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PCR fast test method of sugarcane ratoon stunting germina

A detection method and sugarcane technology, applied in the directions of microorganism-based methods, biochemical equipment and methods, and microorganism determination/inspection, etc., can solve the problems of impractical production, poor repeatability and stability of test results, and immaturity. The results are stable, the electrophoresis results are accurate and reliable, and the detection is fast.

Inactive Publication Date: 2012-06-06
GUANGZHOU SUGARCANE IND RES INST
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  • Abstract
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Problems solved by technology

However, the technology is not mature enough, especially the repeatability and stability of the test results are poor, so it is not practical in production

Method used

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  • PCR fast test method of sugarcane ratoon stunting germina
  • PCR fast test method of sugarcane ratoon stunting germina
  • PCR fast test method of sugarcane ratoon stunting germina

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Embodiment

[0026] The PCR quick detection method of dwarf pathogen of sugarcane perennial root comprises the steps:

[0027] Step 1. Take sugarcane juice: randomly take several sections of sugarcane in the test field, squeeze 1 to 2 mL of sugarcane juice for each and store at -20°C;

[0028] Step 2, sugarcane juice pretreatment: centrifuge at 12000rpm at 4°C for 5 minutes; remove the supernatant of sugarcane juice and leave the precipitate;

[0029] Step 3, extract the genomic DNA of RSD bacteria by improving and optimizing the CTAB method; the specific steps are as follows:

[0030] 1) Add 1 mL of 3% CTAB preheated at 65°C and 10 μL of proteinase K extract to the precipitated sugarcane juice pretreated in step 2, and mix well;

[0031] 2) Incubate at 65°C for 40-60 minutes, during which time shake well;

[0032] 3) Add an equal volume of chloroform-isoamyl alcohol (24:1) mixture, mix gently, centrifuge at 8000rpm, 4°C for 10min;

[0033] 4) Carefully absorb the supernatant, add an eq...

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Abstract

The invention relates to the technical field of propagation and production of sugarcane sprouts and provides a PCR fast test method of sugarcane ratoon stunting germina, comprising the following steps: taking sugarcane juice, preprocessing the sugarcane juice, improving and optimizing CTAB method to extract genome DNA, augmenting PCR, detecting electrophoresis and the like; wherein, in the electrophoresis detection step, the augmented PCR product is treated through electrophoresis with 5% of polyacrylamide gel and dyed in silver nitrate solution for 10min, then the dyed product is cleaned with double distilled water for one or two times and reacted with developer (1.5% of NaOH and 0.15% of formaldehyde) for developing color and finally the result can be observed. The invention has following characteristics and outstanding improvements: 1. the test is fast and the result is stable; the required time for the test result is only 8h after sugarcane juice is taken from a field and the testresult is stable; 2.mass test can be realized, only 1-2ml of sugarcane juice is needed in the invention so that sugarcane juice is easy to collect without special devices and the operation is simple,thus being applicable to mass test by which a skilled technician can test above 800 samples every day. 3. the test result is accurate and the test has high sensitivity; in the preprocess step of the invention, secondary compounds such as cane wax and the like in sugarcane juice can be effectively removed which can affect the DNA of RSD, the obtained DNA has high purity, the PCR process has littleinterference and the electrophoresis result of RSD is accurate and stable and has high sensitivity.

Description

technical field [0001] The invention relates to the technical field of reproduction and production of sugarcane seedlings, in particular to the detection technology of sugarcane perennial root dwarf pathogens. Background technique [0002] Sugarcane (Saccharum.spp) is the main sugar crop and a biomass crop with good development prospects in my country. Sugarcane ratoon dwarf (Ratoon Stunting Disease, RSD) is an important sugarcane disease worldwide, and it is also a major sugarcane disease in my country's sugarcane regions. %, the yield loss of ratoon cane is 20% to 25%, and it affects the life of rattan cane, which is one of the important reasons for the degradation of sugarcane varieties. The pathogenic bacteria of sugarcane RSD live in the vascular bundles of sugarcane stems, and there is no effective chemical control method; at the same time, due to the lack of resistant source materials, sugarcane RSD disease-resistant breeding has not achieved much, and no production v...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12R1/01
Inventor 刘睿邓海华陈仲华沈万宽陈健文
Owner GUANGZHOU SUGARCANE IND RES INST
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