Method for separating and identifying pathogenic bacteria of insects

Abstract

The invention discloses a method for separating and identifying pathogenic bacteria of insects, relating to the field of research for bacterial diseases of insects. The method comprises the followingsteps: S1, disinfecting the body surfaces of the insects carrying pathogenic bacteria, extracting hemolymph, carrying out diluting till the suitable concentration is achieved, coating an LB plate withthe diluent, carrying out culturing, picking out single colonies, thus obtaining pathogenic bacteria, carrying out pure culturing, transferring to liquid LB culture medium, and observing the morphological features of isolated strains under an oil immersion lens; S2, treating the thallus obtained by culturing in S1 by using lysozyme and SDS in sequence, and after the thallus is crushed, purifyingthe total DNA of the isolated strains through phenol extraction; and S3, amplifying the 16S rDNA with overall length of the isolated strains, after sequencing, carrying out clustering analysis, and determining the species of the isolated strain in combination with the morphological features of an isolated strain. The method for separating and identifying pathogenic bacteria of insects is universalfor gram-positive bacteria and gram-negative bacteria, and is suitable for separating and identifying pathogenic bacteria of the insects including bees, cotton bollworms, inchworms, Bombyx mori and the like, and the screened 16S rDNA amplification and sequencing primers are good in universality and high in stability.

Description

technical field [0001] The invention relates to the research field of insect bacterial diseases, in particular to a method for isolating and identifying entomopathogenic bacteria. Background technique [0002] In order to study the mechanism of insect diseases, researchers need to isolate and identify entomopathogenic bacteria. Existing entomopathogens are usually isolated from whole insect tissues, which cannot avoid the influence of insect intestinal symbiotic bacteria, which interferes with the cultivation and identification of pathogenic bacteria. [0003] Chinese patent CN200610054049.0 "A method for separating and purifying entomopathogenic fungal cells from hemolymph of susceptible insects" proposes the idea of ​​isolating pathogenic fungi from the hemolymph of infected insects, physically isolating the pathogenic bacteria mixed with insect guts However, the technology has not yet solved the following problems: [0004] One is that the purpose of this patent is to c...

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Problems solved by technology

[0004] One is that the purpose of this patent is to construct a gene library of pathogenic fungi to reveal the infection mechanism of entomogenous fungi. The isolation method of pathogenic bacteria provided by it requires enzymatic hydrolysis to remove insect tissues, and the removal effect of insect tissues needs to be monitored by PCR and RT-PCR. Human and material resources;

[0005] The second is that the pathogenic bacteria isolated in this method have changed their natural physiological state due to enzymatic treatment, and are mainly used to extract DNA and mRNA of pathogenic fungi. The effect of continuing to culture the isolated pathogenic fungi to obtain pure cultures of pathogenic bacteria is uncertain. Especially for the applicability of bacteria is not optimistic;

[0006] Third, due to the different cell wall structures of Gram-positive bacteria and negative bacteria, the main component of Gram-positive bacteria is peptidoglycan, while the cell wall of Gram-negative bacteria is thinner. In addition to peptidoglycan, there are also lipopolysaccharide and phospholipid bilayers Lipoprotein and lipoprotein, so the two need to adopt different total DNA extraction methods, which increases the workload and labor costs of the extraction and identification steps of entomopathogenic bacteria, and is not convenient for large-scale identification of pathogenic bacteria. The above-mentioned differences are not addressed in the patent method optimization

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