CN109457043A - The Real-timePCR detection primer and detection method of notoginseng root rot pathogenic bacteria Fusarium oxysporum - Google Patents
The Real-timePCR detection primer and detection method of notoginseng root rot pathogenic bacteria Fusarium oxysporum Download PDFInfo
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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Abstract
The invention discloses a kind of Real-time PCR detection primer of notoginseng root rot pathogenic bacteria Fusarium oxysporum and detection method, the nucleotide sequences of detection primer are as follows: upstream primer Fo-QF:5 ' CTCAAACAGTGGTACATGCGAGG3 '；Downstream primer Fo-QR:5 ' CATCTAGGTCTTCCATCCACTTGA3 '；The present invention designs the specific primer of Fusarium oxysporum, establishes qualitative, quantitative Molecular Detection and disease Accurate Diagnosis of the standard curve for Fusarium oxysporum；The Number dynamics that Fusarium oxysporum in complicated notoginseng planting soil and Radix Notoginseng diseased plant can be quickly obtained using technology and methods of the invention are changed, thus can for Radix Notoginseng soil treatment, the early diagnosis of root rot and dynamic monitoring and in spite of illness Panax notoginseng seeds, seedling Molecular Detection technical support is provided.
Description
Technical field
The present invention relates to a kind of Real-time PCR detection primer of notoginseng root rot bacterium Fusarium oxysporum and its detection sides
Method, is exclusively used in the rapid molecular detection of notoginseng root rot bacterium Fusarium oxysporum, while can realize the morning of field notoginseng root rot bacterium
The Monitoring on Dynamic Change of phase diagnosis and germ, can be also used for the seed borne fungi of notoginseng planting soil and seed, seedling, belongs to
The molecular Biological Detection field of plant disease.
Background technique
Radix Notoginseng (Panax notoginseng(Burk) F.H. Chen) alias pseudo-ginseng, mountain knee, Typhonium flagelliforme (Lodd.) Blume, radix notoginseng,
Blood ginseng, Yunnan Radix Notoginseng etc., there is the good reputation of " southern part of the country god grass ", " invaluable ".Radix Notoginseng is used to treat disease the history of centuries, " this
Careless detailed outline " in record Radix Notoginseng there is the effect of styptic powder blood analgesic therapy, have special efficacy to knife wound, arrow wound, traumatic injury, haemophilia,
Radix Notoginseng also has swelling and pain relieving, anti-inflammatory, anti-aging, improves multiple efficacies (Zhang Jingjing, Liu Guiqin, the Wang Qing such as immunity simultaneously
The progress Liaocheng University journal (natural science edition) of saponins chemical component in roc plant Radix Notoginseng, 2018,31 (2):
43-59.).Radix Notoginseng chemical component containing there are many, wherein arasaponin is one of main effective active composition, and content is about
8%~12%.In addition, in Radix Notoginseng also containing the compounds such as alcohols, flavonoids, ucleosides, alkaloid, protein, vitamin C and
A variety of inorganic elements such as potassium, magnesium, calcium have the speciality basis of multiple efficacies for Radix Notoginseng.
Radix Notoginseng has platelet aggregation-against and thrombolytic effect, and arasaponin mainly passes through the function of improving blood vessel endothelium,
Improve blood flow state, improve blood constituent reach inhibit platelet adhesion reaction and assemble antithrombotic purpose (Wang J,
Huang ZG, et al. Screening of anti-platelet aggregation agents from Panax notoginseng using human platelet extraction and HPLC–DAD–ESI‐MS/MS. Journal
of Separation Science , 2015 , 31 (6‐7) :1173-1180.).Notoginsenoside can obviously reduce experimentally
Thrombosis, and the platelet aggregation for inhibiting fibrin ferment to lure in a dose-dependent manner, may also suppress the normal of thrombin induction
Blood pressure and renal hypertensive rat Platelet endothelium cell adhesion molecular-1 concentration increase.Radix Notoginseng has an effect on blood pressure, and arasaponin has direct
Expand blood vessel, the effect of reducing blood pressure.Generally believe that notoginsenoside is calcium channel blocker at present, expanding vascular mechanism may be three
Seven saponin(es have Ca caused by blocking norepinephrine2+The effect of interior stream.Also there is the work for reducing angiosthenia and slightly subtracting heart rate
With, make heart working amount lower (Liu Y, Hao F, et al.Panax notoginseng saponins promote
endothelial progenitor cell mobilization and attenuate atherosclerotic
lesions in apolipoprotein E knockout mice. Cellular Physiology & Biochemistry
, 2013 , 32 (4) :814-826.).Radix Notoginseng also has study of anti-atherogenic effect.It is as main component multiple with Radix Notoginseng
Square XUESHUANTONG JIAONANG has the bore of expansion arteriole and thin vein, improves microcirculation, increases the blood perfusion amount of tissue, extends
Histanoxia life span.
Radix Notoginseng main product is in Yunnan Wenshan Prefecture, and with the fast development of Radix Notoginseng industry in recent years, planting area the sixth of the twelve Earthly Branches is had occurred very
Big variation, Yunnan producing region expand to the areas such as Honghe, Yunnan, Yuxi, Qujing, Kunming, Dali by mountain of papers, Guizhou, Sichuan, etc.
Ground also starts to cultivate.With the increase year by year of notoginseng planting area, problems faced is also therewith in large-scale production for Radix Notoginseng
Come, there are two aspects the main reason for restricting current Radix Notoginseng sustainable development: first is that Soil-sickness Problem is serious, in the reality of Radix Notoginseng
It is suitble to the land resource growing tension of plantation Radix Notoginseng in the production of border, needs to be spaced 10-15 or more ability multiple cropping, continuous cropping obstacle is big
The planting cost of Radix Notoginseng is increased greatly.Second is that unexcellent new varieties, Radix Notoginseng germ plasm resource is seriously degenerated, Radix Notoginseng disease species
Increase year by year, onset area increases year by year, to the large-scale production of Radix Notoginseng cause it is huge loss (Cui Xiuming, Huang Luqi,
Equal China Radix Notoginseng industry Situation and development countermeasure CHINA JOURNAL OF CHINESE MATERIA MEDICA, 2014,39 (4): 553-557).
Notoginseng root rot is disease the most serious during Panax notoginseng Growth, and long-term disease incidence is 5% ~ 20%, serious height
Up to 70% or more, plant catches an illness, and back root part is rotten, and overground part Huang withers, and is commonly called as rotten " smelly seven " of chicken droppings.(Deng Renke, He Yingjie, Zhao
Application Yunnan Prov Agriculture University journal of the tinkling of pieces of jade botanical nematicide " line opposes No. 1 " in prevention and treatment notoginseng root rot shake (certainly
So science), 2016,30 (S1): 57-62.).The cardinal symptom of notoginseng root rot has yellow rotten type, dry and cracked type, the rotten type of marrow, urgency
Property green withered type, web rot type and stem foot dry up type etc., it is more typical with yellow rotten type and the acute withered type of blueness.It is separated from rotten Panax notoginseng root
To include Fusarium oxysporum (Fusarium oxysporum) including reaping hook category fungi, it is a variety of to be also separated to bacterium, nematode etc.
Pathogen, it is believed that notoginseng root rot (Jiang Ni, Qin Liuyan, Ye Yun caused by the compound Combined Infection such as fungi, bacterium, nematode
Peak Radix Notoginseng disease progress agriculture in south journal, 2011,42 (9): 1070-1074).
Fusarium oxysporum is typical soil-borne disease fungi, is an important monoid in Fusarium, dependent territory earth habitat
Bacterium is a kind of saprophytic bacteria active more than quantity in soil and organic matter, and host range is very extensive, can cause more than 100
Wilt disease occurs for kind plant, leads to serious economic loss.Plant wilt disease caused by being infected by Fusarium oxysporum is a kind of generation
The silborne fungal diseases of criticality, germ cause harm plant from root, cause vascular bundle diseases, cause plant withered, in the complete of plant
Breeding time can occur.It is with its spore germination pipe or mycelium directly from the tip of a root, root that Fusarium oxysporum, which invades in plant body,
Wound or enter in plant body in the formation point of lateral root, and this is grown between root skin confluent monolayer cells, then can be by wooden
The pit in portion invades conduit, then grows up in the catheter to the stem of plant and top (Zhang Bin, Yang Xiaoyun, Chen Zhiyi
The research Plant Pathology of the pathogenic sickle-like bacteria Identification of Species of tomato wilt and dominant population, 2016,46 (4):
561-565).
Real-Time Fluorescent Quantitative PCR Technique is as a kind of highly sensitive, high specific detection of nucleic acids means, compared to pathogen
It is fast accurate with microscope observation method to be separately cultured, in the disease for causing plant disease by fungi, bacterium, virus, nematode etc.
It is widely applied in original diagnosis and prevention and treatment control research.In addition, in the mechanism of production and pests occurrence rule of research plant disease
When, moreover it is possible to know in complex environment that plant pathogenic fungi Number dynamics change, to put prevention first, early diagnosing and dynamic monitoring mentions
For technical guarantee.
Summary of the invention
Pathogen morpha is based primarily upon for the detection and identification in the prior art to notoginseng root rot bacterium Fusarium oxysporum
Feature, program is cumbersome, time-consuming, low to identification skill requirement height, accuracy, can not carry out the quantitative analysis of pathogen, it is difficult to
Meet the status that notoginseng root rot bacterium quick and precisely diagnoses, the present invention provides a kind of notoginseng root rot bacterium Fusarium oxysporums
Real-time PCR molecular detection primer and its detection method.
To achieve the above object, this invention takes following technical schemes:
Present invention firstly provides a kind of Real-time PCR detection primer of notoginseng root rot pathogenic bacteria Fusarium oxysporum,
Nucleotide sequence are as follows:
Upstream primer Fo-QF:5 ' CTCAAACAGTGGTACATGCGAGG3 '
Downstream primer Fo-QR:5 ' CATCTAGGTCTTCCATCCACTTGA3 '；
The primers F o-QF and Fo-QR goes out the product of 209 bp to notoginseng root rot bacterium Fusarium oxysporum specific amplification.
The present invention also provides a kind of rapid detection methods of notoginseng root rot bacterium, comprising the following steps:
(1) DNA of Radix Notoginseng test sample or soil is extracted；
(2) real-time fluorescence quantitative PCR amplification, real-time fluorescence are carried out by template of the DNA of the Radix Notoginseng test sample of extraction or soil
Quantitative pcr amplification reaction system is 20 μ L, is drawn comprising 2 × SYBR Green Mix, 10 μ L, 7 μ L of nuclease-free water, upstream
Object Fo-QF (2 μm of ol/L) 1 μ L, downstream primer Fo-QR (2 μm of ol/L) 1 μ L, 1 μ L of DNA profiling；In quantitative fluorescent PCR
It being expanded on instrument, amplification program is 95 DEG C of 2 min, then carries out 45 circular responses, and each circular response is 95 DEG C of 1 min,
62 DEG C of 30 s, 72 DEG C of 1 min measure fluorescent value when 72 DEG C；Respectively collects automatically after circulation terminates and record fluorescence signal；
(3) foundation of real-time fluorescence quantitative PCR standard curve
Prepare the recombinant plasmid pGEM-T- that concentration is 35ng/ μ LCYP505As standard items, it is dilute to carry out 10 times of concentration gradient
It releases, 5 gradients is set altogether, and for concentration within the scope of 0.0035ng/ μ L~35ng/ μ L, dilution medium is nuclease-free water；To 5
The standard items of gradient carry out real-time fluorescence quantitative PCR reaction, and 3 repetitions are arranged in each gradient；Quantitative fluorescent PCR reaction system and
The same step of amplification program (2) measures fluorescent value when 72 DEG C, fluorescence signal is collected and recorded after each circular response, with
The corresponding plasmid quality logarithm of each gradient concentration is abscissa as ordinate, Ct value, obtains equation of linear regression, and establish
Standard curve；
(4) it can determine that there are root rot pathogenic bacteria Fusarium oxysporums in institute's test sample if having fluorescence signal, by upper step institute
The Ct value obtained is brought into the standard curve that the present invention constructs, and the quantity of Fusarium oxysporum in test sample is calculated.
The present invention has the advantages that
(1) accuracy is high: the present invention is the well-conserved and category kind according to fungi DNA sequence in fungi kind
Between changeability the characteristics of design to notoginseng root rot bacterium Fusarium oxysporum have specific amplified effect Real-time PCR draw
Object；Detection point has been carried out to the rotten root of the notoginseng root rot diseased plants of different planting bases and healthy Panax notoginseng root
Analysis only can specifically amplify the band of 209 bp in the rotten root of root rot diseased plant, illustrate designed by the present invention
Primer for detect notoginseng root rot pathogenic bacteria Fusarium oxysporum accurate and reliable；
(2) high specificity: primer pair notoginseng root rot pathogenic bacteria Fusarium oxysporum designed by the present invention has very strong special
Property, it can be used in the pathogen for distinguishing the Radix Notoginseng Common Diseases such as notoginseng root rot, Alternaria panax；
(3) high sensitivity: the special primer of design is subjected to Real-time PCR analysis using the present invention, to notoginseng root rot
The detection sensitivity of pathogenic bacteria Fusarium oxysporum can reach 0.35 pg/ μ L on DNA level；
(4) applicability is wide, practicability is good: the detection method of notoginseng root rot pathogenic bacteria Fusarium oxysporum of the invention, can not only
Germ mycelium is detected, susceptible Panax notoginseng root, blade, seed, seedling and soil can also be detected, it can be real
The early detection of existing notoginseng root rot, i.e., detected before disease shows disease, the large area outburst of controlling disease and prevalence.
Detailed description of the invention
Fig. 1 is the amplification electrophoretogram for verifying Fusarium oxysporum fatty acid Ω '-hydroxylase gene specificity, and wherein swimming lane M is
2000bp DNA Marker, swimming lane 2 are sclerotinite, and swimming lane 3 is tobacco brown spot pathogen, and swimming lane 4 is ginseng rod method, and swimming lane 5 is
Branch sickle-like bacteria is taken turns, swimming lane 6 is Fusarium graminearum, and swimming lane 7 is Fusarium oxysporum, and swimming lane 8 is Fusarium solani, and swimming lane 9 is feminine gender
It compares (no template)；
Fig. 2 is primer pair notoginseng root rot pathogenic bacteria Fusarium oxysporum real-time fluorescence quantitative PCR specific amplification curve of the present invention
(a) and melting curve figure (b) are schemed, wherein eight kinds of DNA profilings are respectively ginseng rod method, tobacco brown spot pathogen, sclerotinite, eggplant
Rotten sickle-like bacteria, Fusarium oxysporum, wheel branch sickle-like bacteria, Fusarium graminearum, grape seat chamber bacterium；
Fig. 3 is the real-time fluorescence that the present invention constructs plasmid using Fusarium oxysporum fatty acid Ω '-hydroxylase gene (CYP505) segment
The amplification curve diagram of quantitative PCR, wherein it is 35 ng/ μ L, 3.5 ng/ μ L, 0.35 ng/ μ L, 0.035 that 1 to No. 5, which is concentration respectively,
Ng/ μ L, 0.0035ng/ μ L recombinant plasmid pGEM-T-CYP505The real time fluorescent quantitative amplification curve of standard items；
Fig. 4 is the canonical plotting established using plasmid control quality logarithm as ordinate using Ct value as abscissa；
Fig. 5 is glimmering in real time to be carried out with primer of the present invention and detection method to 14 Radix Notoginseng samples and notoginseng planting soil DNA
The amplification curve diagram of Fluorescent Quantitative PCR, wherein 4 test samples for generating Ct value are No. 7, No. 8, No. 9, No. 10, it is Roots of Panax Notoginseng
The root samples of maize ear rot diseased plant.
Specific embodiment
Below by drawings and examples, the present invention is further described, but the scope of the present invention is not limited in described
Hold, method operating according to a conventional method unless otherwise specified in the present embodiment, the use of agents useful for same unless otherwise specified is often
Rule reagent or the reagent configured according to a conventional method.
Embodiment 1: Fusarium oxysporum fatty acid Ω '-hydroxylase gene (CYP505) specificity analysis
Fusarium oxysporum fatty acid Ω hydroxylase (P450foxy, AB030037.1) gene conduct is selected according to early-stage study result
Design the gene loci of specific PCR primers；Upstream and downstream primer sequence is respectively Fo-F (5 '
CATCCAACGCCGCCCACTTTATC3 ') and Fo-R (5 ' CAGCAACCACCAACGCTTCTTCG3 ').Primer sequence entrusts elder brother
The synthesis of Ming Shuoqing Biotechnology Co., Ltd.
Take out several stored refrigerated disease fungus from 4 DEG C of refrigerators, they be respectively ginseng rod method (Alternaria panax), tobacco brown spot pathogen (Alternaria alternata), sclerotinite (Sclerotinia sclerotiorum), eggplant
Rotten sickle-like bacteria (Fusarium solani), Fusarium oxysporum (Fusarium oxysporum), wheel branch sickle-like bacteria (Fusarium verticillioides), Fusarium graminearum (Fusarium graminearum), grape seat chamber bacterium (Botryosphaeria dothidea)；Appropriate mycelia is inoculated in fresh PDA culture medium, is subsequently placed in 28 DEG C of incubators and cultivates；It is scraped after five days
Mycelia, and use CTAB method extracts the genomic DNA of several fungies, the purity extracted using UV spectrophotometer measuring DNA
And mass concentration.
PCR is carried out by template of the genomic DNA of above-mentioned several disease fungus, with the specificity of detection primer；PCR reaction
System (20 μ L) is as follows: 2 × Rapid Taq Master Mix, 10 μ L, aseptic double-distilled water 7 μ L, upstream primer Fo-F (2
μM) 1 μ L, downstream primer Fo-R (2 μM) 1 μ L, 1 μ L of DNA profiling；PCR amplification condition are as follows: 95 DEG C of 3 min； 94℃
30 s, 57 DEG C of 30 s, 72 DEG C of 45 s, 28 circulations；72℃ 5 min；PCR product is detected in 1.2% agarose gel electrophoresis,
As a result as shown in Figure 1, this only produces 400 bp's or so in the PCR amplification using Fusarium oxysporum DNA as template to primer
Amplified production, and be consistent with expected 378 bp of amplified production length.It can tentatively assert that the primer that the present invention designs has inter-species
Specificity only generates special band using this primer in Fusarium oxysporum, and does not cause to have in other reaping hook category fungies
Effect amplification, thus can specifically distinguish Fusarium oxysporum and other reaping hook category pathogens.
PCR product is subjected to T-A clone, is selected pGEM-T easy Vector System (Promega, USA)
It as cloning vector, is attached with pcr amplification product of the present invention, connection product is transferred to bacillus coli DH 5 alpha competent cell
In, positive colony sequencing is then selected, and bioinformatic analysis is carried out to sequencing result；Using BLAST (http: //
Blast.ncbi.nlm.nih.gov/Blast.cgi) the base in the sequence and GenBank that on-line analysis tool obtains sequencing
Because sequence carries out homology analysis, (fatty acid Ω is hydroxylated for extension increasing sequence and Fusarium oxysporum CYP505 gene as the result is shown for analysis
Enzyme) with 98% similitude.It can be seen that the fatty acid Ω hydroxylase for the primer energy specific amplification Fusarium oxysporum that the present invention designs
Genetic fragment.
Embodiment 2: real-time fluorescence quantitative PCR design of primers and specificity analysis
According to the gene order in above-described embodiment, the specific primer of real-time fluorescence quantitative PCR is designed, the upstream and downstream of design is drawn
The nucleotide sequence of object is respectively 5 ' CTCAAACAGTGGTACATGCGAGG3 ' (Fo-QF) and 5 '
CATCTAGGTCTTCCATCCACTTGA3 ' (Fo-QR), and Kunming Shuo Qing Biotechnology Co., Ltd is entrusted to synthesize above-mentioned primer.
Real-time PCR is carried out by template of the genomic DNA of several disease fungus in embodiment 1, according to following
System carries out quantitative fluorescent PCR to detect the specificity of primer in the present invention.Quantitative fluorescent PCR reaction system (20 μ L) includes
7 μ L of nuclease-free water, 2 × SYBR Green Mix, 10 μ L, upstream primer Fo-QF (2 μM) 1 μ L, downstream primer Fo-
QR (2 μM) 1 μ L, 1 μ L of DNA profiling；It is expanded on fluorescence quantitative PCR instrument, amplification spectrum is 95 DEG C of 2 min, is then carried out
45 circular responses, each circular response include 95 DEG C of 1 min, and 62 DEG C of 30 s, 72 DEG C of 1 min measure fluorescence when 72 DEG C
Value, and in each collection automatic after circulation terminates and record fluorescence signal.
3 repetitions of each fungal DNA template-setup, and negative control is set and (template DNA is not added, with nuclease-free water generation
For), for check whether there are template pollute and generate primer dimer, specific amplification curve is as shown in Figure 2 a, only with
Fusarium oxysporum DNA is that effective amplification is produced during the real-time PCR of template reacts, and three repetitions obtain three coincidences
Amplification curve, Ct value is respectively 26.33,26.37,26.48, it is seen that Real-time PCR primer specificity of the invention
Very well, without amplification in other disease fungus and negative control.In addition, melting curve figure (2b) also indicates that primer of the invention
Specificity is good, does not generate primer dimer, does not also form the PCR product of mispairing.
Embodiment 3: the foundation of real-time fluorescence quantitative PCR standard curve
The Escherichia coli for carrying the zhib of Fusarium oxysporum fatty acid Ω '-hydroxylase gene segment in embodiment 1 are coated on solid
On LB plating medium, 37 DEG C of inversion 12 h of overnight growth, then with autoclaved toothpick from one list of picking on LB plate
In the LB liquid medium of bacterium colony access addition ampicillin, then 37 DEG C of 200 12 h of rpm/min shaken cultivation is used
The a small amount of extraction agent boxes of SanPrep pillar Plasmid DNA (the raw work in Shanghai) extract plasmid, and measure plasmid with ultraviolet specrophotometer
Concentration, by the plasmid of extracting be placed in -80 DEG C it is spare.
Using the plasmid for measuring concentration as standard items, 10 times of concentration gradient dilution is carried out, 5 gradients are set altogether, point
Not Wei 35 ng/ μ L, 3.5 ng/ μ L, 0.35 ng/ μ L, 0.035 ng/ μ L, 0.0035ng/ μ L, dilution medium is nuclease free
Water.Real-time fluorescence quantitative PCR reaction is carried out to the standard items of 5 gradients, 3 repetitions are arranged in each gradient.Quantitative fluorescent PCR is anti-
System (20 μ L) is answered, includes 7 μ L, 2 × SYBR Green Mix quantitative fluorescent PCR reaction premixed liquid of nuclease-free water, 10 μ L,
Upstream primer Fo-QF (2 μM) 1 μ L, downstream primer Fo-QR (2 μM) 1 μ L, 1 μ L of DNA profiling, wherein template is 5
The plasmid standard of mass concentration gradient.It is expanded on fluorescence quantitative PCR instrument, amplification spectrum is 95 DEG C of 2 min, then carries out 45
A circular response, each circular response are 95 DEG C of 1 min, and 62 DEG C of 30 s, 72 DEG C of 1 min measure fluorescent value when 72 DEG C,
And in each collection automatic after circulation terminates and record fluorescence signal；
The Real-time PCR amplification curve of plasmid standard is shown in Fig. 3, and obtained Real-time PCR Ct value is shown in Table 1, simultaneously
Using the corresponding plasmid quality logarithm of each gradient concentration as ordinate, Ct value is abscissa, generates standard in Excel software
Curve, obtaining equation of linear regression is y=- 0.2904x+4.3445, R2=0.9983, the standard curve of foundation is shown in Fig. 4, the curve
Slope is that -0.2904(is between -1 ~ 0), related coefficient (R2) it is 0.9983.
The Ct value of 1 Fusarium oxysporum plasmid standard of table
；
In addition, further experiment, as the result is shown when template concentrations are lower than 0.35pg/ μ L, Ct value and template concentrations (log value) are no
There is linear relationship again, show that the minimum template concentrations for the Fusarium oxysporum real-time fluorescence quantitative PCR detection that the present invention establishes are
0.35 pg/μL.It can be seen that the real-time fluorescence quantitative PCR detection method sensitivity that the present invention establishes is very high.
Embodiment 4: the application of notoginseng root rot bacterium Fusarium oxysporum Real-time PCR detection method
The Radix Notoginseng disease in notoginseng planting base is based on root rot and black spot, and in Wenshan Prefecture Radix Notoginseng main producing region, acquisition has root-rot
Disease, the Radix Notoginseng diseased plant of black spot classical symptom and notoginseng planting soil, the Fusarium oxysporum designed with the present invention are special
Real-time PCR primer and its detection method analyze multiple samples.
Collected sample is first numbered, No. 1-2 be healthy Radix Notoginseng leaf sample, No. 3-4 is sick leaf sample,
No. 5-6 root samples for healthy Radix Notoginseng, No. 7-10 is root rot sample, No. 11-12 be root rot diseased plant pedotheque,
No. 13-14 is healthy Panax notoginseng root pedotheque, and No. 15 are no Template-negative controls；Each sample total DNA is extracted with CTAB method,
DNA number is identical as sample number into spectrum, then carries out quantitative fluorescent PCR reaction with the DNA of sample, and amplification reaction system is 20 μ L,
Include 7 μ L of nuclease-free water, 2 × SYBR Green Mix, 10 μ L, upstream primer Fo-QF (2 μM) 1 μ L, downstream primer
Fo-QR (2 μM) 1 μ L, 1 μ L of DNA profiling；Expanded on fluorescence quantitative PCR instrument, amplification spectrum be 95 DEG C of 2 min, then into
45 circular responses of row, each circular response are 95 DEG C of 1 min, and 62 DEG C of 30 s, 72 DEG C of 1 min measure fluorescence when 72 DEG C
Value, and in each collection automatic after circulation terminates and record fluorescence signal.
Obtained amplification curve is shown in Fig. 5, and as can be seen from the figure 7-10 sample has amplification, remaining sample is without amplification.
The Ct value average value of 7-10 sample is respectively 29.53,32.20,33.77,33.22 as can be seen from Table 2, Fusarium oxysporum
Content more than 10 pg/g samples, high reaches 177 pg/g samples, illustrates exist in notoginseng root rot diseased plant sample
Fusarium oxysporum.Since the Fusarium oxysporum quantity carried in soil is very small, so this root rot diseased plant collected
Fusarium oxysporum is not detected in pedotheque.In addition, in healthy Panax notoginseng root, blade, the black spot disease leaf of healthy Radix Notoginseng
And Fusarium oxysporum is not detected in healthy Panax notoginseng root soil.
Above-mentioned experimental result is shown, uses primer of the present invention and real-time fluorescence quantitative PCR amplification detection method, the accurate area of energy
Divide the root rot sample of Radix Notoginseng, pathogen DNA of the lowest detection to 0.35 pg/ μ L, it is seen that the primer and build that the present invention designs
Vertical real time fluorescence quantifying PCR method specificity is good, sensitive, reliable, can achieve specific detection notoginseng root rot bacterium point spore
The purpose of sickle-like bacteria.
The real-time fluorescence quantitative PCR result of 2 14 Radix Notoginseng of table and Soil K+adsorption sample
。
Sequence table
<110>Kunming University of Science and Technology
<120>the Real-time PCR detection primer and detection method of notoginseng root rot pathogenic bacteria Fusarium oxysporum
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 23
<212> DNA
<213>artificial sequence (Artificial)
<400> 1
catccaacgc cgcccacttt atc 23
<210> 2
<211> 23
<212> DNA
<213>artificial sequence (Artificial)
<400> 2
cagcaaccac caacgcttct tcg 23
<210> 3
<211> 23
<212> DNA
<213>artificial sequence (Artificial)
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ctcaaacagt ggtacatgcg agg 23
<210> 4
<211> 24
<212> DNA
<213>artificial sequence (Artificial)
<400> 4
catctaggtc ttccatccac ttga 24
Claims (4)
1. a kind of Real-time PCR detection primer of notoginseng root rot pathogenic bacteria Fusarium oxysporum, which is characterized in that primer
Nucleotide sequence are as follows:
Upstream primer Fo-QF:5 ' CTCAAACAGTGGTACATGCGAGG3 '
Downstream primer Fo-QR:5 ' CATCTAGGTCTTCCATCCACTTGA3 '.
2. the detection method of the notoginseng root rot pathogenic bacteria Fusarium oxysporum using detection primer described in claim 1, feature
It is, comprising the following steps:
(1) DNA of Radix Notoginseng test sample or soil is extracted；
(2) real-time fluorescence quantitative PCR amplification is carried out by template of the DNA of extraction, amplification program is 95 DEG C of 2 min, is then carried out
45 circular responses, each circular response are 95 DEG C of 1min, and 62 DEG C of 30s, 72 DEG C of 1min measure fluorescent value when 72 DEG C, often
It is collected after a circular response and records fluorescence signal；
(3) foundation of real-time fluorescence quantitative PCR standard curve
Prepare recombinant plasmid pGEM-T- of the concentration within the scope of 0.0035ng/ μ L~35ng/ μ LCYP505Solution, to different dense
The solution of degree carries out real-time fluorescence quantitative PCR reaction, and the same step of amplification program (2) measures fluorescent value, each circulation when 72 DEG C
Fluorescence signal is collected and recorded after reaction, using the corresponding plasmid quality logarithm of each gradient concentration as ordinate, Ct value
For abscissa, equation of linear regression is obtained, and establishes standard curve；
(4) determine that there are root rot pathogenic bacteria Fusarium oxysporums in institute's test sample if there is fluorescence signal, and by acquisition
Ct value is brought into the standard curve of Fusarium oxysporum, and the quality of Fusarium oxysporum in test sample is calculated.
3. detection method according to claim 2, it is characterised in that: the reaction system of real-time fluorescence quantitative PCR is 20 μ L,
Comprising 2 × SYBR Green Mix, 10 μ L, 7 μ L of nuclease-free water, 1 μ L of upstream primer Fo-QF, 1 μ L of downstream primer Fo-QR,
1 μ L of DNA profiling.
4. detection method according to claim 3, it is characterised in that: upstream primer Fo-QF's and downstream primer Fo-QR
Concentration is 2 μm of ol/L.
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| CN110241242A (en) * | 2019-06-10 | 2019-09-17 | 沈阳农业大学 | The quantitative detecting method of plasmodiophora brassicae in different type sample |
| CN112521443A (en) * | 2021-01-13 | 2021-03-19 | 昆明理工大学 | Preparation method and application of pseudo-ginseng flower protein |
| CN113981144A (en) * | 2021-11-16 | 2022-01-28 | 昆明理工大学 | Real-time fluorescent quantitative PCR detection method for pseudo-ginseng A virus |
| CN116083630A (en) * | 2022-12-31 | 2023-05-09 | 昆明理工大学 | Primer group for detecting pathogenic bacteria Fusarium oxysporum of root rot of pseudo-ginseng by real-time fluorescent LAMP |
| CN117126750A (en) * | 2023-08-30 | 2023-11-28 | 云南农业大学 | Fusarium oxysporum J2 strain and application thereof in biological prevention and control and growth promotion of pseudo-ginseng |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110241242A (en) * | 2019-06-10 | 2019-09-17 | 沈阳农业大学 | The quantitative detecting method of plasmodiophora brassicae in different type sample |
| CN112521443A (en) * | 2021-01-13 | 2021-03-19 | 昆明理工大学 | Preparation method and application of pseudo-ginseng flower protein |
| CN112521443B (en) * | 2021-01-13 | 2024-03-26 | 昆明理工大学 | Preparation method and application of pseudo-ginseng flower protein |
| CN113981144A (en) * | 2021-11-16 | 2022-01-28 | 昆明理工大学 | Real-time fluorescent quantitative PCR detection method for pseudo-ginseng A virus |
| CN116083630A (en) * | 2022-12-31 | 2023-05-09 | 昆明理工大学 | Primer group for detecting pathogenic bacteria Fusarium oxysporum of root rot of pseudo-ginseng by real-time fluorescent LAMP |
| CN116083630B (en) * | 2022-12-31 | 2024-04-12 | 昆明理工大学 | Primer group for detecting pathogenic bacteria Fusarium oxysporum of root rot of pseudo-ginseng by real-time fluorescent LAMP |
| CN117126750A (en) * | 2023-08-30 | 2023-11-28 | 云南农业大学 | Fusarium oxysporum J2 strain and application thereof in biological prevention and control and growth promotion of pseudo-ginseng |
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