Bacillus species level identification method based on high-throughput sequencing technology

A bacillus and sequence technology, applied in the field of molecular biology, can solve the problems of cumbersome analysis, inability to meet rapid detection, low efficiency, etc., and achieve the effects of accurate detection results, saving identification time and identification cost, and high resolution ability.

Active Publication Date: 2022-03-22
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

A Korean study used the MLSA method to concatenate four amplified sequences to achieve accurate detection of Bacillus cereus and Bacillus thuringiensis. Although this detection method can meet the requirements of detection limit, accuracy, and sensitivity, it is based on MLSA The shortcomings of cumbersome analysis and low efficiency in the rapid identification of strains cannot meet the needs of rapid detection, and only two species can be identified, which limits the detection range (KR1020120131784A)

Method used

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  • Bacillus species level identification method based on high-throughput sequencing technology
  • Bacillus species level identification method based on high-throughput sequencing technology
  • Bacillus species level identification method based on high-throughput sequencing technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1: Construction of Bacillus-specific primers based on the high-resolution core gene of Bacillus

[0037] 1. Collected and summarized 85 species of spores that have been reported in the databases of the National Center for Biotechnology Information (NCBI) and the European Molecular Biology Laboratory (EMBL) and whose genomes have been sequenced Bacillus, and according to the completeness of their sequencing, each selected several, and downloaded a total of 202 whole-genome sequences (some species contain only one strain, which is included as an outer branch of the evolutionary tree in the later stage). Prokka software was used to analyze these genomes Re-annotation was performed and pan-genome analysis was performed by Roary software. The phylogenetic tree of the annotated genes was constructed by MEGA7 software, and a fragment on the sigA gene with the ability to distinguish different species was screened. Such as figure 1 It can be seen from the figure that...

Embodiment 2

[0073] Example 2: Validation of Bacillus-specific primers at the genus level

[0074] Select 9 species of bacillus that are frequently reported in raw milk: 1. Bacillus licheniformis, 2. Bacillus cereus, 3. Bacillus subtilis, 4. Thuringiensis Bacillus (B.thuringiensis), 5. Bacillus amyloliquefaciens (B.amyloliquefaciens), 6. Bacillus pumilus (B.pumilus), 7. Bacillus megaterium (B.megaterium), 8. Bacillus mycoides (B. .mycoides), 9. Bacillus anthracis (B. anthracis). And 10 kinds of non-Bacillus raw milk: 1. Flavobacterium maritypicum, 2. Escherichia coli, 3. Lactobacillus fermentum, 4. Enterococcus faecalis, 5. Golden grape Staphylococcus aureus, 6. Pseudomonas fluorescens.

[0075] The genomic DNA of the above-mentioned bacterial strains was extracted respectively, and the microbial genome was extracted with reference to the instructions in the bacterial genomic DNA extraction kit (Tiangen Biochemical Technology (Beijing) Co., Ltd., Beijing, China), using sequences such a...

Embodiment 3

[0082] Example 3: Construction of a rapid detection method for Bacillus and its verification at the species level

[0083] In order to verify the accuracy of primers SEQ ID NO.1 and SEQ ID NO.2 in analyzing Bacillus, Bacillus licheniformis (B.licheniformis), Bacillus cereus (B.cereus), Bacillus subtilis (B.subtilis ), B. thuringiensis, B. amyloliquefaciens, B. pumilus, B. megaterium, B. mycoides, total The genomes of 8 species of Bacillus (as shown in Table 3) are at a concentration of 25-0.005ng / μL (the concentration of the DNA before mixing is shown in Table 3, respectively 0.005, 0.01, 0.02, 0.005, 0.1, 1, 5 and 25ng / μL) Mixing is used to simulate the detection of different species of Bacillus in complex samples. Based on the newly designed bacillus identification primers combined with a next-generation sequencer, then the simulated sample sequencing was carried out according to the method steps described in Example 2, and finally the sequence information of the sigA gen...

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Abstract

The invention discloses a bacillus species level identification method based on a high-throughput sequencing technology, and belongs to the technical field of molecular biology. According to the method provided by the invention, 85 bacillus in a complex sample can be detected and identified, and the detection accuracy rate reaches 100%. High-throughput identification and batch processing of strain information can be realized, the bacillus composition in a complex sample can be comprehensively identified without separation and purification, the bacillus composition can be identified to a seed level, the resolution capability is higher than that of an existing method, meanwhile, the amplification length meets the requirement of next-generation sequencing, and the time and the identification cost can be remarkably saved. The method disclosed by the invention has a good application prospect.

Description

technical field [0001] The invention relates to a Bacillus species-level identification method based on high-throughput sequencing technology, and belongs to the technical field of molecular biology. Background technique [0002] Bacillus can survive in most natural environments and often causes food contamination during acquisition, processing, and handling. Common species mainly include B. licheniformis, B. cereus, B. subtilis, B. thuringiensis and B. amyloliquefaciens . Bacillus has a thick peptidoglycan layer, which improves the adaptability to temperature, humidity and other environments; it can form biofilms within or between genera to enhance its drug resistance and proliferate in large quantities; by forming spores, tolerance Conventional sterilization methods such as thermal processing. Among all food microbial contaminations, it is the most difficult to eliminate and cause continuous impact, leading to the decline of product quality and the loss of enterprises. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/6869C12N15/11C12R1/07
CPCC12Q1/689C12Q1/6869C12Q2531/113C12Q2535/122
Inventor 翟齐啸任泽宇陈卫于雷雷田丰伟王刚陆文伟崔树茂王晨赵建新张灏
Owner JIANGNAN UNIV
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