Improved PCR detection method of dwarfing bacteria of sugarcane persistent roots

A detection method, a dwarfing technology, applied in the directions of microorganism-based methods, biochemical equipment and methods, and microorganism determination/inspection, etc., can solve the problems of low detection sensitivity, low total DNA content, expensive, etc. Improved extraction method, high specificity effect

Inactive Publication Date: 2010-01-27
SUGARCANE RES INST OF YUNNAN ACADEMY OF AGRI SCI
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Problems solved by technology

However, electron microscope negative staining detection requires expensive equipment, which is not suitable for the detection of large quantities of samples in the field; serum detection method is highly sensitive, but RSD is difficult to manually separate, purify, and cultivate, so the preparation of high-purity antibodies has become a bottleneck for the application of this technology; and currently The PCR detection technology applied to RSD mainly uses the conventional CTAB method to extract DNA or the rapid lysis method of bacteria to release DNA and then PCR amplification. Although it is fast, the detection sensitivity is low due to the low content of total DNA released by extraction or lysis. , The experimental system is not easy to stabilize

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Embodiment Construction

[0032] 1. Primer design:

[0033] A pair of specific primers Lxx1 / Lxx2 were designed according to the reported nucleotide sequence of the Lxx 16S-23S rDNA intergenic region of RSD pathogenic bacteria. The size of the amplified target fragment was 438bp. The primer sequences were as follows:

[0034] Lxx1: 5′-CCGAAGTGAGCAGATTGACC-3′

[0035] Lxx2: 5′-ACCCTGTGTTGTTTTCAACG-3

[0036] 2. Sample cane juice collection:

[0037] Randomly take 6 cane stalks from each sample, cut the middle and lower nodes of each cane stalk, cut it into a length of about 7cm with a machete, squeeze about 25ml of sugarcane juice with pliers, mix it in a 50ml centrifuge tube, and store it at -20°C Store in refrigerator until use.

[0038] 3. Sugarcane juice total DNA extraction:

[0039] (1) Get 2000ul sugarcane juice for each sample and put it into a centrifuge tube, centrifuge for 10min on an ultracentrifuge at 12000rpm, and discard the supernatant;

[0040] (2) Add 300 ul of sterilized deionized...

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Abstract

The invention provides an improved PCR detection method of the dwarfing bacteria of sugarcane persistent roots. A pair of specific primers Lxx1 / Lxx2 are designed according to an RSD pathogenic bacterium Lxx 16S-23SrDNA intergenic region nucleotide sequence, and a PCR multiplication method is adopted to detect the dwarfing bacteria of sugarcane persistent roots after the improved method is used for extracting the total DNA of sugarcane juice; the method overcomes the defects of the prior detection technique; and the invention provides the PCR detection method of the dwarfing bacteria of the sugarcane persistent roots, which has the advantages of high speed, stability, accuracy, high sensitivity and strong specificity.

Description

Technical field: [0001] The invention relates to the field of plant protection, in particular to an improved PCR detection method for sugarcane perennial dwarf pathogen. Background technique: [0002] Sugarcane ratoon stunting disease (RSD) is an important worldwide disease that exists in all sugarcane growing regions. The disease is caused by a bacterium of the genus Corynebacterium (Clavibacter xyli subsp.xyli) that parasitizes in the vascular bundles of sugarcane plants, and is mainly spread through sugarcane seedlings, and the spread is extremely strong. Cane plants become shorter after infection, cane stems become thinner, growth retardation, perennial roots grow less, and the degree of damage caused by the disease increases with the increase in the number of perennial roots. Generally, the yield is reduced by 10-30%, and it can reach more than 60% when there is drought and water shortage. It can also lead to species degeneration. Because sugarcane (RSD) has no obviou...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12R1/01
Inventor 李文凤黄应昆王晓燕卢文洁罗志明
Owner SUGARCANE RES INST OF YUNNAN ACADEMY OF AGRI SCI
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