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Method for detecting enterobacter sakazakii in water

A technology for the detection of Enterobacter sakazakii and its detection method, which is applied in the field of detection of Enterobacter sakazakii in water, can solve the problems of unsuitable water sample detection, lower detection rate, cumbersome steps, etc., shorten the inspection report time, improve High detection rate and high sensitivity

Pending Publication Date: 2021-05-11
ICDC CHINA CDC +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Therefore, the use of food testing methods to detect these two types of water samples will cause serious contamination and vigorous growth of miscellaneous bacteria, which will form a competitive advantage with Enterobacter sakazakii, cover up the growth of Enterobacter sakazakii, and affect the subsequent detection of Enterobacter sakazakii. Unfavorable separation will lead to a decrease in the detection rate or even a false negative result; on the other hand, disinfection of the water sample with hypochlorous acid will cause damage to Enterobacter sakazakii and make it difficult to recover, which will eventually cause the isolation rate of Enterobacter sakazakii reduced, even false-negative results
[0004] In addition, the inventors found that the above-mentioned standard method for the detection of food samples undergoes pre-enrichment and selective enrichment steps, but because the existence of microorganisms in water is different from that of food, it is not suitable for the detection of water samples , and according to the inspection method of food samples, it takes 5 to 6 days from the beginning of the detection to the report of the results. The steps are relatively cumbersome and require a lot of manpower and material resources. It is proved that it is not suitable for the growth of some Enterobacter sakazakii (including the standard strain ATCC 29544), which will cause the missed detection of the bacteria, and only pick the yellow colonies on the TSA plate for the identification of Enterobacter sakazakii, which will also cause some non-producing bacteria. Missed or even false-negative results of yellow-pigmented Enterobacter sakazakii

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  • Method for detecting enterobacter sakazakii in water

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Example 1 Detection of Enterobacter sakazakii in reservoir water by serial concentration and dilution method

[0061] Proceed as follows:

[0062] 1. Water sample collection: Use a 50mL sterile centrifuge tube to collect 50mL of reservoir water samples, transport them to the laboratory at room temperature, and wait for inspection.

[0063] 2. Sample dilution: The collected water sample was centrifuged at 5500rpm for 20min, discarded the supernatant, added 10mL of sterile ultrapure water to resuspend and mix well, then took 1mL as the stock solution for gradient dilution, and then took 100μl in the stock solution for 10 times Gradient dilution, according to the degree of contamination, obtain 10 times, 100 times, 1000 times dilutions...

[0064] 3. Isolation of bacterial colonies: Take the above-mentioned appropriate 3 serial dilutions, and apply 100 μl of the dilution on the chromogenic Cronobacter isolation (CCI) agar plate of the Cronobacter genus chromogenic medium ...

Embodiment 2

[0067] Example 2 Detection of Enterobacter sakazakii in self-prepared well water by serial concentration and dilution method

[0068] Proceed as follows:

[0069] 1. Water sample collection: Use a 500mL sterile bottle or water sample collection bag to collect 500mL of self-prepared well water samples, transport them to the laboratory at room temperature, and wait for inspection.

[0070] 2. Dilution of samples: the collected water samples are divided into sterile centrifuge tubes, centrifuged at 5500rpm for 20min, discard the supernatant, add 10mL sterile ultrapure water to each tube to resuspend and mix, and then take 1mL from each tube as a gradient Dilute the stock solution, and then take 100 μl of the stock solution for 10-fold gradient dilution, and obtain 10-fold, 100-fold dilutions according to the degree of contamination...

[0071] 3. Isolation of bacterial colonies: Take the above-mentioned 3 appropriate serial dilutions, combine the dilutions of the centrifuge tube...

Embodiment 3

[0074] Example 3 Membrane filter method for detection of Enterobacter sakazakii in secondary water supply

[0075] Proceed as follows:

[0076] 1. Water sample collection: Collect 200mL of drinking water with a 500mL sterile bottle or water sample collection bag, transport it at room temperature, and wait for inspection.

[0077] 2. Filtration of samples: Use a vacuum pump to filter the collected water samples through the cellulose filter membrane (if the microbial contamination is serious, it can be filtered in 4 times), the size of the filter membrane is 47mm, and the pore size is 0.45μm.

[0078] 3. Recovery of bacteria: Paste the above filter membrane on the TSA plate after filtering the water samples, and incubate at 36±1°C for 4±2 hours to recover the damaged bacteria.

[0079] 4. Isolation of bacterial colonies: transfer the filter membrane of the recovered bacteria to a CCI agar plate, separate and culture at 36±1° C. for 20±2 hours.

[0080] 5. Colony identification...

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Abstract

The invention provides a method for detecting enterobacter sakazakii in water, and belongs to the technical field of microbiological detection and analysis. When a water sample to be detected is source water, the method comprises the steps of sample dilution, bacterial colony separation and bacterial colony identification. When the water sample to be detected is domestic drinking water, the method comprises the following steps of sample filtering, thallus recovering, bacterial colony separation and bacterial colony identification. According to the method for detecting the enterobacter sakazakii in the water, a concentration and dilution method and a filter membrane method detection method are independently designed according to the characteristics of different water samples; and meanwhile, part of existing detection steps are reasonably simplified, and the method for detecting the enterobacter sakazakii in the water has the advantages of being rapid, efficient, good in specificity, high in sensitivity and the like, so that good practical application value is achieved.

Description

technical field [0001] The invention belongs to the technical field of microbial detection and analysis, and in particular relates to a detection method for Enterobacter sakazakii in water. Background technique [0002] The information disclosed in this background section is only intended to increase the understanding of the general background of the present invention, and is not necessarily taken as an acknowledgment or any form of suggestion that the information constitutes the prior art already known to those skilled in the art. [0003] At present, the detection of Enterobacter sakazakii by the US FDA, ISO and China National Standards are all aimed at the detection of food samples, and their benchmark methods are based on pre-enrichment, selective enrichment, isolation and identification There are slight differences in culture medium and identification methods. However, due to the particularity of water samples compared with food samples, they are mainly in liquid state...

Claims

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Application Information

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IPC IPC(8): C12Q1/04C12R1/01
CPCC12Q1/04
Inventor 崔志刚刘铭胡锦瑞刘辉时玉雯胡光春李健关恒云刘岚铮
Owner ICDC CHINA CDC