Method for high throughput screening and separation of bacillus thuringiensis and application

A technology of bacillus and thuringiensis, applied in the field of microbiology, can solve the problems of difficult high-throughput operation, heavy workload, and low separation efficiency, and achieve the effects of high separation efficiency, increased biomass, and avoiding inactivation

Active Publication Date: 2018-10-02
江西省农业科学院农业应用微生物研究所
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The advantage of this method is that it increases the chance of isolating non-spore-forming Bt bacterial strains, but its disadvantage is that the step of spore-forming culture is not selective to Bt bacterial strains, and other spore-like bacteria are also spore-forming at the same time, which cannot be filtered out by high-temperature treatment. It is necessary to pick a large number of colonies for transfer culture and microscopic screening, which is time-consuming and

Method used

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  • Method for high throughput screening and separation of bacillus thuringiensis and application
  • Method for high throughput screening and separation of bacillus thuringiensis and application
  • Method for high throughput screening and separation of bacillus thuringiensis and application

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Embodiment 1

[0043] Screening of Bt strains from simulated soil samples spiked with known Bt strains:

[0044]In order to verify the effectiveness of the separation method provided by the present invention, the present embodiment has carried out the separation of Bt strains to the simulated soil sample added with known Bt strains, and the steps are as follows:

[0045] 1. Preparation of simulated soil samples

[0046] 1.1 Selection of test strains: 100 publicly reported Bt strains were selected from the strains preserved in our laboratory as test strains. These strains were originally isolated from different ages, countries and regions, and the species covered most of the known serotypes. Species (see Table 1 for detailed information), whose genome has been sequenced, and related information can be found in Zheng J, Gao Q, Liu L, et al.Comparative Genomics of Bacillus thuringiensis Reveals a Path to Specialized Exploitation of Multiple Invertebrate Hosts[J].Mbio , 2017, 8(4):e00822-17.). ...

Embodiment 2

[0089] Isolation of Bt strains from freshly collected natural soil samples

[0090] In order to further verify the effectiveness of the separation method provided by the present invention, the present embodiment has carried out the separation of Bt bacterial strains to 200 parts of freshly collected natural soil samples, and its steps are as follows:

[0091] 1. Selective spore culture

[0092] 1.1 Randomly select 50 soil samples collected, weigh 2 grams of each soil sample, and put it into a sterilized 10ml centrifuge tube.

[0093] 1.2 Add 5ml of sterilized water / tube into the soil sample centrifuge tube, shake well to make a soil sample suspension.

[0094]1.3 Take 100ul of the soil sample suspension and spread it on a plate of selective spore formation medium, and place it upside down in an incubator at 30°C for 5 days.

[0095] 2. Selective Germination Inhibition Culture and High Temperature Treatment

[0096] 2.1 Add 5 ml of selective germination inhibition medium to ...

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Abstract

The invention belongs to the field of microbiology and discloses a method for high throughput screening and separation of bacillus thuringiensis (Bt for short) and application. The method is characterized in that absolute biomass and relative proportion of Bt cells in a sample are greatly improved by selective culture of continuous three-step different strategies; colony counts needing to be picked for transfer culture and microscopic examination are reduced, separating efficiency is greatly improved and greater convenience in operation is realized; meanwhile, the needed sample quantity is reduced and high-throughput operation can be realized; the method is particularly suitable for separating Bt strains from samples with small sample amount or samples with small Bt biomass.

Description

technical field [0001] The invention relates to the field of microbiology, in particular to a method and application for high-throughput screening and isolation of Bacillus thuringiensis (abbreviated as Bt). Background technique [0002] Bacillus thuringiensis (Bt) is one of the most successful and widely used biocontrol microorganisms, and has important research and application value in biopesticides and transgenic insect-resistant breeding. The isolation of Bt strains from nature is a long-term basic work and the most important content of bio-control resource mining. There are four existing methods for separating and screening Bt: [0003] 1. Direct separation method [0004] The direct separation method is a common separation method for bacteria. The method is to prepare the sample into a suspension, serially dilute it and spread it on an LB plate to cultivate a single colony visible to the naked eye. Cultivate on the culture medium until the spore stage, and screen th...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12N1/02C12R1/07
CPCC12N1/02C12N1/20
Inventor 王奋山姚英娟刘子荣徐雪亮刘小娟曾绍民
Owner 江西省农业科学院农业应用微生物研究所
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