Novel method for detecting enterobacter sakazakii, application and detection kit

A technology of Enterobacter sakazakii and detection kit, applied in the field of food-borne pathogen detection, can solve the problems of undiscovered patent publications, etc., achieve easy standardization and automatic operation, high reliability and sensitivity, and reduce detection Effects of time and false positive results

Active Publication Date: 2021-09-24
ANIMAL & PLANT & FOOD INSPECTION CENT OF TIANJIN ENTRY EXIT INSPECTION & QUARANTINE BUREAU +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Through the search, no patent publications related to the patent application of the present invention have been found

Method used

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  • Novel method for detecting enterobacter sakazakii, application and detection kit
  • Novel method for detecting enterobacter sakazakii, application and detection kit
  • Novel method for detecting enterobacter sakazakii, application and detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1: Mining of characteristic peptides

[0056] 1) Non-targeted proteomic analysis: UPLC-Q Exactive was used to conduct non-targeted proteomic identification analysis on 21 strains of Enterobacter sakazakii, and then the data was processed by Mascot. Liquid chromatography conditions: flow rate 0.2 mL / mL; injection volume 10 μL; elution program: 0~5 min, keep phase B from 5%; 5~30 min, increase phase B from 5% to 40%; 30~ For 40 min, 95% of phase B was maintained. Mass spectrometry conditions: heated electrospray ion source (HESI); positive ion mode; full scan mode, particle range: m / z 300-1500; resolution 70 000; maximum capacity of Ctrap (AGC target): 100000; maximum injection time of Ctrap : 100 ms; sheath gas velocity: 40 arb; auxiliary gas flow rate: 3 arb; spray voltage: 3.5 kV; capillary temperature: 300 ℃. Mascot software identification conditions: select SwissProt for protein database, trypsin for protease type, 1 maximum missed cleavage site, Carbamidom...

Embodiment 2

[0059] Example 2: Specific verification and optimization of candidate characteristic peptides

[0060] 4) Standard strains of Enterobacter sakazakii, Salmonella and Escherichia coli were cultured in nutrient agar medium (1% peptone, 0.5% NaCl, 0.3% beef extract, 2% agar) respectively, and cultured at 37 °C for 24 h.

[0061] 2) Bacteria collection and sample pretreatment: add 0.5 ml Buffer 1 (C) and (1) treated bacteria liquid, mix well, and ultrasonically break for 10 min. Then, add 100 μL of Buffer 2 (D) and mix quickly, and react in the dark for 15 minutes. Then 4 μL of trypsin (E) was added and digested in a water bath at 50 °C for 15 min. Add 0.5 μL formic acid (F) to terminate trypsin digestion and analyze samples directly or store at -20 °C until analysis.

[0062] 5) MRM pattern targeted analysis of LC-DSI / QQQ. Chromatographic conditions: Vydax C18 column (2.1mm×150mm, 5 μm); flow rate 0.3 ml / min; injection volume 2 μL; mobile phase A is an aqueous solution containi...

Embodiment 3

[0072] Example 3: Application of characteristic peptides in the detection of Enterobacter sakazakii contamination in milk powder samples

[0073] The target pathogenic bacteria liquid was added to pure milk powder (Yili, China) to a concentration of 100 CEU / mL. Take 0.1 mL contaminated milk powder sample, add it to 0.9 mL LB medium, and incubate at 37°C for 12 h. Subsequent steps are consistent with the processing methods (2)-(4) in Example 2. The evaluation results are shown in Table 3.

[0074]

[0075]

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Abstract

The invention discloses a novel method for detecting enterobacter sakazakii, application and a detection kit. According to the method, enterobacter sakazakii pollution in dairy products is detected by analyzing the following characteristic peptides: I1: SEQ ID NO.1; I2: SEQ ID NO.2; I3: SEQ ID NO.3; I4: SEQ ID NO. 4. According to the method, a liquid chromatography triple quadrupole mass spectrometer is used for detecting the characteristic peptide. The detection process of the method does not depend on bacterial colony selection and biochemical identification, the method has high reliability, the detection time is greatly shortened, and a reliable and effective method is provided for detecting enterobacter sakakaakii pollution in food.

Description

technical field [0001] The invention belongs to the technical field of detection of food-borne pathogenic bacteria, in particular to a new method, application and detection kit for detecting Enterobacter sakazakii. Background technique [0002] The existence of foodborne pathogens seriously threatens human health and public health. Pathogenic microorganisms carried by food can reproduce in the host and express virulence factors, thereby damaging the health of the body. Despite increasing levels of public health, foodborne pathogens continue to cause frequent disease outbreaks today. Efficient and rigorous inspection and quarantine methods are extremely important for the control of foodborne pathogenic bacteria and national food safety. [0003] Enterobacter sakazakii ( Cronobacter sakazakii ) is a rod-shaped, non-spore-forming, peri-flagellated, facultative anaerobic Gram-negative foodborne pathogen belonging to the family Enterobacteriaceae. Enterobacter sakazakii was p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02G01N30/06G01N30/32G01N30/72C12Q1/04
CPCG01N30/02G01N30/06G01N30/32G01N30/72C12Q1/04G01N2030/324G01N2333/24Y02A50/30
Inventor 董志珍赵良娟王洪彬王玉迎赵宏庞路宓捷波袁兆霆于敏赵化冰王淞
Owner ANIMAL & PLANT & FOOD INSPECTION CENT OF TIANJIN ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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