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Method of rapidly judging survival state of mycobacterium paratuberculosis in milk and milk product by applying BLU-V PMA technology

A BLU-V, mycobacteria technology, applied in the field of microbial detection, can solve the problems of reduced PMA treatment effect, false positives, short peak brightness time, etc., to achieve the effect of protecting health, rapid development, simple operation, and improving efficiency

Inactive Publication Date: 2015-08-12
巩红霞
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  • Summary
  • Abstract
  • Description
  • Claims
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Problems solved by technology

[0004] However, the type of light source is also an important factor affecting the treatment of PMA. In the previous experiments on PMA, the light source used was a 600W halogen lamp. Although the cost is reasonable and the illumination is sufficient, the time it takes to reach the peak brightness Short, and affected by air medium and sample turbidity, will reduce the treatment effect of PMA, resulting in false positive and false negative results in the test

Method used

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  • Method of rapidly judging survival state of mycobacterium paratuberculosis in milk and milk product by applying BLU-V PMA technology
  • Method of rapidly judging survival state of mycobacterium paratuberculosis in milk and milk product by applying BLU-V PMA technology
  • Method of rapidly judging survival state of mycobacterium paratuberculosis in milk and milk product by applying BLU-V PMA technology

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Effect test

Embodiment 1

[0030] Primer specificity verification

[0031] The genomic DNA of 6 standard strains of Map (P10 and P18), Staphylococcus aureus, Salmonella, Escherichia coli, and Mycobacterium phlei are extracted and amplified by TaqMan fluorescent quantitative PCR to detect the specificity of the method of the invention. (Mycobacterium paratuberculosis ( M.paratuberculosis ) Standard strains: P18 (United States), P10 (Japan) (both are standard strains of bovine paratuberculosis), all provided by Jilin Veterinary Research Institute. Staphylococcus aureus, Salmonella, Escherichia coli, and Mycobacterium phlei are all preserved in the laboratory of the Technical Center of Shanxi Entry-Exit Inspection and Quarantine Bureau. )

[0032] 1. DNA preparation

[0033] Take 50 μL of bacterial solution, add 400 μL of TE, and mix. The specific steps for extracting DNA template are as follows:

[0034] (1) Heat in a water bath at 80 ℃ for 20 min, then cool to room temperature to inactivate;

[0035] (2) Add...

Embodiment 2

[0048] Optimization of the optimal reaction conditions of PMA on MAP standard strains

[0049] 1. Preparation of MAP standard strains (P10 and P18) membrane damage bacteria

[0050] Take 1 mL of P10 and P18 bacterial solution (take as many bacteria as possible) in a 2 mL EP tube with 50 magnetic beads, seal it with a parafilm, and put it in the nucleic acid extractor (act once every 5 s), Suspend the bacterial liquid thoroughly; after suspension, inactivate it in a water bath at 80 ℃ for 20 min.

[0051] 2. Treatment of MAP standard strains (P10 and P18) by PMA

[0052] Dissolve 0.5 mg of PMA in 1 mL of RNase-free water to prepare a 0.5 mg / mL stock solution of PMA and store at -20°C. Place the inactivated bacterial solution in a centrifuge and centrifuge at 14500 r for 5 minutes; after centrifugation, discard the supernatant; then add 500 μL of EB Buffer to the remaining bacterial cells and mix.

[0053] 3. Optimization of PMA mass concentration

[0054] Add a certain amount of PMA to ...

Embodiment 3

[0060] Quantitative detection of viable bacteria in milk and milk products under optimal PMA reaction conditions

[0061] 1. Preparation of membrane-damaging bacteria (P10 and P18) in artificially contaminated MAP milk samples

[0062] Take 1 mL of P10 and P18 bacterial solution (take as many bacteria as possible) in a 2 mL EP tube with 50 magnetic beads, seal it with a parafilm, and put it in the nucleic acid extractor (act once every 5 s), Suspend the bacterial mass as a bacterial suspension; aseptically take a negative milk sample that has not detected MAP by fluorescent PCR, ordinary PCR and electrophoresis, that is, take 1 mL of yogurt and 0.5 g of milk powder in sterilized 2 mL In the EP tube; add 500 μL of bacterial suspension to the obtained milk sample, mix thoroughly on a vortex shaker; after mixing, place it in a water bath at 80 ℃ for 20 min inactivation. Through this treatment, the cell wall and cell membrane of the bacteria are damaged to different degrees to achieve...

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Abstract

The invention discloses a method of rapidly judging the survival state of mycobacterium paratuberculosis in milk and a milk product by applying a BLU-V PMA technology. According to an ISMAV2 genomic sequence (with the accession number of AF286339) of Map published by GenBank, a conserved sequence of the gene is selected, and a TaqMan fluorescence probe and a primer are designed; a BLU-V system, serving as a light source, is applied to a PMA technology, and a method of rapidly detecting Map live bacteria in the milk and the milk product is successfully established with the combination of the advantages of the fluorescence quantitative PCR technology. The method is simple to operate and is rapid and accurate.

Description

Technical field [0001] The present invention is a method for rapidly determining the survival state of Mycobacterium paratuberculosis in milk and milk products by applying BLU-V PMA technology, and belongs to the technical field of microbial detection. Background technique [0002] Paratuberculosis ( Paratuberculosis ) Is composed of Mycobacterium paratuberculosis, namely Mycobacterium avium subspecies paratuberculosis ( Mycobacteriumaviumsubsp.Paratuberculosis,MAP ) A chronic wasting infectious disease mainly caused by ruminants, also known as Johne's disease. The disease is difficult to purify, and there is a potential connection with human Crohn's (Crohn, s) disease. Studies have shown that this serious disease in humans may be due to the fact that milk pasteurization cannot completely kill the disease. The study found that almost 44% of dairy products were positive for Paratuberculosis. With the continuous improvement of people’s living standards in my country, the demand f...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/04C12Q1/68
Inventor 巩红霞巩强张祖维傅英文贾广乐霍乃蕊宋洁王静慧刘晓琳王芬张敏爱姚亚婷
Owner 巩红霞
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