Cronobacter sakazakii CRISPR typing method

A typing method and bacillus technology, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve the problem of no suitable CRISPR typing method for Cronobacter sakazakii, etc., to facilitate the molecular traceability and operation of strains Simple and convenient, low equipment requirements

Active Publication Date: 2019-02-01
GUANGDONG INST OF MICROBIOLOGY GUANGDONG DETECTION CENT OF MICROBIOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But there is currently no CRISPR typi...

Method used

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  • Cronobacter sakazakii CRISPR typing method
  • Cronobacter sakazakii CRISPR typing method
  • Cronobacter sakazakii CRISPR typing method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1: CRISPR typing method of 136 strains of Kronobacter sakazaki, and its resolution comparison with MLST typing method

[0035] (1) Strain

[0036] The strain used in this experiment was selected from 136 strains of Kronobacter sakazaki isolated from various regions of our country preserved in our center. Traditional biochemical identification and molecular testing have been completed and it is determined to be Kronobacter sakazaki.

[0037] (2) Extraction of bacterial DNA

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

[0039] (3) Primer synthesis

[0040] Table 1 Primer list for CRISPR molecule amplification

[0041]

[0042] The primer concentrations used in PCR amplification are all 10 μmol / L.

[0043] (4) PCR amplification

[0044] 4.1 CRISPR1 amplification PCR reac...

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 ST4, ST12 and ST13) isolated from patients and feeding formula after the outbreak of Cronobacter Sakazaki in the French neonatal intensive care unit in 1994 Sequence (Masoodet al. BMC Genomics 16:750), according to the method in Example 1, determine the type and number of CRISPR molecules, and extract the spacer sequence for typing.

[0081] (2) Three ST-type strains of ST4, ST12 and ST13 in Example 1 were selected for comparison of 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 Cronobacter sakazaki strains

[0083]

[0084]

[0085] As shown in Table 3, three ST-type strains isolated from patients in the French neonatal intensive care unit outbreak of Kronobacter Sakazaki infection are ST4 (13 strains), ST12 (5 strain...

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Abstract

The invention discloses a Cronobacter sakazakii CRISPR typing method. The method is as follows: performing DNA sequencing on CRISPR1, CRISPR2, CRISPR3 and CRISPR6 locus sequences of Cronobacter sakazakii, extracting spacer sequences of the four locus sequences, and determining CRISPR type of the Cronobacter sakazakii based on the combination of the four spacer sequences. The method is simple, rapid and low-cost, the resolution of the method is higher than the resolution of MLST typing method, and the Cronobacter sakazakii CRISPR typing method has no environmental pollution, and has low requirements on laboratory equipment and software, CRISPR molecules preserve a lot of information such as historically infected phage and plasmid, by joint with a database, national and even global standardization molecule traceability can be realized, and the method can be extended to the fields of food, inspection and quarantine.

Description

Technical field [0001] The invention belongs to the field of molecular epidemiology, and specifically relates to a CRISPR typing method of Kronobacter sakazaki. Background technique [0002] Cronobacter sakazakii (Cronobacter sakazakii) is an important food-borne pathogen, which can infect people of all ages. Infecting infants and young children, especially premature infants, low birth weight infants or immunocompromised infants can cause meningitis and necrosis Enterocolitis and bacteremia, with a mortality rate as high as 40% to 80%, have attracted worldwide attention. The International Committee for Food Microbiological Standards (ICMSF) defines Cronobacter as "a kind of microorganisms that cause serious life hazards to specific populations or produce serious chronic sequelae." The International Food and Agriculture Organization and the World Health Organization have listed Cronobacterium as a type A pathogen in infant formula. Kronobacter sakazaki is the most important spec...

Claims

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Application Information

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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
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