Universal primers for identifying bacillus and classification method using same

Abstract

The invention discloses universal primers for identifying bacillus. The universal primers comprise a forward degenerate primer composed of a nucleotide sequence shown as SEQID NO:1 and a reverse degenerate primer composed of a nucleotide sequence shown as SEQID NO:2. The invention also discloses a method for classifying and identifying bacillus, which comprises the following steps: utilizing the universal primers to perform PCR (Polymerase Chain Reaction) amplification; sequencing the PCR amplification product; utilizing sequence comparison software to perform sequence comparison in a Genbank data base; and acquiring the variety of a known strain having the highest sequence similarity, namely the variety of the strain. All varieties of bacillus can be classified by the universal primers, and the classification result is accurate and reliable; and the universal primers can be used for classifying bacillus on a variety level and can be also used for classifying bacillus on a subvariety level. Besides, the classification and identification method is simple and has a high identification speed.

Description

technical field [0001] The invention belongs to a method for identifying and classifying bacillus. In particular, it relates to universal primers for identifying bacillus, and a method for using them to classify and identify bacillus Background technique [0002] Bacillus (Bacillus) is a class of Gram-positive bacteria that widely exist in nature and can form spores. Different types of Bacillus have different effects on humans, and some are beneficial. For example, some Bacillus subtilis, Bacillus atrophaeus and Bacillus amyloliquefaciens can produce antifungal substances for biological control of crop diseases, and Bacillus licheniformis can produce Enzymes needed by industry, Bacillus thuringiensis (BT) can produce toxic proteins that can be used as microbial pesticides. And some are harmful, such as Bacillus anthracis can cause lethal anthrax in humans; Bacillus cereus can contaminate food and cause diarrhea in humans. Therefore, it is very important to accurately clas...

AI Technical Summary

Problems solved by technology

[0003] Some species in the genus Bacillus are closely related and have similar morphological characteristics, such as B. subtilis, B. atrophaeus, B. amyloliquefaciens, Bacillus licheniformis (B.licheniformis), Bacillus pumilus (B.pumilus), Bacillus megaterium (B.megaterium), Bacillus firmus (B.firmus), Bacillus cereus (B.cereus), Bacillus thuringiensis ( B.thuringiensis) or Bacillus anthracis (B.anthracis), etc. (Porwal et al.PLoS One2009(4), 4438), it is sometimes difficult to accurately distinguish using existing methods

[0004] The classification and identification methods of Bacillus mainly include: (1) morphological and physiological and biochemical methods. This method is difficult to distinguish between different Bacillus species due to their similar morphological and physiological and biochemical characteristics. Other shortcomings (Ash, C, etc. Letters in Applied Microbiology.1991.13, 202-206)

(2) 16S rDNA sequence comparison method, because the 16S rDNA sequence similarity between different species of Bacillus is extremely high (>98%), it is difficult to accurately classify some Bacillus

(3) RAPD and AFLP amplification marker comparison methods, both of which have the disadvantages of complicated operation or inaccurate classification results (Levy, H. et al. (2010). The Challenge of Highly Pathogenic Microorganisms 191-198. Tourasse, N.J. et al. (2010).Food Microbiology)

Wherein the gyrB gene has been widely used in the phylogenetic analysis and bacterial strain identification of Bacillus (Bavykin, S.G. et al. Journal of Clinical Microbiology.2004.42, 3711), but this gene cannot accurately separate Bacillus thuringiensis (B.thuringiensis) and Differentiation of Bacillus cereus (B. cereus) (Wang, L.T. et al. International journal of systematic and evolutionary microbiology. 2007.57, 1846)

From the above, it can be seen that so far, there is no method that can distinguish all Bacillus species, which makes it difficult to distinguish between some Bacillus species, which limits the application of beneficial Bacillus or lacks a clear understanding of harmful Bacillus

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