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Method for quickly detecting pathogenic bacteria carried by plant seeds of bacterial black rot

A detection method, plant seed technology, applied in the determination/inspection of microorganisms, biochemical equipment and methods, bacteria, etc., can solve the problems of false negative PCR reaction and small reaction system, achieve high sensitivity, overcome limitations, improve Effects of Sensitivity and Reliability

Active Publication Date: 2010-09-22
INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, PCR technology also has its undeniable limitations in the detection of seed bacteria. For example, if the sample soaking solution is directly added to the reaction system, since the concentration of pathogenic bacteria contained in the seed soaking solution is low and the reaction system is small, the pathogenic bacteria may not be involved. Into the PCR reaction, resulting in a false negative PCR reaction

Method used

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  • Method for quickly detecting pathogenic bacteria carried by plant seeds of bacterial black rot
  • Method for quickly detecting pathogenic bacteria carried by plant seeds of bacterial black rot
  • Method for quickly detecting pathogenic bacteria carried by plant seeds of bacterial black rot

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1 10

[0056] Example 1. Obtaining antiserum against Bacterial black spot of cruciferous vegetables

[0057] Refer to the methods in "Veterinary Microbiology and Immunology Technology" and "Plant Disease Research Methods" for the preparation of bacterial cell (de-flagella) antigen, preparation of bacterial whole protein antigen, immunization of rabbits, determination of serum titer and antibody Specificity (test tube agglutination method, indirect ELISA method), indirect ELISA method to determine the accuracy of antigen detection.

[0058] (1) Preparation of bacterial cell (de-flagella) antigen

[0059] The Pseudomonas syringae spotted pathogenic species (3935 in Table 1) cultivated for 48 hours was prepared with normal saline (isotonic saline 0.85%) to a final concentration of 3×10 8 ~3×10 9 CFU / ml bacterial suspension, boiled at 100℃ for 1h.

[0060] (2) Preparation of bacterial whole protein antigen

[0061] The Pseudomonas syringae spotted pathogenic species (3935 in Table 1) cultivated f...

Embodiment 2

[0078] Example 2. Routine PCR detection of bacterial black spot in cruciferous vegetables

[0079] Use bacterial genomic DNA extraction kit to extract chromosomal DNA of all target and non-target bacteria (Table 1 and Table 2), see Figure 4 , 1-8 is part of the chromosomal DNA of the bacteria.

[0080] Table 1 Test bacterial black spot (target) strains

[0081]

[0082] Table 2 Tested non-target strains

[0083]

[0084]

[0085]

[0086] Step 1 Use DNAssist 2.0 to compare the 16S-23S rDNA ITS of each subspecies of Pseudomonas syringae, and use Primer Premier 5.0 and Oligo 6.0 software to design the primers to obtain the specific primers (SEQ ID NO1):

[0087] Forward primer T1: 5’TGCTTTGCACACCCGATTT 3’

[0088] Reverse primer T2: 5’CCCCAAGCAATCTAGGT 3’

[0089] Step 2 Perform specific detection on the designed specific primers of bacterial black spot, using the chromosomal DNA of the target bacteria in Table 1 and the non-target bacteria in Table 2 as templates, using Promega's Taq DNA ...

Embodiment 3

[0091] Example 3. Detection of bacterial black spot pathogen in cruciferous vegetables by immunoadsorption PCR (IMS-PCR)

[0092] Solution preparation:

[0093] Saturated ammonium sulfate solution (SAS): in 990ml H 2 Add 1.219g Tris to O, adjust pH to 7.0, and dilute the final volume to 1L to prepare 0.01mol / L Tris·Cl solution. Weigh 767g(NH 4 ) 2 SO 4 , Stir and heat slightly to dissolve it in 1L 0.01mol / LTris·Cl, adjust the pH to 7.0, and store at 4°C. When stored at 4℃, the bottom of the bottle should be visible (NH 4 ) 2 SO 4 Crystal.

[0094] 33% (v / v) SAS solution: 33ml SAS solution plus 67ml PBS, adjust the pH to 7.0.

[0095] PBS(pH7.4): NaCl 8.10g, NaH 2 PO 4 0.18g, Na 2 HPO 4 1.97g, 1L distilled water, autoclaved at 121°C for 20min, stored at room temperature.

[0096] Immunomagnetic beads (IMBs): Dynabeads M-280 Sheep anti-Rabbit IgG (Dynal, 2ml / bottle).

[0097] PBS-BSA: PBS contains 0.1% bovine serum albumin.

[0098] Step 1. Antiserum purification.

[0099] Under constant s...

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Abstract

The invention relates to a method for quickly detecting pathogenic bacteria carried by plant seeds of bacterial black rot, and relates to the biological detection of plant pathogenic bacteria. The method comprises the following steps of: (1) enriching the pathogenic bacteria of the bacterial black rot in soak solution of the plant seeds to be detected by adopting immunomagnetic beads; and (2) performing PCR amplification detection by taking enrichment solution obtained by the step (1) as a template, wherein antibodies coated on the immunomagnetic beads are antibodies of anti-pseudomonas syringae pv. maculicola; PCR amplification primers comprises a forward primer T1: 5'TGCTTTGCACACCCGATTT 3' and a reverse primer T2: 5'CCCCAAGCAATCTAGGT 3'; and an amplification product is 454bp. The detection method combines the adsorption technology of the immunomagnetic beads and real-time fluorescence quantitative PCR; and compared with detection methods of single planting, selective media and ELISA, the detection method of the invention is time-saving, space-saving, peculiar and sensitive, and is particularly important to the integrated control of plant bacterial black rot of plants. The detection method can provide technical guarantee for safety production, seed import and export trade, and is particularly suitable for the detection of crucifer seeds.

Description

Technical field [0001] The invention relates to the detection of plant pathogens, in particular to a method for rapidly detecting bacterial black spot pathogens carried by plant seeds. Background technique [0002] Cruciferous bacterial black spot is caused by Pseudomonas syringaepv. maculicola. It belongs to the kingdom of Prokaryotic Bacteria, Proteus, and Pseudomonas. The bacterium is short rod-shaped with a size of 1.3-3.0 μm×0.7-0.9 μm, with 1-5 polar flagella, and Gram staining is negative. The host is mainly cruciferous vegetables, such as cabbage, cauliflower, radish, etc. It can also damage peppers and tomatoes. [0003] Pseudomonas syringae pv.maculicola is a special variant, which has close nutritional requirements, pathogenicity and similar origin to other Pseudomonas syringae, especially caused by Pseudomonas syringae tomato which causes bacterial diseases of tomatoes. The diseased species (P.syringae pv.tomato) have similarities in nutritional conditions, pathogenic...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12N1/20
Inventor 赵廷昌王华杰胡俊
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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