CN109371164B - Application of specific sequence of rape black shank bacterium subspecies in rape black shank bacterium detection - Google Patents
Application of specific sequence of rape black shank bacterium subspecies in rape black shank bacterium detection Download PDFInfo
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- CN109371164B CN109371164B CN201811569049.3A CN201811569049A CN109371164B CN 109371164 B CN109371164 B CN 109371164B CN 201811569049 A CN201811569049 A CN 201811569049A CN 109371164 B CN109371164 B CN 109371164B
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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
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
Abstract
The invention belongs to the technical field of plant protection, and discloses application of a specific sequence of a rape black shank bacterium subspecies in rape black shank bacterium detection. The LAMP primer designed aiming at the target can detect the genomic DNA of the L.bioglobosa 'brassicae' at 132 fg/muL, and provides a sensitive and rapid tool for rapidly detecting and identifying the phytophthora parasitica subspecies Lbb of rape.
Description
Technical Field
The invention belongs to the technical field of plant protection, and particularly relates to application of a specific sequence of rape black shank bacterium subspecies in rape black shank bacterium detection.
Background
Rape black shank (Blackleg) is a worldwide rape disease. Worldwide economic losses of more than $ 9 billion per rape production season have been reported (Fernando et al, 2016), and the causative bacteria are mycosphaerella which can severely harm brassicaceous plants such as rape. The mycosphaerella (Leptosphaeria) fungus includes two closely related species: ma cumans and l bilobosa (Shoemaker and brun.,2001) (kaczmarek., 2009). Both pathogens are distributed in canada, australia and europe (West et al, 2001), but only l.biglocosa is reported in our country, and l.maculans is not found yet. Therefore, L.maculans is listed as a plant quarantine pathogenic bacterium prohibited from entry in China in 2007, 5, 28 th.e. (Zhou Liang, 2010; Zhou, etc., 2016).
The blackleg bacterium brassicae includes six subspecies or subclasses, namely, L.biglobosa 'brassicae' (abbreviated as Lbb in the present invention), L.biglobosa 'canadensis,' L.biglobosa 'australesis,' L.biglobosa 'occidentalis,' L.biglobosa 'thlaspii,' and L.biglobosa 'eryimii', wherein L.biglobosa 'canadensis' is the dominant population in Canada, and L.biglobosa 'brassicae' is the dominant population in eastern Europe and Asia (Mendes-Pereira et al 2003; Vincenot et al 2008). The main cause of rape black shank in China is L. The rape black shank fungus mainly infects rape stems, yellow brown split spots are formed at the infected parts, the stems are lodging in severe cases, and the production safety of the rape is seriously threatened. At present, the prevention and control of rape black shank mainly depends on the key monitoring of rape production areas, the disease harm and the variation trend of pathogenic bacteria are mastered in time, and accordingly, a prevention and control system for the traditional prevention and control is established (Rong, pine, cypress and the like, 2018). At present, AFLP, ISSR and other methods are applied to the detection and identification work of rape black shank subspecies (Liu et al, 2014; Huoli fen and other methods, 2014). However, the existing detection method has the disadvantages of long period, high requirements on instruments and equipment and workers, complex detection steps and the like, and is difficult to meet the requirement of real-time and rapid detection in the field. Therefore, a rapid, accurate, sensitive and simple detection technology is particularly important for port detection of rape phytophthora parasitica.
Aiming at the problems, the invention provides a rape blackleg bacterium Lbb specific sequence which specifically exists in rape blackleg bacterium and can well distinguish kindred species and subspecies; meanwhile, specific LAMP detection primers of the rape phytophthora parasitica Lbb are designed according to the sequence, and an LAMP rapid detection technology suitable for the field is established.
Disclosure of Invention
The invention aims to provide application of a specific sequence of a rape black shank subspecies in rape black shank bacterium detection, wherein the specific sequence of the rape black shank bacterium subspecies is shown in SEQ ID NO. 1.
The invention also aims to provide application of the reagent for detecting the sequence shown in SEQ ID NO.1 in preparation of the test reagent for detecting the phytophthora parasitica var nicotianae.
The invention also aims to provide application of the primer designed aiming at the sequence shown in SEQ ID NO.1 in preparing the detection reagent for the phytophthora parasitica var nicotianae.
The invention also aims to provide LAMP primers designed aiming at the specific sequences of the rape phytophthora parasitica subspecies, wherein the primers are F3: 5'-GTATTGGCCGCGAATTCC-3', B3: 5'-GGAGATTGGCCACTATGG-3', FIP 5 ' -GGCGTCTCTTTTATGGCTATTTTCTGGTCAAAAGTTGGTTTGGA, BIP: 5'-AATGTCAGGAAGTCTGAAAAGCTCACGTTCTCTGATCAGGAC-3', LF 5'-CCGAAATGAATTGTACCAGTATCCT-3' and LB 5'-ACTGCCTCATGCAACATGG-3'.
In order to achieve the above purpose, the invention adopts the following technical measures:
the application of the sequence shown in SEQ ID NO.1 in the detection of L.biglobosa 'branched' (Lbb) of Leptosphaeria maculans is protected by the invention as long as the sequence is applied to the detection of the L.biglobosa 'branched'.
The application of the reagent for detecting the sequence shown in SEQ ID NO.1 in the preparation of the test reagent for detecting the phytophthora parasitica, which comprises the application of the primer designed aiming at the sequence shown in SEQ ID NO.1 in the preparation of the test reagent for detecting the phytophthora parasitica, belongs to the protection content of the invention as long as the primer designed aiming at the sequence is used for detecting the phytophthora parasitica.
In the above application, preferably, the LAMP primer designed for the sequence shown in SEQ ID No.1 is: f3: 5'-GTATTGGCCGCGAATTCC-3', B3: 5'-GGAGATTGGCCACTATGG-3', FIP 5 ' -GGCGTCTCTTTTATGGCTATTTTCTGGTCAAAAGTTGGTTTGGA, BIP: 5'-AATGTCAGGAAGTCTGAAAAGCTCACGTTCTCTGATCAGGAC-3', LF 5'-CCGAAATGAATTGTACCAGTATCCT-3' and LB 5'-ACTGCCTCATGCAACATGG-3'.
Compared with the prior art, the invention has the following advantages:
1. the invention provides a specific detection target of L.biglobosa 'brassicae', and no related report exists before the filing date. The target has the characteristics of good specificity, strong stability and high sensitivity, can well distinguish related species and subspecies of rape black shank with higher homology, and avoids the generation of false positive.
2. The LAMP primer is designed aiming at the specific sequence of L.biglobosa 'brassicae', the design of the LAMP primer needs to be combined with six regions on a target sequence, the existence of the Leptosphaeria brasiliensis Lbb can be accurately detected, 132 fg/. mu.L Lbb genomic DNA can be detected, and the conventional PCR (F3: 5'-TATTGGCCGCGAATTCC-3', B3: 5'-GGAGATTGGCCACTATGG-3') can only detect 1.32 ng/. mu.L.
3. The detection primer provided by the invention has the advantages of good specificity, high sensitivity, strong stability, rapidness, simplicity and convenience, the occurrence of false positive is well controlled in the detection process, and the existence of L. The method provides a good tool for field detection of the rape phytophthora parasitica and field monitoring and identification of the disease.
Drawings
FIG. 1 is a visual diagram and a gel electrophoresis diagram for LAMP detection primer specificity detection of Leptosphaeria maculans;
wherein, the upper diagram: 1-21 tubes: 21 strains of the national l. biglobosa 'branched' strain (see table 1 for specific sequence), 22-23 tubes: 2 subspecies l.biglobosa 'canadens', 24-35 tubes: 2 related species L.maculans, 10 pathogenic fungi isolated from Brassica napus (in order: Phoma sp., Phoma macrotoma, Phoma sp., Phomaglomertata, Phoma herbarum, Borrytis cinerea, Sclerotinia sclerotiorum, Colletotrichum sp., Alternaria alternata, Chaetolimumbosum), 36 th tube: negative control;
the following figures: gel electrophoresis pattern: lane M100 bp DNAladder, Lane 1: bioglobosa 'branched' W10, lanes 2-15: corresponding to strain nos. 22-35 in table 1, lane 16: negative control, water;
FIG. 2 is a visual diagram and a gel electrophoresis diagram for LAMP detection primer sensitivity detection of Leptosphaeria maculans;
the upper graph is a visual detection graph of the rape phytophthora parasitica L.bioglobosa 'scientific', wherein 1-9 tubes of the rape phytophthora parasitica L.bioglobosa 'scientific' W10 have the DNA concentrations of: 132 ng/. mu.L, 13.2 ng/. mu.L, 1.32 ng/. mu.L, 132 pg/. mu.L, 13.2 pg/. mu.L, 1.32 pg/. mu.L, 132 fg/. mu.L, 13.2 fg/. mu.L, 1.32 fg/. mu.L, lane 10: negative control;
the lower panel is the gel electrophoresis of the amplification product of L.biglobosa 'branched', wherein lane M is 100bp DNA Ladder, 1-9: 132 ng/. mu.L, 13.2 ng/. mu.L, 1.32 ng/. mu.L, 132 pg/. mu.L, 13.2 pg/. mu.L, 1.32 pg/. mu.L, 132 fg/. mu.L, 13.2 fg/. mu.L, 1.32 fg/. mu.L of the genomic DNA of L.biglobosa 'brassicae' of Leptosphaeria maculans, 10: negative control.
FIG. 3 is a visual image and a gel electrophoresis image of rape stem stalk detection in the field by specific LAMP detection primers of rape phytophthora parasitica;
the upper diagram: 1-7 tubes: collecting diseased rape stem DNA in a field, wherein 8-13 pipes of rape stem DNA without symptoms are collected;
the following figures: lane M is 100bp DNA Ladder,1-7 field collecting diseased rape stem DNA, and 8-13 field collecting non-symptom rape stem DNA.
Detailed Description
The technical solutions according to the present invention are, unless otherwise specified, conventional solutions in the art, and the reagents or materials used are, unless otherwise specified, publicly available.
Example 1:
the application of the sequence shown in SEQ ID NO.1 or a primer designed aiming at the sequence in detecting Leptosphaeria maculans (L.biglobosa 'brassicae'):
aiming at the sequence shown in SEQ ID NO.1, the LAMP primer is designed as follows:
F3:5’-GTATTGGCCGCGAATTCC-3’
B3:5’-GGAGATTGGCCACTATGG-3’
FIP:5’-GGCGTCTCTTTTATGGCTATTTTCTGGTCAAAAGTTGGTTTGGA-3’
BIP:5’-AATGTCAGGAAGTCTGAAAAGCTCACGTTCTCTGATCAGGAC-3’
LF:5’-CCGAAATGAATTGTACCAGTATCCT-3’
LB:5’-ACTGCCTCATGCAACATGG-3’。
the LAMP amplification system is as follows:
bst 2.0WarmStart DNA Polymerase was purchased from New England Biolabs.
The LAMP amplification program was as follows:
reacting at 65 ℃ for 40 min; inactivating at 80 deg.C for 5min
Adding 1000 XSYBR Green I into the reaction product, wherein the positive reaction shows fluorescent Green, which indicates the existence of L.biglobosa 'brassicae' of the rape-oil rape-black shank; the negative control remained orange.
Example 2:
specific detection and sensitivity detection of rape phytophthora parasitica (L.bioglobosa 'brassicae') LAMP primer:
specific detection of the rape phytophthora parasitica (L.bioglobosa 'brassicae') LAMP primer:
35 strain samples are selected in total, and the primers provided by the invention are subjected to specificity detection:
21 L.biglobosa 'brassicae' strains collected from winter rape producing areas throughout the country; 2 subspecies l. biglobosa 'c anadenss'; 2 closely related species l.maculans; 10 pathogenic fungi (in turn: Phoma sp., Phoma macrotoma, Phoma sp., Phoma globrata, Phoma herbarum, Borrytis cinerea, Sclerotinia sclerotiorum, Colletotrichum sp., Alternariaaltana, Chaetomium globosum) isolated from Brassica napus, all strains are shown in Table 1.
The strains described in Table 1 were LAMP detected using the method described in example 1. In Table 1, "+" indicates positive results and "-" indicates negative results.
TABLE 1
Sensitivity detection of rape black shank LAMP primer
The genome DNA of the rape phytophthora parasitica Lbb (W10) is diluted by 10 times of concentration gradient, and the LAMP primer provided by the invention is used for detecting the sensitivity of the rape phytophthora parasitica Lbb. The result shows that the primer group can detect the phytophthora parasitica DNA with the lowest concentration of 132 fg/. mu.L, and the sensitivity is strong (figure 2).
Example 3: field application of rape phytophthora parasitica LAMP (loop-mediated isothermal amplification) specific detection primer
Collecting the rape stalks with morbidity in rape producing areas (Hubei red wall, Hubei Yanning, Neimangsan, Gansu foley, Xining mutual assistance, Neimanyuan river, Shanxi Hanzhong, Hunan Huaihe, Zhejiang Ningbo, Guizhou Guiyang and the like), randomly selecting 7 rape stalks with obvious morbidity and 6 rape stalks without symptoms, extracting DNA from the morbidity tissues, carrying out LAMP detection by using the primer provided by the invention, wherein the result shows that the rape black shank Lbb is detected on the 7 rape stalks with morbidity, 2 rape stalks without symptoms are also detected with the rape black shank Lbb on the 6 rape stalks without symptoms, only 4 rape without the germ is detected (figure 3), and the 2 rape stalks without symptoms which are detected to be positive are cultured in an environment with the humidity of 25 ℃, and the typical brown blotches of the rape stalks with the brown morbidity are observed after 7 days, the LAMP primer can be used for detecting L.bioglobosa 'brassicae' in rape diseased tissues.
Sequence listing
<110> university of agriculture in Huazhong
Application of specific sequence of rape black shank bacterium subspecies in rape black shank bacterium detection
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tataaagggt agttacaatg aaaacttaac ttgagagcag ctcatataag tgttgattga 60
ggaaagtttg atcgaaatag caattataca gtgctttggt cagacaatgt agaagctggc 120
tatgcgagac aagcatatgt tttattgctt gctcttagac atgttccaga atgctgttcc 180
cgcctagacc tggtaggcta gattgctagg gccattgcga tgtgacgcac gcgtttggtc 240
aagctaccaa gatcgtcgtg aaaggtttgt attggccgcg aattccgggt caaaagttgg 300
tttggaagag gatactggta caattcattt cggtagtttt ttccagaaga aaatagccat 360
aaaagagacg ccgtttccaa tgtcaggaag tctgaaaagc tactgcctca tgcaacatgg 420
tcgaaaagta ggatgaaggt cctgatcaga gaacgtgcag ccatagtggc caatctccag 480
cgcttcgtat taggtatgtg tgccaatgaa agttgccgac atggacaaca cgaacacctt 540
gataccaatg ttattagcgt catatccaat ttagtgagca ttcagtgggt ctgttattgc 600
gcagtctgaa gttac 615
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ggcgtctctt ttatggctat tttctggtca aaagttggtt tgga 44
<210>5
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aatgtcagga agtctgaaaa gctcacgttc tctgatcagg ac 42
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Claims (4)
- The application of the sequence shown in SEQ ID NO.1 in detecting the rape phytophthora parasitica L.
- 2. The application of the reagent for detecting the sequence shown in SEQ ID NO.1 in the preparation of the rape blackleg bacterium kit is L.
- 3. The application of the primer designed aiming at the polynucleotide shown in SEQ ID NO.1 in the preparation of the detection reagent for the rape black shank bacterium is L.
- 4. The use of claim 3, wherein the primers are:F3:5’-GTATTGGCCGCGAATTCC-3’、B3:5’-GGAGATTGGCCACTATGG-3’、FIP:5’-GGCGTCTCTTTTATGGCTATTTTCTGGTCAAAAGTTGGTTTGGA-3’、BIP:5’-AATGTCAGGAAGTCTGAAAAGCTCACGTTCTCTGATCAGGAC-3’、LF:5’-CCGAAATGAATTGTACCAGTATCCT-3’、LB:5’-ACTGCCTCATGCAACATGG-3’。
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