A CRISPR typing method for Cronobacter malonate

A technology of Cronobacter malonate and typing method, applied in the direction of microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve the problem of high cost of genome sequencing, and achieve low typing cost and good resolution The effect of high rate and high resolution

Active Publication Date: 2020-10-09
GUANGDONG INST OF MICROBIOLOGY GUANGDONG DETECTION CENT OF MICROBIOLOGY +1
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  • Abstract
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AI Technical Summary

Problems solved by technology

A large number of typing methods based on gene sequencing are applied to the molecular traceability of pathogenic bacteria, such as the widely used multi-locus sequence typing method (multi-locus sequence typing, MLST), the core genome multi-locus sequence typing method (cgMLST). ), whole genome multi-locus sequence typing (wgMLST), etc., based on the whole genome typing effect is the best, but the cost of genome sequencing is high, and operators need to have better bioinformatics analysis foundation, it is difficult to popularize and apply

Method used

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  • A CRISPR typing method for Cronobacter malonate
  • A CRISPR typing method for Cronobacter malonate
  • A CRISPR typing method for Cronobacter malonate

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 164

[0033] Example 1 CRISPR typing method of 64 strains of Cronobacter malonate

[0034] (1) strain

[0035] The 64 strains used in this experiment were isolated from milk powder, edible fungus and vegetable samples in various regions of my country. Traditional biochemical identification and molecular detection have been completed, and they were identified as Cronobacter malonate.

[0036] (2) Bacterial DNA extraction

[0037] Genomic DNA of the above-mentioned 64 strains to be tested was extracted using the bacterial genomic DNA extraction kit from Magen Company, and the extracted DNA was stored at -20°C.

[0038] (3) Primer synthesis

[0039] Table 1 Primer list for CRISPR molecular amplification

[0040]

[0041]

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

[0043] (4) PCR amplification

[0044] 4.1 CRISPR1 amplification PCR reaction system:

[0045]

[0046] The PCR reaction conditions for CRISPR1 amplification were as follows:...

Embodiment 2

[0069] Example 2 CRISPR typing of Cronobacter malonate and comparison of the resolution of MLST typing methods

[0070] (1) MLST classification

[0071] Seven housekeeping genes of Cronobacter malonionum were mainly selected for amplification and sequencing, and the obtained sequence information was uploaded to a special website ( https: / / pubmlst.org / cronobacter / ) to obtain the corresponding MLST type, see the MLST website for specific experimental operation methods.

[0072] (2) Comparison of the resolution of the two typing methods

[0073] Table 3 Comparison of MLST typing and CRISPR typing results of Cronobacter malonobacter

[0074]

[0075]

[0076]

[0077] The resolution evaluation is quantified by the Simpson diversity index (D), and its calculation formula is: D=1-∑[nj(nj-1)] / [N(N-1)], where nj represents the value of the jth band type The number of strains, N represents the total number of experimental strains. The larger the D value, the stronger the...

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Abstract

The invention discloses a cronobacter sakazakii CRISPR classification method. In the method, four CRISPR molecules of cronobacter sakazakii are sequenced to extract the interval sequence in the sequences, and the CRISPR type of the cronobacter sakazakii is determined according to the combination of the interval sequences of the sequences. The method is simple, rapid, low in cost, higher in resolution than an MLST classification method, free of environmental pollution and low in requirements for laboratory equipment, the CRISPR modules preserve lots of information about historically infected phages and plasmids, which can realize national or even global standardized molecular traceability through a joint database, and the method is suitable for promotion 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 malonate. Background technique [0002] The health of infants and young children has always been the focus of national and social attention. For artificially fed newborns, the safety of milk powder and its substitute food is very important. Cronobacter (Enterobactersakazakii), also known as Cronobacter (Enterobactersakazakii), is a highly pathogenic bacterium detected in milk powder, which can cause infantile meningitis, necrotizing enterocolitis and bacteremia, with a high mortality rate 40% to 80%, has attracted global attention. At present, it is believed that the contamination of Cronobacter in infant formula milk powder is the main channel for newborns to be infected with the bacteria. The International Food and Agriculture Organization and the World Health Organization have listed Cronobacter spp. However, recent cas...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/689C12Q1/6869C12Q1/04C12R1/01
Inventor 曾海燕吴清平李程思张菊梅杨小鹃王涓丁郁陈谋通张淑红叶青华雷涛
Owner GUANGDONG INST OF MICROBIOLOGY GUANGDONG DETECTION CENT OF MICROBIOLOGY
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