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Nucleic acid test strip method for detecting bacterial angular leaf spot of cucumber and its application

A technology for bacterial angular spot and cucumber, which is applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., and can solve the problems of unfavorable grassroots promotion, long time consumption, low sensitivity, etc.

Active Publication Date: 2019-08-02
CHINESE ACAD OF INSPECTION & QUARANTINE +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The detection methods of bacterial angular spot of cucumber are mainly based on morphological characteristics, pathogenicity determination, culture traits and serological reaction identification, real-time fluorescent PCR, etc., which are time-consuming, low-sensitivity or require expensive operating instruments, which are not conducive to the establishment of grass-roots units. promotional use of

Method used

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  • Nucleic acid test strip method for detecting bacterial angular leaf spot of cucumber and its application
  • Nucleic acid test strip method for detecting bacterial angular leaf spot of cucumber and its application
  • Nucleic acid test strip method for detecting bacterial angular leaf spot of cucumber and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] Example 1. Design and synthesis of primers

[0070] After a lot of sequence analysis, sequence design, manual screening optimization, and effect verification, a set of specific primers for the identification of Cucumber Bacteroides angularis was finally obtained. The specific primer set consists of primer PSPL-BF, primer PSPL-MB, primer PSPL-DF, primer PSPL-CPR and primer PSPL-BR.

[0071] PSPL-BF (sequence 1): 5'-GCATCCTGCACCACCAATT-3';

[0072] PSPL-MBF (sequence 2): 5'-ACCACCATCCATGCCTACA-3';

[0073] PSPL-DF (sequence 3): 5'-CAACGACCAGAACCTGATT-3';

[0074] PSPL-CPR (sequence 4): 5'-ACCACCATCCATGCCTACATAGGTACGGATCGGTGTGATA-3';

[0075] PSPL-BR (sequence 5): 5'-ATTGATTGCGTCGCCGAAC-3'.

[0076] The 5' end of PSPL-MBF was labeled with biotin, and the 5' end of PSPL-DF was labeled with 6-FAM.

[0077] PSPL-BF is a forward displacement primer. PSPL-MBF is the detection probe 1. PSPL-DF is the detection probe 2. PSPL-CPR is a cross primer. PSPL-BR is a reverse di...

Embodiment 2

[0078] Example 2, the establishment of the method

[0079] 1. The method of identifying whether the bacteria to be tested is Cucumber bacterial keratosis

[0080] 1. Prepare a bacterial suspension of the bacteria to be tested.

[0081] 2. Using the bacterial suspension prepared in step 1 as a template, the primer set designed in Example 1 is used to carry out cross-primer constant temperature amplification.

[0082] Reaction system (20 μl): primer PSPL-CPR 1.5 μmol / L, 5’ end with biotin-labeled primer PSPL-MBF 0.9 μmol / L, 5’ end with 6-FAM labeled primer PSPL-DF 0.9 μmol / L, Primer PSPL-BF 0.3μmol / L, primer PSPL-BR 0.3μmol / L, dNTP 0.4mmol, 10×Thermopol buffer 2μl, Bst DNA polymerase 8 units, MgSO 4 2 mmol, template 2 μl, and the balance is sterile water.

[0083] Reaction conditions: 62°C, 60min.

[0084] 3. Take the product of step 2 and use a disposable nucleic acid detection device for detection (operate according to the instructions). If both the detection band and th...

Embodiment 3

[0090] Example 3. Universal test

[0091] Bacteria to be tested: strain NCPPB540, strain NCPPB542, strain NCPPB277, strain NCPPB467 or strain NCPPB1428, all of which are existing strains of Pseudomonas syringae pv. Lachrymans. References to the above-mentioned strains: Wang Zhe, Chen Qing, Tian Qian, etc. Rapid detection of Cucumber Bacteroides angularis by PCR method [J]. Plant Quarantine, 2011, 25(6): 29-32.

[0092] Using the bacterial suspension of the bacteria to be tested as a template, the detection was carried out according to the method of step 1 of Example 2.

[0093] The results for strain NCPPB540 can be found in figure 1 3. Results of strain NCPPB542 see figure 1 4. For the results of strain NCPPB277 seefigure 1 5, for the results of strain NCPPB467 see figure 1 6. For the results of strain NCPPB1428 see figure 1 7. The results showed that the detection results of each bacteria to be tested were positive.

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Abstract

The invention discloses a nucleinic acid test paper strip method for detecting pseudomonas syringaepv. Lachrymans and application thereof. Firstly, a specific primer group is provided, and consists of a primer PSPL-BF, a primer PSPL-MBF, a primer PSPL-DF, a primer PSPL-CPR and a primer PSPL-BR; each primer is sequentially shown as sequences 1 to 5 in a sequence table. The primer group is used for identifying the pseudomonas syringaepv. Lachrymans or detecting whether the pseudomonas syringaepv. Lachrymans is contained in a plant sample to be tested. The nucleinic acid test paper strip method has the advantages that the specificity is good; the sensitivity is high, the operation is simple and convenient; the detection efficiency is high, and the like; the basic requirements of port and field detection can be met.

Description

technical field [0001] The invention belongs to the technical field of plant quarantine, relates to biological detection technology, and in particular relates to a nucleic acid test strip detection method for Cucumber Bacterial keratosis and application of the method to identify Cucumber Bacterial keratosis or those containing cucumber Bacterial keratosis A method for pathogenic plant material. Background technique [0002] Cucumber bacterial angular spot is one of the important diseases of cucumber, which occurs all over my country. With the large-scale cultivation of cucumbers, cucumber bacterial angular spot has made the cucumber yield reduction aggravated year by year. The pathogen of bacterial angular leaf spot on cucumber is Pseudomonas syringae pv. Lachrymans, the main host of which is cucumber, and also infects gourd, melon, zucchini and loofah. In the early stage of cucumber infection, yellow and chlorotic spots appeared, and the back of the leaves were water-soake...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11C12Q1/689C12Q1/6844C12Q1/04
CPCC12Q1/6844C12Q1/689C12Q2565/625
Inventor 赵文军霍亚云张永江李为民朱罗罗胡林尤其敏
Owner CHINESE ACAD OF INSPECTION & QUARANTINE