Method for rapidly identifying bacillus cereus and bacillus thuringiensis based on MALDI-TOFMS

A technology of Bacillus thuringiensis and Bacillus cereus, applied in the field of microbial detection, can solve the problems of low resolution, inability to distinguish between Bacillus cereus and Bacillus thuringiensis, etc., achieve accurate and reliable results, reduce labor and consumables costs, Simple operation effect

Inactive Publication Date: 2021-08-06
GUANGDONG INST OF MICROBIOLOGY GUANGDONG DETECTION CENT OF MICROBIOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the current commercial data analysis software Biotyper has a low resolution for closely related species, and it is not yet possible to accurately distinguish Bacillus cereus from Bacillus thuringiensis

Method used

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  • Method for rapidly identifying bacillus cereus and bacillus thuringiensis based on MALDI-TOFMS
  • Method for rapidly identifying bacillus cereus and bacillus thuringiensis based on MALDI-TOFMS
  • Method for rapidly identifying bacillus cereus and bacillus thuringiensis based on MALDI-TOFMS

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Example 1: Mining potential MALDI-TOFMS characteristic peaks of Bacillus cereus and Bacillus thuringiensis based on genome-wide data

[0020] Search GTDB (https: / / gtdb.ecogenomic.org / ), rMLST (https: / / pubmlst.org / rmlst) and NCBI (https: / / www.ncbi.nlm.nih.gov / genome / ) three databases Among the taxonomic information of Bacillus cereus and Bacillus thuringiensis, 106 strains of Bacillus cereus and 175 strains of Bacillus thuringiensis had consistent species information in the three databases. After downloading the whole genome sequence of the strain, use Prokka to annotate the whole gene sequence, and use TBtools to extract the ribosomal protein sequence. Analyzes ribosomes by considering N-terminal methionine cleavage when the second amino acid at the N-terminus of the amino acid chain is glycine, alanine, proline, serine, threonine, valine, or cysteine For protein N-terminal methionine removal, use the protein molecular mass calculator (https: / / web.expasy.org / compute_pi...

Embodiment 2

[0024] Example 2 Screening and identification of MALDI-TOF MS characteristic peaks of Bacillus cereus and Bacillus thuringiensis

[0025]35 strains of Bacillus cereus, 44 strains of Bacillus thuringiensis and their corresponding ATCC standard strains were selected for experiments. The strains were streaked on LB agar and cultured at 37°C for 16 hours. Use a sterile toothpick to directly smear the colony on the target plate, add 1 μL of 70% formic acid aqueous solution by volume fraction, wait for it to dry, add 1 μL of standard solution containing 10 mg / mL α-cyano-4-hydroxycinnamic acid (by volume fraction, Consisting of 50% acetonitrile, 47.5% water and 2.5% trifluoroacetic acid), dried for detection. MALDI-TOF MS detection uses a 200Hz smartbeam solid-state laser to collect spectra in the range of 2-20kDa in positive ion linear mode. Each sample is bombarded 240 times by laser, and BTS (bacterial test standard) is used for calibration before each experiment. The collected m...

Embodiment 3

[0029] Example 3 Specificity Evaluation Results of MALDI-TOF MS Characteristic Peaks of Bacillus cereus and Bacillus thuringiensis

[0030] According to the national and international standards of common foodborne pathogenic bacteria in food microbiological examination, 13 strains of common foodborne pathogenic bacteria were selected for the specificity test of MALDI-TOF MS characteristic peaks of Bacillus cereus and Bacillus thuringiensis. The strain obtained the MALDI-TOF MS spectrum through the cultivation and sample preparation steps in Example 2, and the specificity results are shown in Table 3. The MALDI-TOF MS characteristic peaks of Bacillus cereus and Bacillus thuringiensis in the present invention do not overlap with the mass spectrum peaks of common food-borne pathogenic bacteria, so they are specific to Bacillus cereus and Bacillus thuringiensis. The characteristic peak of the present invention can be used as a specific MALDI-TOF MS peak for distinguishing Bacillus...

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Abstract

The invention discloses a method for rapidly identifying bacillus cereus and bacillus thuringiensis based on MALDI-TOFMS. According to the invention, a 50S ribosomal protein L30, a 50S ribosomal protein L31 type B and a 30S ribosomal protein S20 are used as characteristic proteins. According to the method, whole genome sequencing information of the bacillus cereus and the bacillus thuringiensis is used for guiding screening and identification of the MALDI-TOFMS characteristic peaks, the MALDI-TOFMS characteristic peaks capable of accurately distinguishing the bacillus cereus and the bacillus thuringiensis are obtained, the result is accurate and reliable, and a thought is provided for distinguishing related species through the MALDI-TOFMS.

Description

Technical field: [0001] The invention belongs to the field of microorganism detection, and in particular relates to a rapid identification method for Bacillus cereus and Bacillus thuringiensis based on MALDI-TOF MS. Background technique: [0002] Bacillus cereus (Bacillus cereus) is one of the common food-borne pathogens in my country. The enterotoxin and vomitoxin produced by it can cause two kinds of gastrointestinal diseases, diarrhea and vomiting. From 2006 to 2016, Bacillus cereus ranked third among food poisoning incidents caused by foodborne pathogens in my country. Bacillus thuringiensis is an entomopathogenic bacterium that produces insecticidal crystal proteins while forming spores and is therefore used as a biopesticide. Bacillus cereus and Bacillus thuringiensis belong to the Bacillus cereus group, the 16S rRNA gene sequence similarity is greater than 99%, and the dDDH (digital DNA-DNA hybridization) value is greater than the species classification threshold of ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/06G01N30/72
CPCG01N30/06G01N30/72
Inventor 吴清平陈敏玲丁郁王涓张菊梅张俊卉周桓余树波陈谋通薛亮叶青华韦献虎张友雄
Owner GUANGDONG INST OF MICROBIOLOGY GUANGDONG DETECTION CENT OF MICROBIOLOGY
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