青岛农业大学 | L AMP primer and kit for rapidly detecting apple scab germs and detection method thereof |
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| CN111500765B (en) * | 2020-06-11 | 2022-08-05 | 青岛农业大学 | LAMP primer, kit and detection method for rapidly detecting apple scab |
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