A method for detecting seed-borne bacteria based on silica sol density gradient centrifugation
A density gradient centrifugation, silica sol technology, applied in the direction of biochemical equipment and methods, microbial determination/inspection, etc., can solve the problems such as the inability to fully reflect the situation of seed bacteria, the inability to meet the requirements of rapid detection, and the lack of isolated bacterial species, and to overcome the problems of Fewer types of bacteria are separated, fast detection and simple operation are satisfied
Active Publication Date: 2019-07-09
FOOD CROPS RES INST YUNNAN ACAD OF AGRI SCI
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Problems solved by technology
[0008] The technical problem to be solved by the present invention is to overcome the shortcomings of the prior art for the detection of bacteria carried by seeds that there are few types of isolated bacteria, the situation of bacteria carried by seeds cannot be fully reflected, and the separation time is long, which cannot meet the needs of rapid detection.
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Embodiment 1
[0048] The inventive method, the steps are as follows:
[0049] (1) Preparation of rice seeds to be inspected
[0050] Take 50 Moon Valley seeds, first wash the Moon Valley seeds with 200mL sterilized water once, then soak them in 2.5mL of ethanol with a volume fraction of 70% for 3min, then soak with 2.5mL of sodium hypochlorite with a mass fraction of 2.5% for 5min, with a volume fraction of 70% Soak in 2.5mL ethanol for 30s, then rinse the Moon Valley seeds with sterilized water for 3 times, dry the seeds and grind them in a sterilized mortar, filter them through a 50-mesh sieve, and take the ground Moon Valley seeds under the 50-mesh sieve , and then filter through a 100-mesh sieve, and get the ground Moon Valley seeds below the 100-mesh sieve to be placed in a sterilized culture dish with a diameter of 9 cm for subsequent use.
[0051] (2) Preparation of Ludox HS40 isotonic mother solution
[0052] Mix 10×PBS buffer solution and Ludox HS40 silica sol according to the vo...
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Abstract
The invention discloses a method for detecting carried bacteria of seeds based on silica sol density gradient centrifugation. The method includes: preparing to-be-detected rice seeds, preparing a Ludox HS40 isosmotic mother liquid, preparing a discontinuous-density Ludox HS40 separation working liquid, laying the discontinuous-density Ludox HS40 separation working liquid in a centrifugal tube, centrifuging, suspending and precipitating by the aid of a 1xTE buffer solution, performing PCR (polymerase chain reaction) amplification on the carried bacteria of the seeds, constructing a 16S rDNA (deoxyribonucleic acid) clone library, evaluating the 16S rDNA clone library, and analyzing bacteria classifications. By laying the discontinuous-density Ludox HS40 separation working liquid in the centrifugal tube through silica sol, as many as possible bacteria can be separated and purified, accuracy of detecting is increased, a diversity level of the carried bacteria of the seeds in one batch can be reflected, and the defects that few bacteria varieties are separated and the carried bacteria of the seeds cannot be reflected fully in the prior art are overcome.
Description
technical field [0001] The invention belongs to the technical field of seed quality detection, and in particular relates to a method for detecting bacteria carried by seeds. Background technique [0002] At present, seed health testing has become a routine measure in the process of seed "quality assessment" and "phytosanitary inspection" in many countries, and has become an indispensable part of the seed quality control system. Some countries have established special seed health inspections. Assay laboratory. Seed health determination is to determine whether the seeds carry pathogenic bacteria (such as fungi, bacteria and viruses), harmful animals (such as nematodes and pests) and other health conditions, that is, to detect the types and quantities of diseases and insect pests carried by seeds, and the detection of bacteria carried by seeds It is an important content in the detection of seed health. For example, since the establishment of the Seed Health Unit (SEED HEALTH ...
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IPC IPC(8): C12Q1/686C12Q1/689C12Q1/04
CPCC12Q1/686C12Q1/689C12Q2523/32C12Q2527/15
Inventor 谷安宇李小林徐雨然叶昌荣邓伟汪永华张锦文
Owner FOOD CROPS RES INST YUNNAN ACAD OF AGRI SCI