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A method for rapidly determining the survival status of Mycobacterium paratuberculosis in milk and milk products by using 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, improving efficiency, and simple operation

Inactive Publication Date: 2018-10-30
巩红霞
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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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  • A method for rapidly determining the survival status of Mycobacterium paratuberculosis in milk and milk products by using blu-v PMA technology
  • A method for rapidly determining the survival status of Mycobacterium paratuberculosis in milk and milk products by using blu-v PMA technology
  • A method for rapidly determining the survival status of Mycobacterium paratuberculosis in milk and milk products by using blu-v PMA technology

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Experimental program
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Effect test

Embodiment 1

[0030] Primer specificity verification

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

[0032] 1. Preparation of DNA

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

[0034] (1) Heat in a water bath at 80°C for 20 minutes, then cool to room temper...

Embodiment 2

[0048] Optimization of Optimum Reaction Conditions of MAP Standard Strain Acting on PMA

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

[0050] Take 1 mL of P10 and P18 bacterial solutions (take as much bacteria as possible) into 2 mL EP tubes with 50 magnetic beads, seal them with a parafilm, and put them in a nucleic acid extractor (once every 5 s). Fully suspend the bacterial solution; after suspension, inactivate in an 80°C water bath for 20 min.

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

[0052] Dissolve 0.5 mg of PMA in 1 mL of RNase-free water to make a 0.5 mg / mL PMA stock solution and store at -20 °C. Place the inactivated bacterial solution in a centrifuge and centrifuge at 14,500 r for 5 min; after centrifugation, discard the supernatant; then add 500 μL of EB Buffer to the remaining bacterial cells and mix well.

[0053] 3. Optimization of PMA mass concentration

[0054] A certain amount of PMA was ad...

Embodiment 3

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

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

[0062] Take 1 mL of P10 and P18 bacterial solutions (take as much bacteria as possible) into 2 mL EP tubes with 50 magnetic beads, seal them with a parafilm, and put them in a nucleic acid extractor (once every 5 s). Fully suspend the bacterial mass to form a bacterial suspension; aseptically take the negative milk samples with no MAP detected by fluorescent PCR, common PCR and electrophoresis, that is, take 1 mL of yogurt and 0.5 g of milk powder in 2 mL of sterilized EP tube; add 500 μL of bacterial suspension to each of the obtained milk samples, mix thoroughly on a vortex shaker; after mixing, place in an 80 °C water bath for 20 min to inactivate. Through this treatment, the cell wall and cell membrane of the bacteria are damaged to varying degrees to a...

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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 invention discloses a method for quickly judging the survival state of Mycobacterium paratuberculosis in milk and milk products by using BLU-V PMA technology, and belongs to the technical field of microbial detection. Background technique [0002] Paratuberculosis ( Paratuberculosis ) is produced by Mycobacterium paratuberculosis, Mycobacterium avium subsp. Mycobacterium avium subsp. Paratuberculosis, MAP ) is 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 link with Crohn's disease in humans. Studies have shown that this serious disease in humans may be due to the inability of pasteurization of milk to completely kill the disease. bacteria, and the study found that almost 44% of the dairy products were positive for paratuberculosis. With the continuous improvement of our country's living standards, the demand for milk is increasing expo...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/04C12Q1/689
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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