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Detecting gene for tomato ulcer germs and detecting reagent case thereof

A detection kit and tomato canker technology, which is applied in the field of gene detection of tomato canker bacterium and its detection kit, can solve the problems of time-consuming instability, low sensitivity, poor specificity, etc., to improve the quarantine level, simplify the detection procedures, Time-consuming unstable fast effect

Inactive Publication Date: 2006-09-13
NANJING AGRICULTURAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Technical problem The purpose of the present invention is to overcome the shortcomings of the existing plant pathogenic bacteria detection method, such as low sensitivity, poor specificity, time-consuming and unstable, and provide a detection gene and detection kit for tomato canker bacterium. A section of cloned Cmm is located between 16S-23SrDNA A pair of Cmm-specific primers and its detection kit were designed based on this ITS sequence, and the kit was used to achieve rapid and accurate detection of Cmm in the sample

Method used

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  • Detecting gene for tomato ulcer germs and detecting reagent case thereof
  • Detecting gene for tomato ulcer germs and detecting reagent case thereof
  • Detecting gene for tomato ulcer germs and detecting reagent case thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0051] Example 1: PCR amplification of Cmm specific primers CmmF1-CmmR2. The genomic DNA of Cmm and other related bacteria and control bacteria were amplified by PCR using the universal primers U1 / U2 (5'-GTGGATCACCTCCTTC-3'; 5'-TTCGCTCGCCCTAC-3') of prokaryotic ITS, and a length was obtained. It is a PCR product of about 700bp (Figure 1). These bacteria include: Curtobacterium flaccumfaciens, Curtobacterium flaccumfaciens pv.flaccumfaciens (ICMP2584), Cur.f.pv.betae (ICMP2594), Curtobacterium flaccumfaciens pv.flaccumfaciens (ICMP2584), Cur.f.pv.betae (ICMP2594), Curtobacterium flaccumfaciens pv.flaccumfaciens (ICMP2584), Curtobacterium flaccumfaciens pv. pv.oortii (ICMP 2632), Cur.f.pv.poinsettiae (ICMP2566); Corynebacterium michigan Clavibacter michiganensis subsp.Michiganensis (ICMP 2550, JCM1370, 1371) 1372, 1373); Rhodococcus fascians (JCM6165); Corynebacterium michiganensis Cl. michiganensis subsp. nebraskensis (ATCC3298); Corynebacterium michiganensis Cl. michiganensis subs...

Embodiment 2

[0079] Example 2: Detection of diseased tomato seeds intercepted by customs.

[0080] Disinfect the surface of 30 grams of seeds with 2% sodium hypochlorite, break them under aseptic conditions, and add 45 ml of 0.01M PBS buffer (pH 7.2). Each component of the kit of the present invention is added to the sample to perform PCR reaction. Under reaction composition and cycle parameters:

[0081] Reaction composition (25μl system): (1) 10×PCR buffer: 350mM KCl; 10mM MgCl2; 80mM Tris-HCl; 8% PVP; (2) 10×d NTP (Mixture): 8mM; (3) 10× primer CmmF1 / primer CmmR2: 8μM; (4) Tag enzyme: 1U; (5) Template DNA: 5μl extract. Cycle parameters: pre-amplification at 95°C for 5 minutes; 94°C for 30 seconds, 64°C for 30 seconds, 72°C for 40 seconds, 28 cycles; 72°C for 10 minutes extension.

[0082] Electrophoresis detected a clear product of 425bp (positive reaction), and the negative control was a healthy domestic tomato seed of the same weight.

Embodiment 3

[0083] Example 3: Detection of inoculated tomato plants

[0084] Number of samples

[0085] Note: + means Cmm is detected;-means Cmm is not detected.

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Abstract

A detecting gene of Cmm and its reagent box are disclosed. The gene is between 16S-23S rDNA nucleic acid sequence of endogenous transcripting spacer. The reagent box includes: super-pure water, PCR reacting buffer 10X, dNTPs 10mM, Mg2+25mM, positive control DNA 100ng / mul, DNA marker, buffer 5X, CmmF1 20muM, CmmR2 20muM. PCR amplifying 44 bacteria to be tested by reagent box and Cmm producing specific amplified product with molecular weight 425bp. It achieves rapid, simple and accuracy testing effictiveness.

Description

1. Technical Field [0001] The detection gene of the tomato canker pathogen and the detection kit thereof of the present invention belong to the field of detection of plant pathogenic bacteria, especially the detection of the tomato canker pathogen Clavibacter michiganensis subsp. michiganensis (Cmm). It is specially used for field prevention of tomato canker bacteria and rapid detection of Cmm in customs plant quarantine. 2. Technical background [0002] Tomato ulcer disease is the third type of dangerous disease in my country. It was first discovered by Erwin F. Smith in Michigan in the United States in 1909. It is now reported in North America, South America, Africa, Australia, New Zealand, Yezhou, Xinjiang and Beijing in China. In addition to harming tomatoes, it can also be harmful. Solanaceous plants such as pepper and nightshade. Tomato canker is a Gram-positive bacteria that can infect seeds, roots, stems or cotyledons of tomatoes and other Solanaceae plants. There are two...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
Inventor 郭坚华张晓梅
Owner NANJING AGRICULTURAL UNIVERSITY
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