CN103409532B - Specific primer used for analysis of diversity of pathogenic fungi causing root rot of soybean - Google Patents
Specific primer used for analysis of diversity of pathogenic fungi causing root rot of soybean Download PDFInfo
- Publication number
- CN103409532B CN103409532B CN201310353895.2A CN201310353895A CN103409532B CN 103409532 B CN103409532 B CN 103409532B CN 201310353895 A CN201310353895 A CN 201310353895A CN 103409532 B CN103409532 B CN 103409532B
- Authority
- CN
- China
- Prior art keywords
- specific primer
- root rot
- pathogenic fungi
- primer
- diversity
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a specific primer used for analysis of diversity of pathogenic fungi causing root rot of soybean, which relates to a specific primer used for analysis of diversity of pathogenic fungi. The nucleotide sequence of the specific primer is 5'-CGTTGGGTTATTCCCCGGC-3'. The specific primer provided by the invention has the advantages of accurate analysis of diversity, visual, rapid and reliable detection results, etc. and can be applied in the field of microbiological detection.
Description
Technical field
The present invention relates to a kind of Auele Specific Primer for pathogenic fungi diversity analysis.
Background technology
Root rot is a kind of wide, that harm is heavy, control is difficult worldwide disease that distributes.Its initial stage mainly infects soybean cortex, final harm side root, has a strong impact on the absorption of crop to moisture and nutrient.Root rot pathogenic bacteria forms complicated, and the Northeast of China is mainly based on pinch outs (Fusarium Oxysporum), fusarium solani (Fusarium Solani), Fusarium graminearum (Fusarium Graminearum), oat Fusariumsp (Fusarium Avenaceum) and F.semitectum bacterium (Fusarium Semitectum).Although molecular marking technique can be utilized now to identify out all separately by often kind of root rot pathogenic bacteria, but often comprise multiple Pathogens Causing Root Rot Disease in soybean soil, if carry out control just first must carry out diversity analysis, thus know containing several different root rot pathogenic fungi in soybean soil, be which root rot pathogenic fungi respectively.There is no at present and can, for the Auele Specific Primer of root rot pathogenic fungi diversity analysis, bring difficulty therefore to the detection of root rot pathogenic fungi and control.
Summary of the invention
The invention provides a kind of Auele Specific Primer for root rot pathogenic fungi diversity analysis, Auele Specific Primer of the present invention and the coupling of DGGE technology, can detect in the Northeast of China tested soybean soil by means of only two steps and comprise several root rot pathogenic fungi, belong to what bacterial classification respectively.
The nucleotides sequence that the present invention is used for the Auele Specific Primer of root rot pathogenic fungi diversity analysis is classified as 5 '-CGTTGGGTTATTCCCCGGC-3 '.
The present invention is used for the Auele Specific Primer of root rot pathogenic fungi diversity analysis with yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) for model animals, S.cerevisiae 18s rRNA (S.cerevisiae 18s rRNA NCBI accession number J01353) is selected to be template, design primer Auele Specific Primer of the present invention according to S.cerevisiae 18s rRNA 359bp ~ 377bp, and the base being positioned at 376bp and 377bp position is modified.
Auele Specific Primer of the present invention may be used for pinch outs (F.Oxysporum), fusarium solani (F.Solani), Fusarium graminearum (F.Graminearum), oat Fusariumsp (F.Avenaceum), F.semitectum bacterium (F.Semitectum), wheel branch Fusariumsp (F.verticillioides), Fusarium solani (F.solani), blue Fusariumsp (F.coeruleum), scouring rush's Fusariumsp (F.equiseti), soyabean phytophthora (Phytophthora spp.) and dry thread Pyrenomycetes (Rhizoctonia solani) are in the diversity analysis of interior multiple root rot pathogenic fungi and detection.
The result of use of Auele Specific Primer of the present invention has diversity analysis accurately, the advantages such as detected result is directly perceived, quick, reliable.
Accompanying drawing explanation
Fig. 1 be in embodiment two proof test with Auele Specific Primer F.rr and universal primer NS1 for primer pair 12 groups of samples carry out the gel electrophoresis figure of PCR, in Fig. 1, " M " swimming lane standard specimen is Marker, " 1 " swimming lane standard specimen is pinch outs (F.Oxysporum) DNA, " 2 " swimming lane standard specimen is fusarium solani (F.Solani) DNA, " 3 " swimming lane standard specimen is Fusarium graminearum (F.Graminearum) DNA, " 4 " swimming lane standard specimen is oat Fusariumsp (F.Avenaceum) DNA, " 5 " swimming lane standard specimen is F.semitectum bacterium (F.Semitectum) DNA, " 6 " swimming lane standard specimen is wheel branch Fusariumsp (F.verticillioides) DNA, " 7 " swimming lane standard specimen is Fusarium solani (F.solani) DNA, " 8 " swimming lane standard specimen is blue Fusariumsp (F.coeruleum) DNA, " 9 " swimming lane standard specimen is scouring rush's Fusariumsp (F.equiseti) DNA, " 10 " swimming lane standard specimen is soyabean phytophthora (Phytophthora spp.) DNA, " 11 " swimming lane standard specimen is dry thread Pyrenomycetes (Rhizoctonia solani) DNA, " 12 " swimming lane standard specimen is above-mentioned 1 ~ No. 11 swimming lane hybrid dna.
Fig. 2 be in embodiment two proof test with primer YUAN1 and universal primer NS1 for primer pair 12 groups of samples carry out the gel electrophoresis figure of PCR, in Fig. 2, " M " swimming lane standard specimen is Marker, and the standard specimen in 1 ~ No. 12 swimming lane is consistent with Fig. 1.
Fig. 3 be in embodiment two interference test with Auele Specific Primer F.rr and universal primer NS1 for primer pair alternaric bacteria (Alternaria) carries out the gel electrophoresis figure of PCR, in Fig. 3, " M " swimming lane standard specimen is Marker, and " 1 " swimming lane standard specimen is alternaric bacteria (Alternaria) DNA.
Fig. 4 is the gel electrophoresis figure carrying out DGGE with the Auele Specific Primer F.rr of 5 ' end interpolation " GC-clamp ", in Fig. 4, " A1 " swimming lane standard specimen is the PCR primer of primers F .rr and NS1 amplification the 1st swimming lane in embodiment two proof test, and in Fig. 4, " A2 " swimming lane standard specimen is the PCR primer of primers F .rr and NS1 amplification the 12nd swimming lane in embodiment two proof test.
Embodiment
Technical solution of the present invention is not limited to following cited embodiment, also comprises the arbitrary combination between each embodiment.
Embodiment one: the nucleotides sequence that present embodiment is used for the Auele Specific Primer of root rot pathogenic fungi diversity analysis is classified as 5 '-CGTTGGGTTATTCCCCGGC-3 '.
By present embodiment Auele Specific Primer called after F.rr.
The nucleotides sequence of the unmodified base obtained according to S.cerevisiae 18s rRNA 359bp ~ 377bp is classified as 5 '-CGAACCCTTATTCCCCGTT-3 '; By its called after YUAN1.
Embodiment two: in present embodiment with the Auele Specific Primer F.rr of embodiment one and contrast primer YUAN1 for doing reverse primer, match use with the NS1 (5 '-GTAGTCATATGCTTGTCTC-3 ') of universal primer centering as forward primer respectively and test.
Proof test: respectively with pinch outs (F.Oxysporum), fusarium solani (F.Solani), Fusarium graminearum (F.Graminearum), oat Fusariumsp (F.Avenaceum), F.semitectum bacterium (F.Semitectum), wheel branch Fusariumsp (F.verticillioides), Fusarium solani (F.solani), blue Fusariumsp (F.coeruleum), scouring rush's Fusariumsp (F.equiseti), the biased sample of soyabean phytophthora (Phytophthora spp.) and dry thread Pyrenomycetes (Rhizoctonia solani) 11 kinds of root rot pathogenic fungi DNA sample and above-mentioned 11 kinds of root rot pathogenic fungi DNA is that template carries out proof test.
Interference test: alternaric bacteria (Alternaria) is a kind of common disease fungi of the Northeast, although be not root rot pathogenic fungi, but very close with the 18s rRNA affinity of root rot pathogenic fungi, usually interference detection results in testing process in the past.
One, PCR
PCR amplification system and reaction conditions as follows: amplification reaction system is 20 μ L, and 2.0 μ L DNA profilings, 1.6 μ L concentration are that the dNTP of 2.5mmol/L, 2.0 μ L 10 × damping fluids are (containing Mg
2+), 2.0 μ L concentration are the primer of 1.0 μm of ol/L, 0.2 μ L concentration is the Taq enzyme (archaeal dna polymerase) of 5U/ μ L and the double distilled water composition of surplus; Amplification reaction condition is denaturation 94 DEG C of 3min, and sex change 94 DEG C of 30sec, annealing 57 DEG C of 1min, extension 72 DEG C of 1min, totally 35 circulations, extend 72 DEG C of 10min.
The PCR result being primer pair root rot pathogenic fungi with Auele Specific Primer F.rr and universal primer NS1 as shown in Figure 1.Can find out, the Auele Specific Primer F.rr of embodiment one, to all root rot pathogenic fungies, is independent or biased sample all has excellent specificity and accuracy, and the band length amplified is all 430bp.
The PCR result being primer pair root rot pathogenic fungi with primer YUAN1 and universal primer NS1 as shown in Figure 2.Can find out, the natural unmodified primer YUAN1 obtained according to S.cerevisiae 18s rRNA 359bp ~ 377bp only can amplify the band of part root rot pathogenic fungi, its poor specificity.
With Auele Specific Primer F.rr and universal primer NS1 for primer pair alternaric bacteria (Alternaria) carries out the result of PCR as shown in Figure 3.Primers F .rr does not amplify false positive band, proves that primers F .rr has excellent specificity.
Two, DGGE
Add " GC-clamp " at the 5 ' end of Auele Specific Primer F.rr, the sequence of GC folder is: 5 '-CGCCCGCCGCGCGCGGCGGGCGGGGCGGGGGCCCGGGGG-3'.With the PCR primer of pcr amplification the 1st swimming lane and the 12nd swimming lane for sample carries out DGGE.
PCR primer applied sample amount 5 μ l, polyacrylamide gel used (acrylamide: bisacrylamide=37.15:1, v/v) concentration is 8%, and denaturing agent gradient scope is 40% ~ 65% (containing 7mon/L urea and 40% deionized formamide in the denaturing agent of 100%).Electrophoresis run conditions is: in 1 × TAE damping fluid, 60 DEG C, run 540min under 130V.Silver stain, Umax scanner scanning gel.
DGGE result as shown in Figure 4.Can be clearly seen that 11 clear, bright bands in Fig. 4 swimming lane A2, equal with the quantity of DNA in primary sample.After being connected with carrier T respectively by the DNA of the band amplified in A2 swimming lane, transformation of E. coli DH5 α competent cell checks order, sequencing result is: sequence length is all 430bp, and analyze and 11 kinds of root rot pathogenic fungi gene order one_to_one corresponding in original mixed sample through blast, and the root rot pathogenic fungi in A2 swimming lane corresponding to each band can be determined according to sequencing result.
Using the experimental result of present embodiment acquisition as reference, kind quantity and the kind of root rot pathogenic fungi in detected soybean soil (the Northeast of China) just can be known like a cork.
Auele Specific Primer F.rr of the present invention has identical accuracy for sample that is independent or mixing, and reliable results, stable.
Claims (1)
1., for the Auele Specific Primer of root rot pathogenic fungi diversity analysis, it is characterized in that the nucleotides sequence of reverse primer in the Auele Specific Primer for root rot pathogenic fungi diversity analysis is classified as 5 '-CGTTGGGTTATTCCCCGGC-3 '; The nucleotides sequence of forward primer is classified as 5 '-GTAGTCATATGCTTGTCTC-3 '.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310353895.2A CN103409532B (en) | 2013-08-14 | 2013-08-14 | Specific primer used for analysis of diversity of pathogenic fungi causing root rot of soybean |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310353895.2A CN103409532B (en) | 2013-08-14 | 2013-08-14 | Specific primer used for analysis of diversity of pathogenic fungi causing root rot of soybean |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103409532A CN103409532A (en) | 2013-11-27 |
| CN103409532B true CN103409532B (en) | 2015-01-07 |
Family
ID=49602573
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310353895.2A Active CN103409532B (en) | 2013-08-14 | 2013-08-14 | Specific primer used for analysis of diversity of pathogenic fungi causing root rot of soybean |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103409532B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104498589B (en) * | 2014-08-29 | 2016-06-29 | 兰州大学 | A pair specific molecular detection primer of prairie milk vetch yellow dwarf's root rot and detection method |
| CN106755416B (en) * | 2016-12-23 | 2018-10-30 | 四川农业大学 | Specific primer group, kit and its application for analyzing soybean fusarium root-rot fungal diversity |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103014171A (en) * | 2013-01-07 | 2013-04-03 | 福州大学 | Method for analyzing fungus microbial community structure by utilizing specific rDNA long fragment |
-
2013
- 2013-08-14 CN CN201310353895.2A patent/CN103409532B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN103409532A (en) | 2013-11-27 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103409546B (en) | Kit for detecting streptococcus suis type 2 and application of kit | |
| CN102417929B (en) | Specific PCR detection method of Riemerella anatipestifer | |
| CN104087654A (en) | Multiple PCR identification kit of salmonella and five serotypes of salmonella | |
| CN104059975B (en) | To Providence O3, the Nucleotide that O4, O8, O12, O13 and O20 are special and application thereof | |
| CN103898108A (en) | Nucleotide specific to Vibrio fluvialis O2, O4, O13, O15 and O18 and application thereof | |
| Foongladda et al. | Multi-probe real-time PCR identification of four common Candida species in blood culture broth | |
| CN110117671A (en) | Riemerellosis Anatipestifer Specific PCR primers are to, PCR detection method and kit | |
| CN102304559A (en) | Fluorescence quantitative polymerase chain reaction (PCR) method for detecting bacillus coagulans quickly | |
| CN103409532B (en) | Specific primer used for analysis of diversity of pathogenic fungi causing root rot of soybean | |
| CN102864226A (en) | LAMP (loop-mediated isothermal amplification) kit for rapid detection of staphylococcus aureus | |
| CN103103258A (en) | Method for analyzing generating trend of aflatoxin B1 in peanut meal by multiple PCR (Polymerase Chain Reaction) technology | |
| CN110734993A (en) | nucleic acid detection test paper strip for distinguishing Brucella vaccine strain S19 from naturally infected strain | |
| CN102465173A (en) | Specific PCR detection method of Ralstonia solanacearum race 2 | |
| Semighini et al. | Molecular identification of Paracoccidioides brasiliensis by 5′ nuclease assay | |
| CN104611439B (en) | A kind of detect the method for feature fungus Amorphotheca resinae in jet fuel | |
| CN103184283A (en) | PCR enzyme digestion type kit for detecting legionella bacteria and application of kit | |
| CN104293903B (en) | For detecting primer combination, test kit and the detection method of dog babesia | |
| CN101368203A (en) | Primer, reagent kit and detection method for monotonic increasing Listeria hymenial veil mediated isothermality amplification technique fast detection | |
| CN102851383A (en) | LAMP kit for rapid detection of Salmonella | |
| CN104313169A (en) | Triple PCR detection method for rapidly distinguishing non-LP (legionella pneumophila), LP and LP I | |
| CN103993090A (en) | Specific nucleotides for providencia O31, O41, O42, O43 and O50 and application of specific nucleotides | |
| CN105177138A (en) | Primer and method used for detecting Oidium heveae and application | |
| CN103215358A (en) | Diarrheogenic escherichia coli detection kit and detection and typing method thereof | |
| CN111088377B (en) | Rapid constant temperature detection method for staphylococcus aureus, primer set and application | |
| CN115029460A (en) | Instant visual detection method for haemophilus parasuis |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20200915 Address after: 151600 Light Industry Park, Qinggang Economic Development Zone, Suihua City, Heilongjiang Province Patentee after: Heilongjiang hejiafu Agricultural Technology Co., Ltd Address before: 150080 Harbin, Heilongjiang, Nangang District Road, No. 74 Patentee before: Heilongjiang University |
|
| TR01 | Transfer of patent right |