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A kind of detection method of Salmonella enteritidis and its detection test strip

A technology for detection of Salmonella enteritidis and test strips, which is applied in the fields of measuring devices, instruments, and biological material analysis, and can solve the problems of monoclonal antibody screening process such as tedious, complicated, and inability to guarantee consistency

Inactive Publication Date: 2019-08-09
NORTHWEST A & F UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Unfortunately, it has been reported that polyclonal antibodies (PcAbs) are not commensurate with monoclonal antibodies (McAbs) in terms of specificity, and that even different batches of polyclonal antibodies prepared from the same antigen cannot guarantee their consistency
Although cell fusion is a classic technique for monoclonal antibody production, the screening process of monoclonal antibodies is tedious and complicated, and there is a lucky factor in producing high-quality antibodies, so that these two high-quality antibodies form a perfect Sandwich pairings are more difficult

Method used

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  • A kind of detection method of Salmonella enteritidis and its detection test strip
  • A kind of detection method of Salmonella enteritidis and its detection test strip
  • A kind of detection method of Salmonella enteritidis and its detection test strip

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Embodiment 1

[0033] Embodiment 1: Preparation method of Salmonella enteritidis universal monoclonal antibody

[0034] The preparation method of Salmonella enteritidis universal monoclonal antibody comprises the following steps:

[0035] 1) Animal immunity

[0036]Purchase 6-week-old BALB / c mice, and use the Salmonella enteritidis preserved in our laboratory provided by Yang Baowei's laboratory to extract flagellin for immunization. For the first immunization, 0.8 mg / mL Salmonella enteritidis flagellin was mixed and emulsified with an equal amount of Freund's complete adjuvant, and the mice were injected intraperitoneally with the emulsified antigen. The second immunization was carried out 4 weeks later, using 0.8 mg / mL Salmonella enteritidis flagellin mixed with an equal amount of Freund's incomplete adjuvant to emulsify, and the mice were intraperitoneally injected with the emulsified antigen. The third immunization was carried out 4 weeks later, and the immunization method was the same...

Embodiment 2

[0053] Embodiment 2: each condition optimization of the test strip of rapid detection Salmonella enteritidis

[0054] 1) Preparation of nitrocellulose membrane

[0055] Coating of detection line: Dissolve Salmonella enteritidis monoclonal antibody in the coating solution to prepare a 1mg / mL solution; use the streaking method to coat the coating solution laterally at the edge of the nitrocellulose membrane at a speed of 1μL / cm 30mm position (that is, the detection line), and then dried at 37°C for 30 minutes. The coating liquid is: 0.02g sodium azide, 0.8g sodium chloride, 0.29g disodium hydrogen phosphate dodecahydrate, 0.02g potassium chloride, 0.02g potassium dihydrogen phosphate and dilute to 100mL with water.

[0056] 2) Preparation of the sample pad: cut the glass fiber membrane to a size of 15mm in length and 3mm in width, soak it in the blocking solution, and dry it at 37°C for 10-16 hours to obtain a sample pad, and then store it in a desiccator at room temperature. ...

Embodiment 3

[0074] Embodiment 3: Sensitivity determination of test strips for rapid detection of Salmonella enteritidis

[0075] The test strip preparation and bacterial culture process steps are the same as 1)-6) in Example 2.

[0076] 8) Detection process: dissolve crystal violet in water to make a solution with a concentration of 0.001%, and then dilute the bacterial solution with 0.01M phosphate buffer to 20-10 8 For the concentration of CFU / mL, take 100 μL solution for each concentration as the detection solution, mix with 90 μL crystal violet and incubate for 1 minute, then add dropwise to the sample pad of the test strip, and take 100 μL 0.01M phosphate buffer solution as the negative control solution. The operation is the same as above, add the sample pad of another test strip drop by drop, and read the result after 10 minutes.

[0077] Test results: (1) Positive: When the detection line of the test strip shows a blue-purple line, it is judged as positive, indicating that the con...

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Abstract

The invention discloses a detection method and a detection test strip of salmonella enteritidis. Based on a Gram staining principle, crystal violet is used for staining bacteria into bluish violet firstly; then under the action of capillary effect, the stained bacteria are transferred along a nitrocellulose membrane; based on an antigen and antibody specific binding principle, the bacteria are captured by an antibody of a T line so that the T line becomes a visible strong bluish violet color. The bright bluish violet color can be obtained only if the bacteria are simply stained, and any nano-material labeled antibody is not used so that a complicated labeling process is omitted. According to the detection method and the detection test strip of the salmonella enteritidis, only one antibodyis scratched on the nitrocellulose membrane to detect directly and a traditional sandwich detection method adopting two antibodies is broken through, so that the cost is greatly saved and the difficulty of matching the antibodies is solved; the detection method and the detection test strip are simpler, more convenient and more novel.

Description

technical field [0001] The invention belongs to the field of biological detection, and in particular relates to a detection method for Salmonella enteritidis and a detection test strip thereof. Background technique [0002] Salmonella is one of the most important food-borne pathogens that cause food infection and food poisoning. People who eat too much live bacteria will produce symptoms such as diarrhea, vomiting, and fever. In severe cases, they will cause sepsis, dehydration, acidosis, anuria, Heart failure, etc., if the first aid is not timely, it will be life-threatening. Salmonella typhimurium, Salmonella choleraesuis, and Salmonella enteritidis are the most common. Salmonella is a test item in many food standards in my country, and it is required not to be detected. Rapid, accurate, sensitive, and simple detection of Salmonella is of great significance in medical hygiene, food hygiene, and animal disease monitoring. To date, several feasible methods for the detecti...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/577G01N33/569G01N33/532
CPCG01N33/532G01N33/56916G01N33/577G01N2333/255
Inventor 张道宏补彤王建龙黄琼闫灵芝窦磊娜赵兵欣赵东阳
Owner NORTHWEST A & F UNIV
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