CN109504799A - A kind of the primer combination and method of Rapid identification sickle-like bacteria strain - Google Patents
A kind of the primer combination and method of Rapid identification sickle-like bacteria strain Download PDFInfo
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Abstract
The invention discloses a kind of combinations of the primer of Rapid identification sickle-like bacteria strain, including being selected from least one of the first primer group, the second primer sets, and/or it is selected from least one of third primer sets, the 4th primer sets, wherein the first primer group sequence shown in SEQ ID NO.1 and SEQ ID NO.2 forms, second primer sets sequence shown in SEQ ID NO.1 and SEQ ID NO.3 forms, third primer sets sequence shown in SEQ ID NO.4 and SEQ ID NO.5 forms, and the 4th primer sets sequence shown in SEQ ID NO.4 and SEQ ID NO.6 forms.Primer combination of the invention is reproducible, and specificity is high.The invention also discloses the methods using above-mentioned primer combination identification sickle-like bacteria strain, and accuracy is high, easy to operate, the Rapid identification suitable for sickle-like bacteria strain.
Description
Technical field
The invention belongs to molecular biology experiment technical fields, and in particular, to a kind of Rapid identification sickle-like bacteria strain
Primer combination and method.
Background technique
Sickle-like bacteria (Fusarium spp.) belongs to Deuteromycotina in asexual period, and sexual period belongs to Ascomycotina.
At present, it has been found that sickle-like bacteria be no less than 44 kinds.Fusarium in and parasitic or saprogenesis, it is widely distributed in the whole world, can
It is grown on animals and plants organism, the root of plant, stem can be caused and the rotting for tissues such as spends, seriously affected grain, industrial crops
And the yield and quality of medicinal plant, harmfulness with higher.
Fusarium is a biggish category in fungi, is filamentous fungi, distributes widely in nature.Sickle-like bacteria can be with
Various plants, such as cereal crops, oil crops, industrial crops are infected, serious production loss is caused.Sickle-like bacteria and its cause
Disease prevention and control be always research hotspot both domestic and external.With the variation of global climate and the change of cropping system, frequent state
Border trade activity, suitable temperature and humidity, extensive route of transmission and advantageous host etc. make sickle-like bacteria promptly trans-regional biography
It broadcasts, significant changes have occurred in the population structure of sickle-like bacteria in the world.Sickle-like bacteria Identification of Species is to carry out its two packing spaces
The basis of the work such as Journal of Sex Research, disease prevention and control and plant inspection quarantine, exploitation Identification of The Genus Fusarium new method are current urgent are essential
It asks.
Since sickle-like bacteria type is more, distribution is wide, and traditional morphological classification method is difficult accurately to identify it, utilizes
The technology of molecular biology identifies sickle-like bacteria that not only speed is fast, but also qualification result is accurate from molecular level, is sickle
The sort research of knife bacterium provides effective ways.Variety classes sickle-like bacteria translation elongation factor gene (translation
Elongation factor 1 α, TEF) conservative with height, TEF gene sequencing compares analysis can precise Identification reaping hook
Special sickle-like bacteria TEF geneseq database has had been established in bacterium at present.There is researcher to be based on trichoderma reesei in the past
The TEF gene order of (Trichoderma reesei) and Histoplasma capsulatum (Histoplasma capsulatum)
(GenBank searching number is respectively Z23012 and U14100) devises the degenerate primer pair for expanding filamentous fungi TEF gene:
EF-1 (5'-ATGGGTAAGGA (A/G) GACAAGAC-3') and EF-2 (5'-GGA (G/A) GTACCAGT (G/C) ATCATGTT-
3')(O'Donnell K.,Kistler H.C.,Cigelnik E.,Ploetz R.Multiple evolutionary
origins of the fungus causing Panama disease of banana:concordant evidence
From nuclear and mitochondrial gene genealogies.PNAS, 1998,95:2044-2049, herein entirely
Text is incorporated herein by reference), which plays the role of good in most of filamentous fungi identifications.But utilize primer pair
Often occur amplification failure when EF-1/2 carries out sickle-like bacteria genome amplification or have the case where miscellaneous band, illustrates the use of the primer pair
It, may be not applicable to certain strains with certain limitation.It is worth noting that, primer pair EF-1/2 is according to trichoderma reesei
The design of the gene order of (Trichoderma reesei) and Histoplasma capsulatum (Histoplasma capsulatum) is opened
Hair, the position of design of primers may be not conservative in sickle-like bacteria genome.
Summary of the invention
In order to solve the above-mentioned technical problem, the TEF gene order of 10 kinds of sickle-like bacteria is compared in inventor, as a result
It was found that, it has unexpectedly been found that, the 15th bit base C of primer EF-1 is G in most sickle-like bacteria genomes, this illustrates primer EF-1 not
Suitable for most sickle-like bacteria.Therefore, the present invention finds conservative sequence by comparing analysis to sickle-like bacteria TEF gene order not of the same race
Then column, the primer pair for devising high specific amplification sickle-like bacteria TEF gene are counted by the way that amplified fragments are sequenced
It compares and analyzes according to library, thereby completing the present invention to the purpose of sickle-like bacteria precise Identification.
For this purpose, one aspect of the present invention provides a kind of primer combination of Rapid identification sickle-like bacteria strain, including it is selected from first
At least one of primer sets, second primer sets, and/or at least one of third primer sets, the 4th primer sets are selected from,
In,
The first primer group includes having the upstream primer TEF-1F of nucleotide sequence shown in SEQ ID NO.1 and with SEQ
The downstream primer TEF-2R of nucleotide sequence shown in ID NO.2；
Second primer sets include having the upstream primer TEF-1F of nucleotide sequence shown in SEQ ID NO.1 and with SEQ
The downstream primer TEF-3R of nucleotide sequence shown in ID NO.3；
Third primer sets include having the upstream primer EF-LF1 of nucleotide sequence shown in SEQ ID NO.4 and with SEQ
The downstream primer EF-LR1 of nucleotide sequence shown in ID NO.5；
4th primer sets include having the upstream primer EF-LF1 of nucleotide sequence shown in SEQ ID NO.4 and with SEQ
The downstream primer EF-LR2 of nucleotide sequence shown in ID NO.6.
In some embodiments of the invention, the primer combination includes in the first primer group, the second primer sets
At least one, or it is selected from least one of third primer sets, the 4th primer sets.
In some specific embodiments of the invention, the primer combination includes the first primer group.Of the invention another
In some specific embodiments, the primer combination includes the second primer sets.In other specific embodiment of the invention,
The primer combination includes third primer sets.In other specific embodiment of the invention, primer combination includes the
Four primer sets.
In some specific embodiments of the invention, the primer combination includes the first primer group and the second primer sets.
In other specific embodiments of the invention, the primer combination includes third primer sets and the 4th primer sets.
In some embodiments of the present invention, the primer combination includes in the first primer group, the second primer sets
At least one, and be selected from least one of third primer sets, the 4th primer sets.
In some specific embodiments of the invention, the primer combination includes the first primer group and third primer sets.
In other specific embodiments of the invention, the primer combination includes the first primer group and the 4th primer sets.In this hair
In bright other specific embodiment, primer combination includes the second primer sets and third primer sets, it is of the invention again
In some specific embodiments, the primer combination includes the second primer sets and the 4th primer sets.
In some specific embodiments of the invention, primer combination include the first primer group, the second primer sets and
Third primer sets.In other specific embodiments of the invention, the primer combination includes the first primer group, the second primer
Group and the 4th primer sets.
In some specific embodiments of the invention, primer combination include the first primer group, third primer sets and
4th primer sets.In other specific embodiments of the invention, the primer sets include the second primer sets, third primer sets
With the 4th primer sets.
In some specific embodiments of the invention, the primer sets include the first primer group, the second primer sets, third
Primer sets and the 4th primer sets.
In embodiments of the invention, if in primer combination simultaneously including the first primer group and the second primer sets, or
Simultaneously include third primer sets and the 4th primer sets, then wherein share upstream primer content or concentration be downstream primer at least
2 times.
The second aspect of the present invention provides a kind of method of Rapid identification sickle-like bacteria strain, comprising the following steps:
Obtain the genomic DNA of sickle-like bacteria to be identified；
Using the DNA as template, PCR amplification is carried out using the primer combination of first aspect present invention；
Amplified production is subjected to detected through gel electrophoresis, and target PCR product is sequenced；
Sequencing result is compared by BLAST,
If the sequence of sequencing result and the similitude of reference sickle-like bacteria are not less than 98%, the sickle-like bacteria to be identified and ginseng
Fusarium is examined in same.Wherein, similitude is not less than 98% such as 98%, 99%, 100%.
In some embodiments of the invention, the primer combination includes in the first primer group, the second primer sets
At least one, or it is selected from least one of third primer sets, the 4th primer sets.
In some specific embodiments of the invention, the primer combination includes the first primer group.Of the invention another
In some specific embodiments, the primer combination includes the second primer sets.In other specific embodiment of the invention,
The primer combination includes third primer sets.In other specific embodiment of the invention, primer combination includes the
Four primer sets.
In some specific embodiments of the invention, the primer combination includes the first primer group and the second primer sets.
In other specific embodiments of the invention, the primer combination includes third primer sets and the 4th primer sets.
In some embodiments of the present invention, the primer combination includes in the first primer group, the second primer sets
At least one, and be selected from least one of third primer sets, the 4th primer sets.
In some specific embodiments of the invention, the primer combination includes the first primer group and third primer sets.
In other specific embodiments of the invention, the primer combination includes the first primer group and the 4th primer sets.In this hair
In bright other specific embodiment, primer combination includes the second primer sets and third primer sets, it is of the invention again
In some specific embodiments, the primer combination includes the second primer sets and the 4th primer sets.
In some specific embodiments of the invention, primer combination include the first primer group, the second primer sets and
Third primer sets.In other specific embodiments of the invention, the primer combination includes the first primer group, the second primer
Group and the 4th primer sets.
In some specific embodiments of the invention, primer combination include the first primer group, third primer sets and
4th primer sets.In other specific embodiments of the invention, the primer sets include the second primer sets, third primer sets
With the 4th primer sets.
In some specific embodiments of the invention, the primer sets include the first primer group, the second primer sets, third
Primer sets and the 4th primer sets.
In embodiments of the invention, if in primer combination simultaneously including the first primer group and the second primer sets, or
Simultaneously include third primer sets and the 4th primer sets, then shares the content of upstream primer in PCR reaction system or concentration is downstream
2 times of primer.
In embodiments of the invention, if the primer sets more than one for including in primer combination, corresponding to expand
Segment is more than one.So, if at least one sequence in sequencing result is not less than with the similitude with reference to sickle-like bacteria
98%, then the sickle-like bacteria to be identified is with reference Fusarium in same.
In embodiments of the invention, if the primer sets more than one for including in primer combination, corresponding to expand
Segment is more than one.So, if at least one sequence in sequencing result is not less than 98% to reference to the similar of sickle-like bacteria,
Then the sickle-like bacteria to be identified is with reference Fusarium in same.
In embodiments of the invention, if the primer sets more than one for including in primer combination, corresponding to expand
Segment is more than one.So, if all sequences in sequencing result are more than 98% with the similitude of reference sickle-like bacteria,
Sickle-like bacteria to be identified is with reference Fusarium in same.
In the present invention, it is described with reference to sickle-like bacteria be the known reaping hook category sickle-like bacteria for being accredited kind.
In some specific truth schemes of invention, the wherein system of PCR amplification are as follows: 50 μ L of total volume: contain Mg2+5 ×
PCRBuffer10 μ L, 10mmol/L dNTPs 4 μ L, 10 μm of ol/L upstream primers 1 μ L, 10 μm of 1 μ L, 5U/ μ of ol/L downstream primer
1 μ L of L KD enzyme, DNA profiling 50ng, ddH2O are mended to 50 μ L.
In some specific truth schemes of invention, the wherein program of PCR amplification are as follows: 94 DEG C of initial denaturation 2min；94 DEG C of changes
Property 30s, 60 DEG C of renaturation 40s, 72 DEG C of extension 90s, 35 circulation；72 DEG C of extension 5min.
Beneficial effects of the present invention
Primer combination of the invention is reproducible, and specificity is high.
Method accuracy of the invention is high, easy to operate, the Rapid identification suitable for sickle-like bacteria strain.
Quickly and accurately reaping hook bacteria strain can be identified using the present invention, thus it is anti-to implement field specific aim to it
Control, carry out biodiversity research and plant inspection quarantine etc. be of great significance.
Detailed description of the invention
Fig. 1 shows primer TEF-1F and TEF-2R to the amplified production electrophoretogram of 14 parts of sickle-like bacteria DNA；
Fig. 2 shows primer TEF-1F and TEF-3R to the amplified production electrophoretogram of 14 parts of sickle-like bacteria DNA；
Fig. 3 shows primer EF-LF1 and EF-LR1 to the amplified production electrophoretogram of 14 parts of sickle-like bacteria DNA；
Fig. 4 shows primer EF-LF1 and EF-LR2 to the amplified production electrophoretogram of 14 parts of sickle-like bacteria DNA.
Specific embodiment
In order to which the technical problems, technical solutions and beneficial effects solved by the present invention is more clearly understood, below in conjunction with
Embodiment, the present invention will be described in further detail.
Embodiment
Following example is used herein to demonstration the preferred embodiments of the invention.Those skilled in the art, it will be appreciated that under
State the technology disclosed in example represent inventor discovery can be used for implementing technology of the invention, therefore can be considered as implementation this
The preferred embodiment of invention.But those skilled in the art should be understood that specific reality disclosed herein according to this specification
Many modifications can be made by applying example, still can be obtained identical or similar as a result, rather than away from the spirit or scope of the present invention.
Unless otherwise defined, the term of all technologies as used herein and science, and the technology in fields of the present invention
Personnel institute is normally understood equivalent in meaning, and being disclosed reference and their materials of reference will all be incorporated.
Those skilled in the art will recognize or just will appreciate that by routine test many described here
Invention specific embodiment many equivalent technologies.These will equally be comprised in claims.
Embodiment 1
1 material and instrument
Experimental material is 14 parts of sickle-like bacteria samples to be identified (specifying information is shown in Table 1), and wherein bacterial strain PH-1 is to have completed
Gene order-checking (Genome Accession AACM02000000) (Cuomo et al., The Fusarium graminearum
genome reveals a link between localized polymorphism and pathogen
Specialization.Science, 2007,317:1400-1402) positive control, belongs to Fusarium
graminearum.Experimental material is provided by Academy of Agricultural Sciences, Shanghai City.Laboratory apparatus mainly have water-bath, supercentrifuge,
NanoDrop 2000, PCR instrument, agarose gel electrophoresis instrument, ultraviolet gel imager.PCR product nucleic acid sequence is sequenced by Shanghai
Sheng Gong biotech firm completes.
The type of 1 sickle-like bacteria of table and source
| Number | Affiliated kind of Morphological Identification | Molecular Identification result | Separate source |
| 1 | Fusarium graminearum(PH-1) | Fusarium graminearum | The U.S. |
| 2 | Fusarium avenaceum | Fusarium avenaceum | Shanghai |
| 3 | Fusarium lateritium | Fusarium lateritium | Shanghai |
| 4 | Fusarium oxysporum | Fusarium oxysporum | Shandong |
| 5 | Fusarium proliferatum | Fusarium proliferatum | Shandong |
| 6 | Fusarium sacchari | Fusarium sacchari | Shandong |
| 7 | Fusarium solani | Fusarium solani | Shanghai |
| 8 | Fusarium equiseti | Fusarium equiseti | Shanghai |
| 9 | Fusarium asiaticum | Fusarium asiaticum | Hubei |
| 10 | Fusarium kyushuense | Fusarium kyushuense | Anhui |
| 11 | Fusarium acuminatum | Fusarium acuminatum | Shaanxi |
| 12 | Fusarium fujikuroi | Fusarium fujikuroi | Henan |
| 13 | Fusarium verticillioides | Fusarium verticillioides | Hubei |
| 14 | Fusarium concentricum | Fusarium concentricum | Hubei |
The culture of 2 sickle-like bacteria
14 parts of reaping hook bacteria strains (table 1) are inoculated into PDA culture medium, 26 DEG C are cultivated three days, sickle-like bacteria hyphae length is observed,
Subsequent experimental can be carried out after hyphae length meets extracting genome DNA amount.
The rapidly extracting of 3 sickle-like bacteria genomic DNAs
Sickle-like bacteria genomic DNA is extracted using the genome DNA rapid extraction method of inventor's invention, detailed step is such as
Under:
Solution A: 50mM Tris-HCl, 50mM EDTA, 10%SDS, 2M NaCl, N- sodium lauroyl sarcosine 3g/
100mL；
Solution B: 3M NaAC, PH 5.2；
Solution C: isopropanol；
Solution D: 75% ethyl alcohol；
Solution E: the ddH containing 50 μ g/mL RNA enzyme2O。
(1) divide the mycelia for taking 20~50mg sickle-like bacteria, be added into 1.5mL centrifuge tube,
(2) solution A of 400 μ L is added；
(3) the heating water bath 10min at 94 DEG C, is mixed by inversion once every 3min；
(4) after being cooled to room temperature, 12000rpm is centrifuged 1min, takes 350 μ L of supernatant into new centrifuge tube；
(5) solution B of 35 μ L is added, is mixed by inversion, 350 μ L solution Cs are added, is mixed by inversion, is placed at room temperature for 2min；
(6) 750 μ L mixed liquors are fully transferred in adsorption column, 9000rpm is centrifuged 30s, outwells the waste liquid in collecting pipe；
(7) 650 μ L solution Ds are added in adsorption column, 9000rpm is centrifuged 30s, outwells the waste liquid in collecting pipe, 9000rpm
It is centrifuged 30s again, adsorption column is transferred in new centrifuge tube, opens adsorption column lid, is placed at room temperature for 2min；
(8) solution E of 50 DEG C of 30 μ L preheatings is added into adsorption column, after standing 3min, 12000rpm is centrifuged 1min, obtains
Obtain fungal genomic DNA, -20 DEG C of preservations.
The pcr amplification reaction of 4 specific primers
14 parts of sickle-like bacteria genomic DNAs that the above method extracts are diluted to 100ng/ μ L as template, with TEF-1F with
TEF-2R, TEF-1F and TEF-3R, EF-LF1 and EF-LR1, EF-LF1 and EF-LR2 tetra- are to primer respectively to 14 parts of sickle-like bacteria
DNA carries out PCR amplification.
Wherein, the sequence (5'-3') of each primer is as follows:
TEF-1F:CTTAACGTCGTCGTCATCG (SEQ ID NO.1)
TEF-2R:CACTTGGTGGTGTCCATCTT (SEQ ID NO.2)
TEF-3R:TTGTAGCCGACCTTCTTGAT (SEQ ID NO.3)
EF-LF1:CCTTAACGTCGTCGTCATCG (SEQ ID NO.4)
EF-LR1:ACGGACTTGACTTCAGTGGT (SEQ ID NO.5)
EF-LR2:GAGCTGCTCGTGGTGCATCT (SEQ ID NO.6)
With TEF-1F, TEF-2R and TEF-1F, TEF-3R two carries out PCR to primer pair sickle-like bacteria genomic DNA, can get
About 860bp amplified fragments；With EF-LF1, EF-LR1 and EF-LF1, EF-LR2 two expands primer, can get about
1300bp amplified fragments.
PCR system and response procedures are as follows:
PCR amplification system (50 μ L of total volume): 5 × PCR Buffer (contains Mg2+) 10 μ L, 10mmol/L dNTPs, 4 μ L,
10 μm of ol/L upstream primers 1 μ L, 10 μm of 1 μ L, 5U/ μ L KD enzymes of ol/L downstream primer 1 μ L, DNA profiling (50ng/ μ L) 1 μ L,
ddH2O 32μL.Replace DNA profiling as negative control using water when PCR amplification.
PCR response procedures are as follows: 94 DEG C of initial denaturation 2min；(94 DEG C of denaturation 30s, 60 DEG C of renaturation 40s, 72 DEG C are prolonged 35 circulations
Stretch 90s)；Last 72 DEG C of extensions 5min.
The Ago-Gel of 5PCR product detects
1.2g agarose is accurately weighed in conical flask, 100mL TAE buffer is added and shakes up, is placed in micro-wave oven and heats
All melt to agarose, GelStain nucleic acid dye liquor is added, after shaking up, pours into gel slab, is placed at room temperature for 20min to solidifying
Gu.3 μ L pcr amplification product loadings are taken, 150V constant pressure electrophoresis 30min is seen whether under gel imager as single bright item
Band.
The sequencing of 6 target fragments
The single and bright PCR product of band is sent to sequencing company and is sequenced, after obtaining base sequence, in NCBI
GenBank(https://www.ncbi.nlm.nih.gov/)、Fusarium-ID(http://www.fusariumdb.org/
Index.php) and Fusarium MLST database (http://www.westerdijkinstitute.nl/ fusarium/) website progress BLAST base sequence comparison, three database comparison result similitudes of being subject to are highest, thus
Determine the strain of the sickle-like bacteria.
By sequencing, after bacterial strain 1 (Fusarium graminearum PH-1) is using primer TEF-1F and TEF-2R amplification
Sequencing result it is following (SEQ ID NO.7):
CTTAACGTCGTCGTCATCGGCCACGTCGACTCTGGCAAGTCGACCACTGTGAGTACCACCGCATCCCAA
CCCCGCCGACACTTGGCGGGGTAGTTTCAAATTTCCAATGTGCTGACATACTTTGATAGACCGGTCACTTGATCTAC
CAGTGCGGTGGTATCGACAAGCGAACCATCGAGAAGTTCGAGAAGGTTGGTCTCATTTTCCTCGATCGCGCGCCCTT
TCCCTTTCGAAATATCATTCGAATCGCCCTCACACGACGACTCGATACGCGCCTGTTACCCCGCTCGAGGTCAAAAA
TTTTGCGGCTTTGTCGTAATTTTTTTCCCGGTGGGGCTCATACCCCGCCACTCGAGCGACAGGCGTCTGCCCTCTTC
CCACAAACCATTCCCTGGGCGCTCATCATCACGTGTCAACCAGTCACTAACCACCTGTCAATAGGAAGCCGCCGAGC
TCGGTAAGGGTTCCTTCAAGTACGCCTGGGTTCTTGACAAGCTCAAAGCCGAGCGTGAGCGTGGTATCACCATTGAT
ATCGCCCTCTGGAAGTTCGAGACTCCTCGCTACTATGTCACCGTCATTGGTATGTTGTCACCACTGCTGTCATCACA
TTCTCATACTAACATGGCTATCAGACGCTCCCGGTCACCGTGATTTCATCAAGAACATGATCACTGGTACTTCCCAG
GCCGATTGCGCCATTCTCATCATTGCCGCCGGTACTGGTGAGTTCGAGGCTGGTATCTCCAAGGATGGCCAGACCCG
TGAGCACGCTCTCCTTGCCTACACCCTTGGTGTCAAGAACCTCATTGTTGCCATCAACAAGATGGACACCACCAAGT
G
Bacterial strain 2 is following (SEQ ID NO.8) using the sequencing result after primer EF-LF1 and EF-LR2 amplification:
GGGGAGTCACGGGGCAGTCGACCACTGTAAGTACAACCATCTGCGAGTCGCTTATCTGCACTCGGAACC
CGTCAGATTTGGCGGGGTATCACCGCAACATCTTGCTAACTCTTGACAGACCGGTCACTTGATCTACCAGTGCGGTG
GTATCGACAAGCGAACCCATCGAGGAAGTTCGAGGAAGGTTAGTCAATATCCCTTTCGATTACGCGCGCTCCCATCG
ATTCCCACGACTCGCTCCCTCATTCGAAACGCATCCATTACCCCGCTCGAGCCCGAAAATTTTGCGGTGCGACCGTG
ATTTTTTTGGTGGGGTATCTTACCCCGCCACTCGAGTGACGGATGCGCTTGCCCTGTTCCCACAAAACCCTACCACA
CTGTTGCGCACTATGTCTTGCAGTCACTAACCACTGGACAATAGGAAGCCGCCGAGCTCGGAAAGGGTTCCTTCAAG
TACGCCTGGGTTCTTGACAAGCTCAAAGCCGAGCGTGAGCGTGGTATCACCATTGATATCGCTCTCTGGAAGTTCGA
GACTCCTCGCTACTATGTCACCGTCATTGGTATGTTGTCACCGTCTCACACCACCATGTCTTCATCATGCTAACATC
CCTCTCAGATGCCCCCGGTCATCGTGATTTCATCAAGAACATGATTACTGGTACTTCCCAGGCTGATTGTGCCATTC
TCATCATTGCCGCCGGTACTGGTGAGTTCGAGGCTGGTATCTCCAAGGATGGCCAGACCCGTGAGCACGCTCTTCTT
GCCTACACCCTTGGTGTCAAGAACCTCATCGTCGCCATCAACAAGATGGACACCACCAAGTGGTCTGAGGCCCGTTA
CCAGGAGATCATCAAGGAGACCTCCTCTTTCATCAAGAAGGTCGGCTACAACCCCAAGGCTGTCGCTTTCGTCCCCA
TCTCCGGTTTCAACGGTGACAACATGCTTGCTGCCTCCACCAACTGCCCCTGGTACAAGGGATGGGAGCGTGAGATC
AAGTCCGGCAAGCTCTCCGGAAAGACCCTCCTTGAGGCCATCGACTCCATCGAGCCCCCCAAGCGCCCCAACGACAA
GCCCCTCCGACTTCCCCTCCAGGATGTCTACAAGATTGGTGGTATTGGAACGGTTCCTGTCGGCCGTATCGAGACTG
GTATCATCAAGCCCGGTATGGTCGTCACCTTCGCCCCTGCTGGCGTACCACGAGACTCTAC
Bacterial strain 3 is following (SEQ ID NO.9) using the sequencing result after primer TEF-1F and TEF-3R amplification:
ACTGTGAGTACTACCCTCTCGACAATGTGCTTATCTGCATACGTCAAAACCCGCCAGAGACCGGCGAGG
TATTTCACAGTCACATCGTGCTGACAGCTTTGGATAGACCGGTCACTTGATCTACCAGTGCGGTGGTATCGACAAGC
GAACCATCGAGAAGTTCGAGAAGGTTGGTCCATTTCTCCCCCGATCGCGCGCCCTTTGCCCATCGATTTGTCCTTCG
AATCGCTACCTCACGACTCGCATGCGCCCATTACCCCGCTCGAGCTCAAAATTTTGCGGTGCAACCGTAATTTTTTT
TTTCGGTGGGGCATCACACCCCGCCACTCGAGCGATGGGCGCTTGCCCTGGTCCCAGCACACAAAACTCAATACCCA
TTCCAGGCGCGCGTCATCATGTGATCGAAAGTTGCTAACCACCATCGACAATAGGAAGCCGCCGAGCTCGGAAAGGG
TTCTTTCAAGTACGCCTGGGTTCTTGACAAGCTCAAAGCCGAGCGTGAGCGTGGTATCACCATCGACATTGCCCTGT
GGAAGTTCGAGACTCCCCGCTACTATGTCACCGTCATTGGTATGTTGTCACTACTGTTCTCATTCACACGCGTCATC
TAACACATCACACAGACGCTCCCGGCCACCGTGACTTCATCAAGAACATGATCACTGGTACTTCCCAGGCCGACTGC
GCTATTCTCATCATTGCTGCCGGTACTGGTGAGTTCGAGGCTGGTATCTCCAAGGATGGCCAGACCCGTGAGCACGC
TCTCCTTGCCTACACCCTTGGTGTCAAGAACCTCATTGTCGCCATCAACAAGATGGACACCACCAAGTGGTCTGAGG
CCCGTTACCAGGAGATCATCAAGGAGACCTCCTCTTTCATCAAGAAGGTCGGCTACAA
Bacterial strain 9 is following (SEQ ID NO.10) using the sequencing result after primer EF-LF1 and EF-LR1 amplification:
CCCCCTTGGCAGTCGACACTGTGAGTACCACCGCATCCCAACCCCGCCGACACTTGGCGGGGTAGTTTC
AAATTTCCAATGTGCTGACATACTTTGATAGACCGGTCACTTGATCTACCAGTGCGGTGGTATCGACAAGCGAACCA
TCGAGAAGTTCGAGAAGGTTGGTCTCATTTTCCTCGATCGCGCGCCCTTTTCCTTTCGAAATATCATTCGAATCGCA
CTCACACGACGACTCGATACGCGCCTGTTACCCCGCTCGAGGTCAAAAATTTTGCGGCTTTGTCGTAATTTTTTTTC
CTAGTGGGGCTCATACCCCGCCACTCGAGCGACAGGCGTTTGCCCTCTTCACACAAACCATTCCCTGGGCGCTCATC
ATCACGTGTCAACCAGTCACTAACCACCTGTCAATAGGAAGCCGCCGAGCTCGGTAAGGGTTCCTTCAAGTACGCCT
GGGTTCTTGACAAGCTCAAAGCCGAGCGTGAGCGTGGTATCACCATTGATATCGCCCTCTGGAAGTTCGAGACTCCT
CGCTACTATGTCACCGTCATTGGTATGTTGTCACCACTGCTGTCATCACATTCTCATACTAACATGGCTATCAGACG
CTCCCGGTCACCGTGATTTCATCAAGAACATGATCACTGGTACTTCCCAGGCCGATTGCGCCATTCTCATCATTGCC
GCCGGTACTGGTGAGTTCGAGGCTGGTATCTCCAAGGATGGCCAGACCCGTGAGCACGCTCTCCTTGCCTACACCCT
TGGTGTCAAGAACCTCATTGTTGCCATCAACAAGATGGACACCACCAAGTGGTCTGAGGCCCGTTACCAGGAGATCA
TCAAGGAGACCTCTTCTTTCATCAAGAAGGTCGGCTACAACCCTAAGGCTGTCGCTTTCGTCCCCATCTCCGGTTTC
AACGGTGACAACATGCTTACTGCCTCCACCAACTGCCCCTGGTACAAGGGTTGGGAGCGTGAGATCAAGTCTGGCAA
GCTCTCCGGAAAGACCCTCCTTGAGGCCATTGACTCCATCGAGCCCCCCAAGCGTCCCAACGACAAGCCCCTCCGAC
TTCCCCTCCAGGATGTCTACAAGATTGGCGGTATTGGAACGGTTCCTGTCGGCCGTATCGAGACTGGTGTCATCAAG
CCCGGTATGGTCGTTACCTTCGCTCCTCCAACTCACCACGATTACCTCCCG
Above-mentioned sequence is by comparing knot after Fusarium MLST database or NCBI GenBank carries out BLAST
Fruit is as shown in table 2.
The Molecular Identification result of 2 sickle-like bacteria of table
All references mentioned in the present invention is incorporated herein by reference, independent just as each document
It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can
To make various changes or modifications to the present invention, such equivalent forms equally fall within model defined by the application the appended claims
It encloses.
Sequence table
<110>Academy of Agricultural Sciences, Shanghai City
<120>the primer combination and method of a kind of Rapid identification sickle-like bacteria strain
<130> XY-2019-1-W-002
<160> 10
<170> SIPOSequenceListing 1.0
<210> 1
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
cttaacgtcg tcgtcatcg 19
<210> 2
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
cacttggtgg tgtccatctt 20
<210> 3
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
ttgtagccga ccttcttgat 20
<210> 4
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
ccttaacgtc gtcgtcatcg 20
<210> 5
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
acggacttga cttcagtggt 20
<210> 6
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 6
gagctgctcg tggtgcatct 20
<210> 7
<211> 799
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 7
gccacgtcga ctctggcaag tcgaccactg tgagtaccac cgcatcccaa ccccgccgac 60
acttggcggg gtagtttcaa atttccaatg tgctgacata ctttgataga ccggtcactt 120
gatctaccag tgcggtggta tcgacaagcg aaccatcgag aagttcgaga aggttggtct 180
cattttcctc gatcgcgcgc cctttccctt tcgaaatatc attcgaatcg ccctcacacg 240
acgactcgat acgcgcctgt taccccgctc gaggtcaaaa attttgcggc tttgtcgtaa 300
tttttttccc ggtggggctc ataccccgcc actcgagcga caggcgtctg ccctcttccc 360
acaaaccatt ccctgggcgc tcatcatcac gtgtcaacca gtcactaacc acctgtcaat 420
aggaagccgc cgagctcggt aagggttcct tcaagtacgc ctgggttctt gacaagctca 480
aagccgagcg tgagcgtggt atcaccattg atatcgccct ctggaagttc gagactcctc 540
gctactatgt caccgtcatt ggtatgttgt caccactgct gtcatcacat tctcatacta 600
acatggctat cagacgctcc cggtcaccgt gatttcatca agaacatgat cactggtact 660
tcccaggccg attgcgccat tctcatcatt gccgccggta ctggtgagtt cgaggctggt 720
atctccaagg atggccagac ccgtgagcac gctctccttg cctacaccct tggtgtcaag 780
aacctcattg ttgccatca 799
<210> 8
<211> 1208
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 8
ggggagtcac ggggcagtcg accactgtaa gtacaaccat ctgcgagtcg cttatctgca 60
ctcggaaccc gtcagatttg gcggggtatc accgcaacat cttgctaact cttgacagac 120
cggtcacttg atctaccagt gcggtggtat cgacaagcga acccatcgag gaagttcgag 180
gaaggttagt caatatccct ttcgattacg cgcgctccca tcgattccca cgactcgctc 240
cctcattcga aacgcatcca ttaccccgct cgagcccgaa aattttgcgg tgcgaccgtg 300
atttttttgg tggggtatct taccccgcca ctcgagtgac ggatgcgctt gccctgttcc 360
cacaaaaccc taccacactg ttgcgcacta tgtcttgcag tcactaacca ctggacaata 420
ggaagccgcc gagctcggaa agggttcctt caagtacgcc tgggttcttg acaagctcaa 480
agccgagcgt gagcgtggta tcaccattga tatcgctctc tggaagttcg agactcctcg 540
ctactatgtc accgtcattg gtatgttgtc accgtctcac accaccatgt cttcatcatg 600
ctaacatccc tctcagatgc ccccggtcat cgtgatttca tcaagaacat gattactggt 660
acttcccagg ctgattgtgc cattctcatc attgccgccg gtactggtga gttcgaggct 720
ggtatctcca aggatggcca gacccgtgag cacgctcttc ttgcctacac ccttggtgtc 780
aagaacctca tcgtcgccat caacaagatg gacaccacca agtggtctga ggcccgttac 840
caggagatca tcaaggagac ctcctctttc atcaagaagg tcggctacaa ccccaaggct 900
gtcgctttcg tccccatctc cggtttcaac ggtgacaaca tgcttgctgc ctccaccaac 960
tgcccctggt acaagggatg ggagcgtgag atcaagtccg gcaagctctc cggaaagacc 1020
ctccttgagg ccatcgactc catcgagccc cccaagcgcc ccaacgacaa gcccctccga 1080
cttcccctcc aggatgtcta caagattggt ggtattggaa cggttcctgt cggccgtatc 1140
gagactggta tcatcaagcc cggtatggtc gtcaccttcg cccctgctgg cgtaccacga 1200
gactctac 1208
<210> 9
<211> 645
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 9
gacaatgtgc ttatctgcat acgtcaaaac ccgccagaga ccggcgaggt atttcacagt 60
cacatcgtgc tgacagcttt ggatagaccg gtcacttgat ctaccagtgc ggtggtatcg 120
acaagcgaac catcgagaag ttcgagaagg ttggtccatt tctcccccga tcgcgcgccc 180
tttgcccatc gatttgtcct tcgaatcgct acctcacgac tcgcatgcgc ccattacccc 240
gctcgagctc aaaattttgc ggtgcaaccg taattttttt tttcggtggg gcatcacacc 300
ccgccactcg agcgatgggc gcttgccctg gtcccagcac acaaaactca atacccattc 360
caggcgcgcg tcatcatgtg atcgaaagtt gctaaccacc atcgacaata ggaagccgcc 420
gagctcggaa agggttcttt caagtacgcc tgggttcttg acaagctcaa agccgagcgt 480
gagcgtggta tcaccatcga cattgccctg tggaagttcg agactccccg ctactatgtc 540
accgtcattg gtatgttgtc actactgttc tcattcacac gcgtcatcta acacatcaca 600
cagacgctcc cggccaccgt gacttcatca agaacatgat cactg 645
<210> 10
<211> 800
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 10
gccacgtcga ctctggcaag tcgaccactg tgagtaccac cgcatcccaa ccccgccgac 60
acttggcggg gtagtttcaa atttccaatg tgctgacata ctttgataga ccggtcactt 120
gatctaccag tgcggtggta tcgacaagcg aaccatcgag aagttcgaga aggttggtct 180
cattttcctc gatcgcgcgc ccttttcctt tcgaaatatc attcgaatcg cactcacacg 240
acgactcgat acgcgcctgt taccccgctc gaggtcaaaa attttgcggc tttgtcgtaa 300
tttttttccc tggtggggct cataccccgc cactcgagcg acaggcgttt gccctcttcc 360
cacaaaccgt tccctgggcg ctcatcatca cgtgtcaacc agtcactaac cacctgtcaa 420
taggaagccg ccgagctcgg taagggttcc ttcaagtacg cctgggttct tgacaagctc 480
aaagccgagc gtgagcgtgg tatcaccatt gatatcgccc tctggaagtt cgagactcct 540
cgctactatg tcaccgtcat tggtatgttg tcaccactgc tgtcatcaca ttctcatact 600
aacatggcta tcagacgctc ccggtcaccg tgatttcatc aagaacatga tcactggtac 660
ttcccaggcc gattgcgcca ttctcatcat tgccgccggt actggtgagt tcgaggctgg 720
tatctccaag gatggccaga cccgtgagca cgctctcctt gcctacaccc ttggtgtcaa 780
gaacctcatt gttgccatca 800
Claims (10)
1. a kind of primer of Rapid identification sickle-like bacteria strain combines, which is characterized in that the primer combination includes drawing selected from first
At least one of object group, second primer sets, and/or it is selected from least one of third primer sets, the 4th primer sets, wherein
The first primer group includes having the upstream primer TEF-1F of nucleotide sequence shown in SEQ ID NO.1 and with SEQ
The downstream primer TEF-2R of nucleotide sequence shown in ID NO.2；
Second primer sets include having the upstream primer TEF-1F of nucleotide sequence shown in SEQ ID NO.1 and with SEQ
The downstream primer TEF-3R of nucleotide sequence shown in ID NO.3；
The third primer sets include having the upstream primer EF-LF1 of nucleotide sequence shown in SEQ ID NO.4 and with SEQ
The downstream primer EF-LR1 of nucleotide sequence shown in ID NO.5；
4th primer sets include having the upstream primer EF-LF1 of nucleotide sequence shown in SEQ ID NO.4 and with SEQ
The downstream primer EF-LR2 of nucleotide sequence shown in ID NO.6.
2. primer according to claim 1 combination, which is characterized in that the primer combination include selected from the first primer group,
At least one of second primer sets, and it is selected from least one of third primer sets, the 4th primer sets.
3. primer according to claim 2 combination, which is characterized in that the primer combination include selected from the first primer group,
One of second primer sets, and it is selected from one of third primer sets, the 4th primer sets.
4. a kind of method of Rapid identification sickle-like bacteria strain, which comprises the following steps:
Obtain the genomic DNA of sickle-like bacteria to be identified；
Using the DNA as template, PCR amplification is carried out using primer combination described in claim 1；
Amplified production is subjected to detected through gel electrophoresis, and target PCR product is sequenced；
Sequencing result is compared by BLAST,
If the sequence of sequencing result and be more than 90% with reference to the similitude of sickle-like bacteria, the sickle-like bacteria to be identified with reference to reaping hook
Pseudomonas is in same.
5. according to the method described in claim 4, it is characterized in that, primer combination includes being selected from the first primer group, second
At least one of primer sets, and it is selected from least one of third primer sets, the 4th primer sets.
6. according to the method described in claim 5, it is characterized in that, primer combination includes being selected from the first primer group, second
One of primer sets, and it is selected from one of third primer sets, the 4th primer sets.
7. according to the method described in claim 6, it is characterized in that, if at least one sequence in sequencing result with refer to sickle
The similitude of knife bacterium is not less than 98%, then the sickle-like bacteria to be identified is with reference Fusarium in same.
8. the method according to the description of claim 7 is characterized in that if two sequences in sequencing result with refer to reaping hook
The similitude of bacterium is not less than 98%, then sickle-like bacteria to be identified is with reference Fusarium in same.
9. according to any method of claim 4-8, which is characterized in that the wherein system of PCR amplification are as follows: 50 μ of total volume
L: contain Mg2+5 × PCR Buffer10 μ L, 10mmol/L dNTPs 4 μ L, 10 μm of ol/L upstream primers 1 μ L, 10 μm of ol/L under
Swim 1 μ L, 5U/ μ L KD enzyme of primer 1 μ L, DNA profiling 50ng, ddH2O is mended to 50 μ L.
10. according to any method of claim 4-8, which is characterized in that the wherein program of PCR amplification are as follows: 94 DEG C of pre- changes
Property 2min；94 DEG C of denaturation 30s, 60 DEG C of renaturation 40s, 72 DEG C of extension 90s, 35 recycle；72 DEG C of extension 5min.
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| CN111363685A (en) * | 2020-03-26 | 2020-07-03 | 广东省林业科学研究院 | Fusarium verticillatum and application thereof |
| CN111378776A (en) * | 2019-10-25 | 2020-07-07 | 安徽省农业科学院植物保护与农产品质量安全研究所 | RPA primer and detection method for detecting rice bakanae disease Gibberella fujikuei |
| CN116024364A (en) * | 2022-08-25 | 2023-04-28 | 上海市农业科学院 | Primer and method for identifying Fusarium goolgardi toxigenic genotype of PCR-RFLP |
| CN116926235A (en) * | 2023-09-18 | 2023-10-24 | 三亚中国检科院生物安全中心 | Fusarium RPA-CRISPR/Cas detection kit and method |
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| CN116926235B (en) * | 2023-09-18 | 2023-12-05 | 三亚中国检科院生物安全中心 | Fusarium RPA-CRISPR/Cas detection kit and method |
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