CN105349655A - Peronophythora litchii molecular detection primers and detection method thereof - Google Patents
Peronophythora litchii molecular detection primers and detection method thereof Download PDFInfo
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Abstract
The invention discloses peronophythora litchii molecular detection primers and a detection method thereof, and is dedicated to specific molecular detection of peronophythora litchi. A pair of peronophythora litchii specific primers PvYF1:5'-GTCCGAGTTTCTAGCAGATTG-3' and PvYR1:5'-ACGATAATACCGTGAGCGCC-3' are mainly designed; through PCR amplification and agarose gel electrophoresis, a specific amplification product with the fragment length of 249 bp is specifically amplified in peronophythora litchii pure DNA and peronophythora litchii-carried plants. The specific molecular detection primers and a usage method thereof can be used for rapid, sensitive and specific detection of peronophythora in the peronophythora litchii infected plants in production practice, at the same time, can be used for early diagnosis of field diseases and monitoring and identification of pathogenic bacteria, and provides a reliable technology and theoretical basis for diseases caused by peronophythora litchii.
Description
Technical field
A kind of peronophythora litchi molecular detection primer of the present invention and detection method thereof, be exclusively used in peronophythora litchi highly sensitive rapid molecular to detect, can be used for monitoring and the qualification of field Peronophythora Litchii disease early diagnosis and germ simultaneously, belong to that corps diseases detects, qualification and the field of Prevention Technique.
Background technology
Litchi flavor is good, look beautiful, nutritious, be subtropical and tropical zones fruit, distribute throughout Hainan, Fujian, Guangxi, Guangdong, the province such as Taiwan, there is Important Economic and be worth.By peronophythora litchi (
peronophythoralitchii) peronophythora litchi that causes is one of most important disease of lichee, has a strong impact on the storing of Yield of Litchi, quality and fresh fruit and export trade.This disease finds as far back as the Taiwan Province of China, at present in Fujian, all there is generation on Guangdong, Guangxi, Hainan, Yunnan, Guizhou, Sichuan, the ground such as Taiwan, not only seriously cause harm close to mellow fruit, also to cause harm the tender tip, blade, Hua Sui, result sprig, carpopodium and young fruit, cause a large amount of shedding and decayed fruit, production loss, up to more than 80%, is even had no harvest, and causes tremendous economic to lose.This sick popular frequency and popularity degree had the trend increased the weight of gradually in recent years, had become the significant obstacle that lichee is produced.In view of the considerable damage of peronophythora litchi, spread for preventing this disease and spread, set up detection method fast and effectively very necessary.
Mostly still continue to use tradition to the detection of peronophythora litchi at present to cultivate and serological identification method.Traditional detection of pathogens technology is on the basis that separation and Culture obtains corresponding pathogen, is judged the kind of pathogen by morphological observation and Koch's Postulates.Whole process usually needs labor force and the time of at substantial, general needs just can complete for several days, and require that operator possesses professional pathogenicbacteria separation, Morphological Identification knowledge and rich experience, therefore traditional diagnosis method of judgement basis is characterized as with Pathogens, because of its length consuming time, efficiency is low, is difficult to meet the actual needs to peronophythora litchi diagnosis, because it easily misses the best period of disease control.Though immunoserology authentication method is set up, the preparation process of serum takes time and effort, and may there is cross reaction, easily causes false positive, and the application of these methods is all subject to certain restrictions.
In recent years along with the development of Protocols in Molecular Biology, be that the molecular detecting method of representative obtains and develops rapidly and apply with round pcr, especially polymerase chain reaction (PCR) technology be applied in the deficiency that compensate for traditional method to a great extent.These class methods generally have quick, accurate, sensitive and do not need the feature of the separation and Culture of carrying out pathogenic bacteria, control Plant diseases propagation, cause disaster in started to play a significant role.Chinese scholars utilizes the Molecular Detection based on ITS base sequence design primer pair oomycetes to conduct a research, but there is for target sequence designs primer the problem being difficult to distinguish allied species with ITS.There are some researches show, GTP (guanosine triphosphate) (GTP) binding-protein gene (
ypt1) be a gene relevant to proto-oncogene Ras (Ratsarcoma), in yeast with, this genes encoding one and Ras correlative GTP bindin.
ypt1gene contains multiple exon and intron, and multiple exon has conservative property, and in these, aobvious son has polytropy between not of the same race, its conserved sequence and evolution region spaced, be suitable as very much the target of white phytophthora Molecular Detection.Therefore, peronophythora litchi been has originally has been researched and analysed and other 30 kinds of oomycetes exist
ypt1difference in gene order, devises Auele Specific Primer, and establishes the PCR rapid molecular detection system of peronophythora litchi on this basis.This technology can be applicable to peronophythora litchi cause disease show disease before early monitoring, for determining that disease control best period has important effect, for the formulation of control strategy provides scientific basis.
Summary of the invention
The object of the invention is there is very high requirement for peronophythora litchi traditional detection method in prior art to the experimental skill of operator and practical experience, and it is very consuming time, the problem of quick test cannot be reached, provide the special molecular of peronophythora litchi to detect primer and reliable results, easy handling, high specificity, the rapid molecular detection system of highly sensitive peronophythora litchi and program.The method can be used for carrying disease germs highly sensitive rapid molecular of plant detects, this technology for peronophythora litchi cause disease show disease before early monitoring, determine that disease control best period tool is of great significance.
Realize object of the present invention to comprise the following steps:
1. according to peronophythora litchi
ypt1the spaced feature of the conserved regions of gene order and evolution district, devise one couple of PCR primers peronophythora litchi to specific amplified effect respectively, sequence is as follows:
PvYF1：5’-GTCCGAGTTTCTAGCAGATTG-3’；
PvYR1：5’-ACGATAATACCGTGAGCGCC-3’。
2. the foundation of peronophythora litchi rapid detection system
(1) from peronophythora litchi, the plant that infected by peronophythora litchi, DNA is extracted;
(2) foundation of peronophythora litchi specific PCR rapid detection system
Described PCR reaction system 25.0 μ l, comprise 2 ×
taqthe each 0.5 μ L of primer (PvYF1/PvYR1) of PCRMasterMix12.5 μ L, 10 μm of ol/L, DNA profiling 25ng, insufficient section is by ddH
2o supplies.PCR reaction conditions is: 95 DEG C of denaturation 3min; 94 DEG C of sex change 1min, 59 DEG C of annealing 30sec, 72 DEG C extend 1min, totally 28 circulations; 72 DEG C extend 10min.
(3) foundation of peronophythora litchi nest-type PRC rapid detection system
First round pcr amplification is carried out, reaction system 25.0 μ L with phytophthora universal primer (Yph1F/Yph2R), comprise 2 ×
taqthe each 1.0 μ L of primer (Yph1F/Yph2R) of PCRMasterMix12.5 μ L, 10 μm of ol/L, DNA profiling 25ng, insufficient section is by ddH
2o supplies.PCR reaction conditions is: 95 DEG C of denaturation 3min, 94 DEG C of sex change 1min, 55 DEG C of annealing 30sec, and 72 DEG C extend 1min, totally 30 circulations, and 72 DEG C extend 10min.
Get 1 μ L first round PCR primer respectively as DNA profiling, carry out second with primer (PvYF1/PvYR1) and take turns pcr amplification.Reaction system 25.0 μ l, comprise 2 ×
taqthe each 0.5 μ L of primer (PvYF1/PvYR1) of PCRMasterMix12.5 μ L, 10 μm of ol/L, DNA profiling 1 μ L, insufficient section is by ddH
2o supplies.PCR reaction conditions is: 95 DEG C of denaturation 3min; 94 DEG C of sex change 1min, 59 DEG C of annealing 30sec, 72 DEG C extend 1min, totally 28 circulations; 72 DEG C extend 10min.
Then get pcr amplification product agarose gel electrophoresis to be separated, observe under ultraviolet lamp after ethidium bromide staining, according to the size result of determination of amplified production, if the product of 249bp can be amplified specifically, can judge to there is peronophythora litchi in described Plant samples.
Guardian technique of the present invention is primer sequence and the amplification method thereof of the efficient specific amplified of peronophythora litchi.In order to detect peronophythora litchi special primer accuracy, the 21 strain peronophythora litchis that the present invention economizes with China Fujian, Guangdong, Guangxi, Taiwan etc. and other 13 kinds of different oomycetes and 9 kinds of fungies are for for examination material, CTAB method is adopted to extract strains tested genomic dna, concrete grammar is as follows: get the hypha powder after 50mg lyophilize in 1.5ml centrifuge tube, adds 900 μ l2%CTAB (cetyl trimethylammonium bromide) extracting solution (2%CTAB, 100mmol/LTris-HCl, pH8.0, 20mmol/LEDTA, pH8.0, 1.4mol/LNaCl) He 90 μ l10%SDS(Sodium dodecylbenzene sulfonatees) mixing of vibrating afterwards, in 55 ~ 60 DEG C of water-bath 1.5h, every 10min puts upside down mixing once, after water-bath 1.5h centrifugal (12, 000rpm) 8min, get supernatant liquor and add isopyknic phenol/chloroform/primary isoamyl alcohol (25:24:1), put upside down and make abundant mixing, centrifugal (12, 000rpm) 12min, get supernatant liquor (aqueous phase), add equal-volume chloroform/primary isoamyl alcohol (24:1), put upside down and make abundant mixing, centrifugal (12, 000rpm) 5min, suct clearly, add the 3mol/LNaAC solution of 0.1 volume and the ice dehydrated alcohol of 2 volumes, put in-20 DEG C of refrigerators and precipitate more than 30min, 12, the centrifugal 5min of 000rpm, remove supernatant liquor lightly, adding 700 μ l ice 70% ethanol carries out washing (slightly centrifugal, incline and fall supernatant), Bechtop dries naturally after alcohol-free taste with 1 × TE(10mmol/LTris-HCl, 0.1mmol/LEDTA, pH8.0) solution dissolves, obtain DNA solution, by UV spectrophotometer measuring DNA concentration and to be diluted to 50ng/ μ L stand-by.
Through carrying out PCR checking to the specificity of strains tested and 21 strain peronophythora litchis.PCR reaction system 25.0 μ L, comprise 2 ×
taqthe each 0.5 μ L of primer (PvYF1/PvYR1) of PCRMasterMix12.5 μ L, 10 μm of ol/L, 25ngDNA template, insufficient section is by ddH
2o supplies.PCR reaction conditions is: 95 DEG C of denaturation 3min; 94 DEG C of sex change 1min, 59 DEG C of annealing 30sec, 72 DEG C extend 1min, totally 28 circulations; 72 DEG C extend 10min.This special primer amplifies the product of 249bp specifically in peronophythora litchi.This illustrates that this primer can be used to detection that in production practice, in disease plant, peronophythora litchi is fast and reliable and qualification.
When there is peronophythora litchi in for plant, adopt NaOH rapid cleavage method to extract the DNA of peronophythora litchi, detailed process is as follows: litchi sickness leaf or sick stem are cleaned, dried by (1), clip site of pathological change; (2) 10uL(0.5mol/LNaOH, 0.5%PVP is added by the sick leaf of 1mg) metering, be fully milled to paste by organizing, centrifugal 5min in 12,000g whizzer; (3) Tris-HCl(pH8.0 of supernatant 20uL and isopyknic 0.1mol/L is got) mix; (4) dilute 10 times, 100 times, 1000 times liquid, get 1 μ l stoste, 10 times, 100 times, 1000 times liquid respectively as pcr template, carry out pcr amplification by following PCR reaction system and reaction conditions.PCR reaction system 25.0 μ L, comprise 2 ×
taqthe each 0.5 μ L of primer (PvYF1/PvYR1) of PCRMasterMix12.5 μ L, 10 μm of ol/L, DNA profiling 1 μ l, insufficient section is by ddH
2o supplies.PCR reaction conditions is: 95 DEG C of denaturation 3min; 94 DEG C of sex change 1min, 59 DEG C of annealing 30sec, 72 DEG C extend 1min, totally 28 circulations; 72 DEG C extend 10min.
2. then get 5 μ LPCR products in 1.5% agarose gel electrophoresis containing 0.5 μ g/mLEB, gel imaging system detects and takes pictures, according to the size result of determination of amplified production.
If when 3. can amplify 249bp product specifically, can judge to there is peronophythora litchi in described Plant samples; Otherwise there is not peronophythora litchi in described Plant samples.
beneficial effect of the present invention:the inventive method is applicable to fast and reliable detection and the qualification of peronophythora litchi in plant, and the disease control caused for peronophythora litchi in agriculture production has important practical value.The present invention compared with prior art, has following technical superiority and positively effect:
1, high specificity: detection method is
ypt1the spaced feature of the conserved regions of gene order and evolution district devises a pair peronophythora litchi special primer and detects.Verify the peronophythora litchi oomycetes different from other of economizing from China Fujian, Guangdong, Guangxi, Taiwan etc. and fungi, result has very strong specificity;
2, practicality is good: a pair Auele Specific Primer designed by the present invention, the highly sensitive rapid molecular that can be used for the plant being with peronophythora litchi detects, therefore present method is practical, can meet and carry out fast and reliable detection and the needs of qualification to the peronophythora litchi existed in the plant that carries disease germs;
3, fast easy and simple to handle: application the inventive method, get final product result of determination, without the need to carrying out digestion with restriction enzyme to amplified production after the agarose gel electrophoresis of germ DNA extraction, pcr amplification and routine is carried out to the plant of band peronophythora litchi.General whole testing process can complete within a few hours.
Accompanying drawing explanation
the specific PCR amplification figure of the peronophythora litchi that Fig. 1 will detect for the present invention
In figure: M is DL2000DNAMarker; 1-4 is different sources Peronophythora Litchii; 5: Phytophthora capsici; 6: soybean phytophthora; 7: phytophthora infestans; 8: melon and fruit corruption is mould; 9: Colletotrichum capsici; 10: Fusarium oxysporum; 11: Pyricularia oryzae; 12: the burnt maize ear rot bacterium of piscidia; 13: botrytis; 14: negative control.
fig. 2 is that the susceptibility of peronophythora litchi of the present invention detects amplification figure
In figure: M is DL2000DNAMarker; 1-8 is respectively 10ng/ μ L, 1ng/ μ L, 100pg/ μ L, 10
Pg/ μ L, 1pg/ μ L, the different concns peronophythora litchi DNA of 100fg/ μ L, 10fg/ μ L, 1fg/ μ L.
fig. 3 is the detected result figure of disease plant of the present invention
In figure: M is DL2000DNAMarker; 1 is negative control; 2 is positive control; 3-5,7-10 are the lichee frost epidemic disease plant of morbidity; 6 is healthy lichee plant.
embodiment:
Technology contents of the present invention comprises the specific detection primer of peronophythora litchi, and designed primer and sequence thereof are: PvYF1(5 '-GTCCGAGTTTCTAGCAGATTG-3 ') and PvYR1(5 '-ACGATAATACCGTGAGCGCC-3 ').Utilize this primer from peronophythora litchi, the product of 249bp can be gone out by specific amplified.
embodiment 1: the specific amplification of primer pair peronophythora litchi
1. the specific detection of peronophythora litchi
PCR reaction system 25.0 μ L, comprise 2 ×
taqthe each 0.5 μ L of primer (PvYF1/PvYR1) of PCRMasterMix12.5 μ L, 10 μm of ol/L, DNA profiling 25ng, insufficient section is by ddH
2o supplies.PCR reaction conditions is:: 95 DEG C of denaturation 3min; 94 DEG C of sex change 1min, 59 DEG C of annealing 30sec, 72 DEG C extend 1min, totally 28 circulations; 72 DEG C extend 10min.
2. detected result
The specificity detected: except the peronophythora litchi DNA economized from China Fujian, Guangdong, Guangxi, Taiwan etc. can amplify except the product of 249bp specifically, have detected 13 kinds of different oomycetes and 8 kinds of fungal DNAs all fail to amplify spawn, there is very strong specificity.
embodiment 2: the sensitivity technique of primer pair peronophythora litchi
1.DNA concentration dilution: the peronophythora litchi genomic dna of extraction, after spectrophotometric determination concentration, adopts series concentration dilution.
2. the sensitivity technique of peronophythora litchi
10 times of concentration series dilution methods are adopted the peronophythora litchi DNA of extraction to be diluted to 10ng/ μ L successively, 1
Ng/ μ L, 100pg/ μ L, 10pg/ μ L, 1pg/ μ L, 100fg/ μ L, 10fg/ μ L, 1fg/ μ L is totally 8 different concns gradients, adopts the sensitivity of nest-type PRC to primer to measure.The peronophythora litchi genomic dna getting 1 μ L different concns is respectively template, carries out first round pcr amplification with phytophthora universal primer (Yph1F/Yph2R), reaction system 25.0 μ L, comprise 2 ×
taqthe each 1.0 μ L of primer (Yph1F/Yph2R) of PCRMasterMix12.5 μ L, 10 μm of ol/L, DNA profiling 1 μ L, insufficient section is by ddH
2o supplies.PCR reaction conditions is: 95 DEG C of denaturation 3min, 94 DEG C of sex change 1min, 55 DEG C of annealing 30sec, and 72 DEG C extend 1min, totally 30 circulations, and 72 DEG C extend 10min.Get 1 μ L first round PCR primer respectively as DNA profiling, carry out second with primer (PvYF1/PvYR1) and take turns pcr amplification, reaction system 25.0 μ L, comprise 2 ×
taqthe each 0.5 μ L of primer (PvYF1/PvYR1) of PCRMasterMix12.5 μ L, 10 μm of ol/L, DNA profiling 1 μ L, insufficient section is by ddH
2o supplies.PCR reaction conditions is:: 95 DEG C of denaturation 3min; 94 DEG C of sex change 1min, 59 DEG C of annealing 30sec, 72 DEG C extend 1min, totally 28 circulations; 72 DEG C extend 10min.
2. detected result: in 25.0 μ L reaction systems, peronophythora litchi genomic dna can obtain obvious amplified band, and detection sensitivity can reach 100fg/25 μ L.
embodiment 3: the detection of peronophythora litchi in disease plant sample.
1. sample collecting: Plant tissue samples picks up from lichee production base, Fujian Province.
2.DNA isolation and determination
Morbidity plant tissue adopts NaOH rapid cleavage method to extract DNA, carries out pcr amplification by the following method:
PCR reaction system 25.0 μ L, comprise 2 ×
taqthe each 0.5 μ L of primer (PvYF1/PvYR1) of PCRMasterMix12.5 μ L, 10 μm of ol/L, DNA profiling 1 μ L, insufficient section is by ddH
2o supplies.PCR reaction conditions is:: 95 DEG C of denaturation 3min; 94 DEG C of sex change 1min, 59 DEG C of annealing 30sec, 72 DEG C extend 1min, totally 28 circulations; 72 DEG C extend 10min.
Agarose gel electrophoresis detects amplified production.
3. detected result
The results are shown in Figure 3, can see one clearly molecular weight be the specific band of 249bp, thus judge incidence tissue infect peronophythora litchi.
The foregoing is only preferred embodiment of the present invention, all equalizations done according to the present patent application the scope of the claims change and modify, and all should belong to covering scope of the present invention.
SEQUENCELISTING
<110> Inst. of Plant Protection, fujian Academy of Agricultural Science
<120> peronophythora litchi molecular detection primer and detection method thereof
<130>2
<160>2
<170>PatentInversion3.3
<210>1
<211>21
<212>DNA
<213> artificial sequence
<400>1
gtccgagtttctagcagattg21
<210>2
<211>20
<212>DNA
<213> artificial sequence
<400>2
acgataataccgtgagcgcc20
Claims (3)
1. a PCR primer for peronophythora litchi Molecular Detection, is characterized in that, primer sequence is:
PvYF1：5’-GTCCGAGTTTCTAGCAGATTG-3’；
PvYR1：5’-ACGATAATACCGTGAGCGCC-3’；
Above-mentioned primer PvYF1/PvYR1 goes out the product of 249bp to peronophythora litchi specific amplification.
2. the detection method of peronophythora litchi Molecular Detection PCR primer as claimed in claim 1, comprises
(1) from peronophythora litchi, the plant that infected by peronophythora litchi, DNA is extracted;
(2) with this DNA for the primer PvYF1/PvYR1 described in template claim 1 is by polymerase chain reaction (PCR) amplification: PCR reaction system 25.0 μ L, comprise 2 ×
taqthe each 0.5 μ L of primer of PCRMasterMix12.5 μ L, 10 μm of ol/L, DNA profiling 25ng, insufficient section is by ddH
2o supplies; PCR reaction conditions is: 95 DEG C of denaturation 3min; 94 DEG C of sex change 1min, 59 DEG C of annealing 30sec, 72 DEG C extend 1min, totally 28 circulations; 72 DEG C extend 10min;
(3) the pcr amplification product agarose gel electrophoresis then getting step (2) is separated, observe under ultraviolet lamp after ethidium bromide staining, according to the size result of determination of amplified production, if the product of 249bp can be amplified specifically, can judge to there is peronophythora litchi in described Plant samples.
3. the application of PCR primer in the monitoring and qualification of field Peronophythora Litchii disease early diagnosis and germ of peronophythora litchi Molecular Detection as claimed in claim 1.
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| CN106434385A (en) * | 2016-10-31 | 2017-02-22 | 华南农业大学 | Convenient method for extracting oospores of Peronophythora litchi from solid culture medium |
| CN107058609A (en) * | 2017-06-29 | 2017-08-18 | 福建省农业科学院植物保护研究所 | A kind of Peronophythora Litchii PCR primer and its molecular detecting method |
| CN112280890A (en) * | 2020-11-18 | 2021-01-29 | 福建省农业科学院植物保护研究所 | Primer and probe combination for detecting peronophythora litchi based on RPA-lateral flow chromatography technology and detection method thereof |
| CN114097510A (en) * | 2021-11-09 | 2022-03-01 | 海南大学 | Comprehensive prevention and control method for downy mildew of litchi |
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| CN106434385B (en) * | 2016-10-31 | 2019-08-13 | 华南农业大学 | A kind of convenient method extracting peronophythora litchi egg spore from solid medium |
| CN107058609A (en) * | 2017-06-29 | 2017-08-18 | 福建省农业科学院植物保护研究所 | A kind of Peronophythora Litchii PCR primer and its molecular detecting method |
| CN107058609B (en) * | 2017-06-29 | 2020-07-17 | 福建省农业科学院植物保护研究所 | Peronophythora litchi PCR primer and molecular detection method thereof |
| CN112280890A (en) * | 2020-11-18 | 2021-01-29 | 福建省农业科学院植物保护研究所 | Primer and probe combination for detecting peronophythora litchi based on RPA-lateral flow chromatography technology and detection method thereof |
| CN114097510A (en) * | 2021-11-09 | 2022-03-01 | 海南大学 | Comprehensive prevention and control method for downy mildew of litchi |
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