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Salmonella and bacteria total number rapid synchronous multiple detection method and kit

A technology for Salmonella and total bacterial counts, applied in biochemical equipment and methods, microbiological-based methods, measuring devices, etc., can solve problems such as difficult multiple detection of Salmonella and total bacterial counts, inability to identify all types of bacteria, time-consuming and labor-intensive problems, etc.

Pending Publication Date: 2021-03-26
NAT INST OF METROLOGY CHINA
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The traditional culture method for the measurement of Salmonella is time-consuming and laborious, such as the national standard GB 4789.4-2016 "National Food Safety Standard Food Microbiology Examination of Salmonella", this method requires a series of steps such as pre-enrichment, enrichment, cultivation, and biochemical identification. It takes more than 60 hours, and BPW, TTB, SC enrichment solution, XLD, BS, HE, chromogenic medium, and subsequent biochemical identification also need a lot of reagents
[0004] The plate counting method of the total number of bacteria also has many shortcomings, such as the national standard GB 4789.2-2016 "Determination of the total number of colonies", this method requires sampling, dilution, pouring plate, culturing, etc. A series of steps, took 48 hours
[0005] These two methods require a lot of manual operation, time-consuming and labor-intensive, and the detection of the two indicators of Salmonella and the total number of bacteria must be carried out separately
[0006] Although many new technologies are currently used for the rapid detection of Salmonella or total bacterial counts, these technologies are difficult to use for multiple detection of Salmonella and total bacterial counts
For example, the existing biochip method cannot identify all types of bacteria; the PCR method can only realize multiple detection of different types of bacteria, but cannot realize the detection of the total number of bacteria
[0007] Flow cytometry technology has the potential to achieve multiple detection of Salmonella and total bacterial count indicators, but due to many technical difficulties in the development of Salmonella-specific fluorescent probes, there have been no relevant reports so far.

Method used

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  • Salmonella and bacteria total number rapid synchronous multiple detection method and kit
  • Salmonella and bacteria total number rapid synchronous multiple detection method and kit
  • Salmonella and bacteria total number rapid synchronous multiple detection method and kit

Examples

Experimental program
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Embodiment 1

[0084] The preparation of embodiment 1 Salmonella antibody

[0085] 1. Materials and methods

[0086] 1. A total of 2 experimental rabbits, with an initial weight of 2.3-2.5kg. Immunization method: multiple intradermal injections on the back. Immunogen: Salmonella-specific membrane protein. Process: A total of three immunizations were carried out with an interval of half a month. Then carry out the potency test. Then the fourth immunization was carried out, and the serum was collected after the titer test was suitable, and the antibody was purified. Got 2 Salmonella antibodies.

[0087] 2. Use the ELISA endpoint method to detect the titer of the obtained 2 Salmonella antibodies. Antibody concentration was detected by SDS-PAGE method.

[0088] 2. Experimental results

[0089] Preliminary preparation of 2 Salmonella, antibody 1 titer 1:1.25×10 6 , antibody 2 titer 1:3.125×10 5 . The electrophoresis results are as follows figure 1 shown.

[0090] Table 1 Antibody Tit...

Embodiment 2

[0095] Evaluation of Example 2 Salmonella Antibody

[0096] 1. Materials and methods

[0097] 1. Use the FITC labeling kit to label the prepared 8 Salmonella antibodies (Ab-1 to Ab-8) with fluorescein FITC.

[0098] 2. Salmonella paratyphi A, Salmonella typhimurium, Salmonella choleraesuis, Salmonella bonarene, Salmonella Kentucky, Salmonella enteritidis, Salmonella Sanftenburg, Salmonella abatetus. were prepared at a concentration of approximately 1×10 7 cells / mL of the above-mentioned Salmonella bacterium liquid.

[0099] 3. Add FITC-labeled antibodies (Ab-1 to Ab-8) at a concentration of 1 μg / mL to the bacterial liquids of the above eight strains of Salmonella, and incubate for 15 minutes in the dark. They were detected by fluorescence microscope and flow cytometer respectively.

[0100] 2. Experimental results

[0101] When labeled with Ab-1, Ab-4, Ab-6, and Ab-7, the eight strains of Salmonella all detected green fluorescence ( figure 2 ). When labeled with Ab-2 a...

Embodiment 3

[0106] Example 3 Affinity Analysis of Salmonella Antibody and Salmonella Membrane Protein

[0107] 1. Materials and methods

[0108] 1. Salmonella paratyphi A, Salmonella typhimurium, Salmonella choleraesuis, Salmonella bonarene, Salmonella Kentucky, Salmonella enteritidis, Salmonella Sanftenburg, Salmonella abatetus.

[0109] 2. Dilute the 4 Salmonella antibodies (Ab-1, Ab-4, Ab-6, Ab-7) obtained from the preliminary screening and 5 commercially purchased Salmonella antibodies (No. Ab-9 to Ab-13) to the same initial concentration, and 2-fold serial dilutions were performed.

[0110] 3. Separately prepare a concentration of about 1×10 7 Cells / mL of the above-mentioned Salmonella bacterium liquid was killed by heating to lyse its cell membrane, and then use the antibody to the structural antigen of Salmonella to carry out the fishing reaction to capture the specific membrane protein of Salmonella.

[0111] 4. Using the EDC / NHS reaction, link Salmonella-specific membrane prot...

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Abstract

The invention discloses a method and detection kit for rapidly and synchronously detecting the total number of salmonella and bacteria in a sample in multiple times. A red fluorescent probe capable ofmarking all bacteria is used for marking the total number of bacteria to distinguish the bacteria and other background particles in the sample, and meanwhile, by a green fluorescent probe, a salmonella antibody cross-linked through a chemical group is used for distinguishing salmonella and non-salmonella in the sample; and red and green fluorescence signals are simultaneously counted through a flow analyzer, so that the synchronous detection of the total number of salmonella and bacteria is realized. The method can be used for simultaneously detecting the total number of salmonella and bacteria, is simple and convenient to operate and short in consumed time, and can be used for detecting most samples within 0.5 hour.

Description

Technical field: [0001] The invention belongs to the field of food microbiology detection, and in particular relates to rapid synchronous multiple detection and a kit for two indicators of salmonella and total number of bacteria in food samples. Background technique: [0002] The total number of colonies (usually referring to the total number of bacteria) and salmonella indicators are the most basic and common testing items in the GB4789 series of food microbiological testing. [0003] The traditional culture method for the measurement of Salmonella is time-consuming and laborious, such as the national standard GB 4789.4-2016 "National Food Safety Standard Food Microbiology Examination of Salmonella", this method requires a series of steps such as pre-enrichment, enrichment, cultivation, and biochemical identification. It takes more than 60 hours, and BPW, TTB, SC enrichment solution, XLD, BS, HE, chromogenic medium, and subsequent biochemical identification also need a lot ...

Claims

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Application Information

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IPC IPC(8): C12Q1/10C12Q1/06G01N33/569G01N15/14C12R1/42
CPCC12Q1/10C12Q1/06G01N33/56916G01N15/1434G01N2333/255G01N2015/1486
Inventor 隋志伟刘思渊王梓权
Owner NAT INST OF METROLOGY CHINA
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