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A CRISPR typing method for Cronobacter sakazakii

A typing method and bacillus technology, applied in biochemical equipment and methods, microbial measurement/inspection, etc., can solve problems such as no suitable CRISPR typing method for Cronobacter sakazakii, and facilitate molecular traceability and operation of strains Simple and convenient, easy-to-operate effects

Active Publication Date: 2021-06-15
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

But there is currently no CRISPR typing method suitable for Cronobacter sakazakii

Method used

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  • A CRISPR typing method for Cronobacter sakazakii
  • A CRISPR typing method for Cronobacter sakazakii
  • A CRISPR typing method for Cronobacter sakazakii

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1: The CRISPR typing method of 136 strains of Cronobacter sakazakii, and its resolution comparison with the MLST typing method

[0035] (1) strain

[0036] The strains used in this experiment were selected from 136 strains of Cronobacter sakazakii isolated from various regions in my country preserved by our center. Traditional biochemical identification and molecular detection have been completed, and they have been identified as Cronobacter sakazakii.

[0037] (2) Bacterial DNA extraction

[0038] Genomic DNA of the above-mentioned 136 bacterial strains to be tested was extracted using Magen’s bacterial genomic DNA extraction kit, and Cronobacter sakazakii ATCC 29544 was used as a positive control. ddH 2 O is a negative control. The extracted DNA was stored at -20°C.

[0039] (3) Primer synthesis

[0040] Table 1 Primer list for CRISPR molecular amplification

[0041]

[0042] The concentration of primers used in PCR amplification was 10 μmol / L.

[0043]...

Embodiment 2

[0079] Example 2: Comparison of Molecular Traceability of CRISPR Typing and MLST Typing Methods

[0080] (1) Download the full-length genomes of 23 strains (including ST types ST4, ST12 and ST13) isolated from patients and feeding formula after the outbreak of Cronobacter sakazakii infection in French neonatal intensive care units in 1994 Sequence (Masood et al. BMC Genomics 16:750), determine the type and quantity of CRISPR molecules according to the method in Example 1, and extract the spacer sequence for typing.

[0081] (2) Three ST-type strains ST4, ST12 and ST13 in Example 1 were selected to compare the CRISPR typing and MLST typing methods, and the results are shown in Table 3 below.

[0082] Table 3 Comparison of CRISPR and MLST typing results of 38 strains of Cronobacter sakazakii

[0083]

[0084]

[0085] As shown in Table 3, three ST-type strains isolated from patients in the outbreak of Cronobacter sakazakii infection in the French neonatal intensive care u...

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PUM

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Abstract

The invention discloses a CRISPR typing method for Cronobacter sakazakii. This method performs DNA sequencing on the CRISPR1, CRISPR2, CRISPR3 and CRISPR6 site sequences of Cronobacter sakazakii, extracts the spacer sequences in the four site sequences, and determines the Cronobacter sakazakii according to the combination of the four spacer sequences. CRISPR type. The method of the present invention is simple, fast, low-cost, and has higher resolution than the MLST typing method, has no environmental pollution, and has low requirements for laboratory equipment and software, and the CRISPR molecule has many information such as historically infected phages and plasmids, and can The joint database realizes national or even global standardized molecular traceability, which can be extended to food, inspection and quarantine and other fields.

Description

technical field [0001] The invention belongs to the field of molecular epidemiology, and in particular relates to a CRISPR typing method for Cronobacter sakazakii. Background technique [0002] Cronobacter sakazakii (Cronobacter sakazakii) is an important food-borne pathogen that can infect people of all ages. Infecting infants, especially premature infants, low birth weight infants or immunocompromised infants can lead to meningitis and necrosis Enterocolitis and bacteremia, with a mortality rate as high as 40% to 80%, have attracted global attention. The International Committee on Microbiological Standards for Food (ICMSF) defines Cronobacter as "a dangerous microorganism that poses a serious threat to the life of a specific population, or produces serious chronic sequelae." The Food and Agriculture Organization of the United Nations and the World Health Organization have listed Cronobacter as a group A pathogenic bacteria in infant formula. Cronobacter sakazakii is the ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6869C12Q1/689C12Q1/04
CPCC12Q1/6869C12Q1/689C12Q2531/113C12Q2535/122
Inventor 曾海燕李程思吴清平张菊梅何文静
Owner GUANGDONG INST OF MICROBIOLOGY GUANGDONG DETECTION CENT OF MICROBIOLOGY