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PCR primers and methods for the identification of different subgroups of cucurbit Bacteroides fruit spot

A fruit spot fungus and bacterial technology, which is applied in the field of PCR primers for identifying different subgroups of melon bacterial fruit spot fungus, can solve the problem of not being able to distinguish strains of group I and group II, achieve high practical application value, and be easy to operate Ease of use and high accuracy

Active Publication Date: 2021-04-16
INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these reports are limited to the rapid detection of B. fruit blotch of melons, and the primers used cannot distinguish between group I and group II strains of B. fruit blotches of cucurbits.

Method used

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  • PCR primers and methods for the identification of different subgroups of cucurbit Bacteroides fruit spot
  • PCR primers and methods for the identification of different subgroups of cucurbit Bacteroides fruit spot
  • PCR primers and methods for the identification of different subgroups of cucurbit Bacteroides fruit spot

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Embodiment 1. screening distinguishes the specific primers of I and II bacterial strains of cucurbit bacterial fruit blotch

[0063] 1. DNA extraction of melon bacterial fruit spot

[0064] Genomic DNA of group I strains and group II strains of bacterial fruit spot bacteria of melons were extracted respectively, and the adopted strains were as follows:

[0065] Group I strains: Fc247, Fc520, AAC92-300, AAC200-23, Saticoy.B.H, pslb2, pslb15, pslb19, pslb36, pslb37, pslb39, pslb48, pslb55, pslb68, pslb74, pslb88, pslb93, pslb97, pslb998, pslb97, pslb998, pslb101, pslb103, aacw1;

[0066] Group II strains: Aac5, Aac13, Aac14, Fc356, Fc376, Fc380, Fc491, AAC94-95, AAC94-39, AAC94-48, AAC208-27, ATCC29625, pslb27, pslbtw14, pslbtw23, pslbtw26, pslbtw33, pslpsbt pslbtw40, pslbtw41, pslbtw38, pslbtw43.

[0067] Genomic DNA extraction method is as follows:

[0068] Take 50 μl of the bacterium liquid of Phytophthora spp. preserved in glycerin, draw lines on the KB solid medi...

Embodiment 2

[0083] Example 2. One-step identification of different subgroups of melon bacterial fruit spot

[0084] 1. Construction of one-step PCR method

[0085] The present invention constructs the PCR method of one-step amplification detection of different subgroups of melon bacterial fruit spot bacteria, that is, the specific primers of Group I and Group II are placed in a PCR amplification system at the same time, and I can be directly identified by one amplification. Group and Group II strains.

[0086] The reaction system and amplification conditions are as follows:

[0087] PCR reaction system: 25 μl of PCR reaction system is used, and the ratio is as follows: KOD-Plus-Neo 10×PCR buffer 2.5 μl, MgSO 4 (25mM) 1.5μl, dNTPs (2mM) 2.5μl, primers BFB / BFB1 / BFB2 (10mM) 0.5μl each, KODPlus Neo enzyme 0.5μl (1.0U / μl), bacterial solution 0.5μl (10 8 CFU / ml), make up 25 μl with sterilized double distilled water.

[0088] PCR amplification conditions were 94°C for 2min; 94°C for 15sec, 5...

Embodiment 3

[0094] Example 3. Sensitivity detection of strain-specific primers for group I and group II strains of melon bacterial fruit spot

[0095] 1. Prepare bacterial solution

[0096] Dilute the bacterium solution of Pseudomonas spp. I group strain pslb2 into 8 concentration gradients: 10 8 CFU / ml, 10 7 CFU / ml, 10 6 CFU / ml, 10 5 CFU / ml, 104 CFU / ml, 10 3 CFU / ml, 10 2 CFU / ml, 10 1 CFU / ml.

[0097] Dilute the bacterial solution of Pseudomonas spp. II group strain pslbtw14 into 8 concentration gradients: 10 8 CFU / ml, 10 7 CFU / ml, 10 6 CFU / ml, 10 5 CFU / ml, 10 4 CFU / ml, 10 3 CFU / ml, 10 2 CFU / ml, 10 1 CFU / ml.

[0098] 2. Sensitivity detection

[0099] Using pslb2 bacterial liquid, pslbtw14 bacterial liquid and pslb2+pslbtw14 bacterial liquid (mixing pslb2 and pslbtw14 bacterial liquid according to the volume ratio of 1:1) as templates, and using BFB / BFB1 / BFB2 primer set to carry out PCR, reaction system and amplification Increase condition with embodiment 2 step 1. As a p...

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Abstract

The invention relates to inspection and quarantine of melon bacterial fruit spot disease, in particular to PCR primers and methods for identifying different subgroups of melon bacterial fruit spot disease. The PCR primer set used to identify different subgroups of melon bacterial fruit spot bacteria provided by the present invention includes forward primer BFB and reverse primer BFB1, BFB2, and its nucleotide sequence is as follows: BFB: 5'-ATGAAGCGTACTGTTCAG-3 ’, BFB1: 5′‑ACCATCGATATTAGCATC‑3′, BFB2: 5′‑TTAAGGAGCAAACGTGC‑3′.

Description

technical field [0001] The invention relates to inspection and quarantine of melon bacterial fruit spot disease, in particular to PCR primers and methods for identifying different subgroups of melon bacterial fruit spot disease. Background technique [0002] Bacterial fruit blotch of melons (BFB) is a kind of seed-borne bacterial disease that occurs on cucurbit crops such as watermelon and muskmelon. Negative bacteria (Schaad, N.W., Postnikova E, Sechler A, et al. Reclassification of subspecies of Acidovorax avenae as A. avenae (Manns 1905) emend., A. cattleyae (Pavarino, 1911) comb. nov., A. citrulli (Schaad et al., 1978)comb.nov., and proposal of A.oryzaesp.nov.Syst Appl Microbiol, 2008,31:434~446), mainly infecting Cucurbitaceae crops such as watermelon, muskmelon, pumpkin, cucumber, etc., seriously threatening The development of the melon industry in various countries. Since it was first reported in the U.S. in 1965, the disease has successively occurred in many waterm...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/689C12Q1/686C12Q1/04C12N15/11C12R1/01
CPCC12Q1/686C12Q1/689C12Q2537/143
Inventor 杨玉文赵廷昌乔培张晓晓白雪关巍季苇芹
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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