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Salmonella enteritidis bacteriophage SEP37 and electrochemical impedance spectroscopy sensor and detection method thereof

A Salmonella Enteritidis and Salmonella technology, applied in the field of biosensors, can solve the problems of time-consuming and laborious, prone to false positive results, inability to distinguish live and dead cells, etc., and achieves improved sensitivity, good linear response, repeatability and stability improvement. Effect

Active Publication Date: 2022-02-01
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] The foodborne disease caused by Salmonella is a serious public health problem worldwide, which brings a heavy burden to human health and economic development. However, the existing detection methods have more or less shortcomings, such as microbial culture and physiological and biochemical Identification is time-consuming and laborious. Immunological methods need to obtain high-affinity antibodies. Molecular biological methods cannot distinguish living and dead cells, and are prone to false positive results. Therefore, the rapid and accurate detection of Salmonella has always been a research hotspot.

Method used

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  • Salmonella enteritidis bacteriophage SEP37 and electrochemical impedance spectroscopy sensor and detection method thereof
  • Salmonella enteritidis bacteriophage SEP37 and electrochemical impedance spectroscopy sensor and detection method thereof
  • Salmonella enteritidis bacteriophage SEP37 and electrochemical impedance spectroscopy sensor and detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0074] Example 1: Phage Separation and Screening, Purification and Value Value and Morphological Analysis

[0075] Phage Sep37 separation and screening

[0076] The sewage samples in Baishazhou, Wuhan, were collected and filtered using filter paper. Take 5 ml of filtrate and 5 mL of cultured to the colitis Salmonella ATCC 13076 in 20 ml of LB. Methods were repeated once to obtain phage stock solution. The separation and identification of the bilateral plate method was used to identify the double-layer plate method, and the cultured Salmonella ATCC 13076 100 μL, the phage raw liquid 100 μl was gently mixed with a semi-solid medium of 3.8 mL 45 to 50 ° C to the lower layer medium, and After solidification, it was inverted at a 37 ° C incubator culture for about 6 h.

[0077] like figure 1 As shown: The transparent circle appears in the figure, it demonstrates the presence of the phage.

[0078] 2. Phage Purification value

[0079] Phage Sep37 is passed with OD 600 The culture of ent...

Embodiment 2

[0084] Example 2: Analysis of Biological Characteristics of Phage Sep37

[0085] Phage Sep37 host spectrum analysis

[0086] Determination of phagebar binder spectrometry adopts a sample method:

[0087] Take 100μL OD 600 = 0.6 to be tested into the warm semi-solid medium, mixed into a pre-prepared LA plate after mixing, and taking 5 μl of titer is 1 × 10 after being solidified. 9 Phage dropped in PFU / mL was added to the upper plate surface, and the dried was dried in a 37 ° C incubator culture 4 to 6 hours, and the cleavage is observed, and the results are shown in Table 1.

[0088] Table 1 Phage Sep37 Subject

[0089]

[0090]

[0091] Note: ATCC a American Type Culture Collection; SJTU b SHA NGHAI JIAO Tonguniversity; CMCC c CENTER for Medical Culture Collectes; CVCC d China Veterinaryculture Collection Center; "++" Strong Intensity (Clear Plaque); "+" WeailinTensity (Opaque Plaque); "-" NO LYTIC Activity (No Plaque).

[0092] 2. Best infection of phage (MOI)

[0093] Mul...

Embodiment 3

[0104] Example 3: Electrochemical impedance spectrum biosensor based on phage detection Salmonella

[0105] 1. Preparation of the working electrode (GDE-AUNPS-CYS-PHAGESEP37-BSA) composite electrode

[0106] 1) Pretreatment of the gold disk electrode (GDE) to obtain an amino functionalized surface:

[0107] Naked GDEs were performed on the polishing cloth, and naked GDE was used to mechanically polish the naked GDE, and then thoroughly cleaned with deionized water, acetone and ethanol, respectively; then in 0.5 mol / l h, respectively. 2 SO 4 In the solution, the voltage of 100 mV / s was scanned 100 times between -0.2 to 1.0 V, electrochemically cleaning the electrode was electrochemically cleaned, and then washed with deionized water and used n. 2 Dry airflow;

[0108] 2) Gold nanoparticles with 25 ~ 30 nm diameter to the electrode work surface to get GDE-AUNPS

[0109] 3) After drying in a clean environment, the gold disk electrode GDE-AUNPS carrying AUNPS is immersed in 4 ° C,...

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Abstract

The invention discloses a salmonella enteritidis bacteriophage SEP37, an electrochemical impedance spectroscopy sensor and a detection method thereof, wherein the preservation number of the salmonella enteritidis bacteriophage SEP37 is CCTCC NO: M 20211127. According to the invention, the sensor takes the bacteriophage as a biological recognition element and consists of a measuring device comprising three electrodes, a nitrogen supply device and an electrochemical analyzer, wherein the electrochemical analyzer is communicated with a superior computer; the bacteriophage SEP37 has the characteristic of high specificity; the electrochemical impedance spectroscopy sensor obtained by utilizing the bacteriophage SEP37 is a potential tool capable of quickly and accurately detecting salmonella in various samples; the detection method of the sensor has the characteristics of low detection limit, high specificity, good stability and high detection speed; and when the sensor is used for detecting labeled lake water, lettuce and chicken samples, the result shows that the sensor can quickly and accurately detect and quantify the concentration of bacteria in the samples.

Description

Technical field [0001] The present invention relates to the technical field of biosensor, and more particularly to the SEP37 and electrochemical impedance spectroscopy sensors and detection methods thereof. Background technique [0002] Salmonella is a serious public health issue in the world, which gives a heavy burden on the health and economic development of human beings, and there are currently more or less shortcomings, such as microbial culture and physiological and biochemical. Identification, time-consuming, immunological methods need to obtain high affinity antibodies, molecular biological methods cannot distinguish between live-dead cells, prone to false positive results, etc., Salmonella's rapid, accurate detection, has always been research hotspots. In recent years, phage-based biosensor is considered to be efficient and simple tools for detecting a source of foodborne pathogens, and increasingly causing concern about researchers. It is expected to be a good choice, h...

Claims

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Application Information

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IPC IPC(8): C12N7/00C12Q1/10C12Q1/00G01N27/26G01N27/02C12R1/92
CPCC12N7/00C12Q1/10C12Q1/001G01N27/26G01N27/02C12N2795/10121Y02A50/30
Inventor 王小红王吉李慧慧丁一峰朱文娟
Owner HUAZHONG AGRI UNIV
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