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Application of specific sequence of subspecies of leptosphaeria biglobosa to detection of leptosphaeria biglobosa

A technology for blackleg bacteria and rapeseed, which is applied in the application field of specific sequences in the detection of blackleg bacteria in rapeseed, can solve the problems of difficulty in meeting the real-time rapid detection in the field, high requirements for instrument and equipment staff, and long detection method cycle, etc. Fast and easy stability, good specificity, and high sensitivity

Active Publication Date: 2019-02-22
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the existing detection methods have disadvantages such as long cycle time, high requirements for equipment and staff, and complicated detection steps, which make it difficult to meet the requirements of real-time and rapid detection in the field.

Method used

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  • Application of specific sequence of subspecies of leptosphaeria biglobosa to detection of leptosphaeria biglobosa
  • Application of specific sequence of subspecies of leptosphaeria biglobosa to detection of leptosphaeria biglobosa
  • Application of specific sequence of subspecies of leptosphaeria biglobosa to detection of leptosphaeria biglobosa

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028]Application of the sequence shown in SEQ ID NO.1 or the primers designed for the sequence in the detection of rapeseed blackleg bacteria (L.biglobosa 'brassicae'):

[0029] For the sequence shown in SEQ ID NO.1, LAMP primers were designed as follows:

[0030] F3:5'-GTATTGGCCGCGAATTCC-3'

[0031] B3:5'-GGAGATTGGCCACTATGG-3'

[0032] FIP: 5'-GGCGTCTCTTTTATGGCTATTTTCTGGTCAAAAAGTTGGTTTGGA-3'

[0033] BIP: 5'-AATGTCAGGAAGTCTGAAAAGCTCACGTTCTCTGATCAGGAC-3'

[0034] LF:5'-CCGAAATGAATTGTACCAGTATCCT-3'

[0035] LB: 5'-ACTGCCTCATGCAACATGG-3'.

[0036] The LAMP amplification system is as follows:

[0037]

[0038] Bst 2.0 WarmStart DNA Polymerase was purchased from New England Biolabs.

[0039] The LAMP amplification procedure is as follows:

[0040] React at 65°C for 40 minutes; inactivate at 80°C for 5 minutes

[0041] Add 1000×SYBR Green I to the reaction product, the positive reaction shows fluorescent green, indicating the presence of L.biglobosa 'brassicae' in rapes...

Embodiment 2

[0043] Specific detection and sensitivity detection of LAMP primers for L.biglobosa ‘brassicae’:

[0044] Specific detection of LAMP primers for L.biglobosa 'brassicae':

[0045] A total of 35 bacterial strain samples were selected, and the primers provided by the invention were specifically detected:

[0046] 21 strains of L.biglobosa 'brassicae' collected from winter rapeseed producing areas in China; 2 strains of subspecies L.biglobosa 'c anadensis'; 2 strains of closely related species L.maculans; 10 strains of pathogenic fungi isolated from rapeseed ( In order: Phoma sp., Phoma macrostoma, Phoma sp., Phoma glomerata, Phoma herbarum, Botrytis cinerea, Sclerotinia sclerotiorum, Colletotrichum sp., Alternaria alternata, Chaetomium globosum), all strains are shown in Table 1.

[0047] Using the method described in Example 1, the strains described in Table 1 were tested for LAMP. In Table 1, "+" indicates a positive result, and "-" indicates a negative result.

[0048] Tabl...

Embodiment 3

[0054] Example 3: Field Application of LAMP-specific Detection Primers for Rapeseed Blackleg Germ

[0055] Rapeseed stalks with diseases were collected in rapeseed producing areas (Chibi, Hubei, Xianning, Inner Mongolia, Fansan, Inner Mongolia, Minle, Gansu, Xining Huzhu, Inner Mongolia, Wuduhe, Hanzhong, Shaanxi, Huaihua, Hunan, Ningbo, Zhejiang, Guiyang, Guizhou, etc.) and selected randomly. The stalks of 7 rapeseed black shanks with obvious symptoms and 6 rapeseeds without symptoms were extracted from the diseased tissues, and the LAMP primers provided by the present invention were used for LAMP detection. The results showed that the 7 rapeseeds with diseased The presence of Lbb of rapeseed blackleg was detected, and the existence of rapeseed blackleg Lbb was also detected in 2 rapeseeds of 6 asymptomatic rapeseed plants, and only 4 plants did not detect the existence of the pathogen ( image 3 ), cultured the stalks of 2 rapeseed plants with no symptoms and tested positive...

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Abstract

The invention belongs to the technical field of plant protection, and discloses application of a specific sequence of a subspecies of leptosphaeria biglobosa to detection of the leptosphaeria biglobosa. The invention provides a specific detection target of L.biglobosa 'brassicae' (Lbb), and the target has the characteristics of good specificity, strong stability and high sensitivity, and can welldistinguish between a closely related species and the subspecies of the leptosphaeria biglobosa with relatively high homology, thereby avoiding the occurrence of false positives. An LAMP primer designed for the target can detect L.biglobosa 'brassicae' genomic DNA of 132fg / [mu]L, and provides a sensitive and rapid tool for the rapid detection and identification of Lbb, namely the subspecies of theleptosphaeria biglobosa.

Description

technical field [0001] The invention belongs to the technical field of plant protection, and in particular relates to the application of the specific sequence of the rapeseed blackleg subspecies in the detection of the rapeseed blackleg. Background technique [0002] Rapeseed blackleg (Blackleg) is a worldwide disease of rapeseed. According to reports, in each rapeseed production season, the global economic loss is as high as 900 million U.S. dollars (Fernando et al., 2016). The pathogen is the fungus of the genus Micrococcomus, which can seriously damage Brassicaceae plants such as rapeseed. Leptosphaeria fungi include two closely related species: L. ma culans and L. biglobosa (Shoemaker and Brun., 2001) (Kaczmarek., 2009). These two pathogenic bacteria are distributed in Canada, Australia, and Europe (West et al., 2001). However, only L.bigl obosa has been reported in my country, and L.maculans has not been found yet. Therefore, on May 28, 2007, my country listed L. macu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12Q1/04C12R1/645
CPCC12Q1/6895
Inventor 李国庆杜然杨龙张静吴明德
Owner HUAZHONG AGRI UNIV
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