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Extraction method for microbial DNA in milk

An extraction method and microbial technology, applied in the field of microbial DNA extraction in milk, can solve the problems of time-consuming, limited application, loss of DNA, etc., and achieve the effect of improving sensitivity

Inactive Publication Date: 2017-02-08
CHINTEM TECH CONSULTING BEIJING CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In order to shorten the detection time, the current domestic methods for directly extracting the DNA of pathogenic bacteria in milk include: ①The phenol-chloroform method, whose operation steps are relatively complicated and time-consuming, and while DNA is lost, it is also easy to cause contamination among milk samples; Organic substances such as phenol remaining in DNA have a certain inhibitory effect on DNA polymerase
In addition, this method requires corrosive and toxic organic solvents, such as phenol, etc., which have adverse effects on personnel and the environment
②The nucleic acid purification column method, due to its complexity and high cost, makes it difficult to apply it to the detection of large batches of milk samples
③Magnetic bead method, the cost of extracting bacterial DNA from milk samples is relatively high, which limits its application in the detection of a large number of milk samples

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] In an embodiment of the present invention, a method for extracting microbial DNA in milk comprises the following steps:

[0027] (1) Take 0.8mL milk sample and add it to 0.4mL ES solution (0.5mol / L EDTA (pH7.5), 0.2% SDS), mix well, then centrifuge at 12000r / min for 10min, discard the supernatant;

[0028] (2) Add 1mL PBS buffer, shake well, then centrifuge at 10000r / min for 5min, discard the supernatant;

[0029] (3) Add 25 μL of lysostaphin with a mass concentration of 0.02 mg / mL; add 175 μL of a 10% Chelex-100 aqueous solution by mass, shake vigorously for 8 seconds, place in boiling water at 100°C and heat for 10 minutes; Centrifuge at a speed of 5 min for 5 min, and finally take the supernatant for PCR reaction.

Embodiment 2

[0031] In an embodiment of the present invention, a method for extracting microbial DNA in milk comprises the following steps:

[0032] (1) Take 0.8mL milk sample and add it to 0.3mL ES solution (0.5mol / L EDTA (pH7.5), 0.2% SDS), mix well, then centrifuge at 10000r / min for 12min, discard the supernatant; The ES solution contains 0.5mol / L of EDTA and 0.2wt% SDS, and the pH of the EDTA is 7.5;

[0033] (2) Add 0.8mL PBS buffer solution, shake well, then centrifuge at 8000r / min for 8min, discard the supernatant;

[0034] (3) Add 20 μL of lysostaphin with a mass concentration of 0.02 mg / mL; add 150 μL of a 10% Chelex-100 aqueous solution, vibrate vigorously for 5 seconds, and heat in boiling water at 100°C for 8 minutes; Centrifuge at a speed of 8 min for 8 min, and finally take the supernatant for PCR reaction.

Embodiment 3

[0036] In an embodiment of the present invention, a method for extracting microbial DNA in milk comprises the following steps:

[0037] (1) Take 0.8mL milk sample and add it to 0.5mL ES solution (0.5mol / L EDTA (pH7.5), 0.2% SDS), mix well, then centrifuge at 15000r / min for 8min, discard the supernatant; The ES solution contains 0.5mol / L of EDTA and 0.2wt% SDS, and the pH of the EDTA is 7.5;

[0038] (2) Add 1.2mL PBS buffer solution, shake well, then centrifuge at 12000r / min for 3min, discard the supernatant;

[0039] (3) Add 30 μL of lysostaphin with a mass concentration of 0.02 mg / mL; add 1200 μL of a 10% Chelex-100 aqueous solution, vibrate vigorously for 10 seconds, and heat in boiling water at 100°C for 12 minutes; Centrifuge at a speed of 3 min for 3 min, and finally take the supernatant for PCR reaction.

[0040] The invention adopts the Chelex-100 method to economically, rapidly and effectively extract bacterial DNA directly from milk samples and use it for PCR detec...

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Abstract

The invention discloses an extraction method for microbial DNA in milk. The method comprises the following steps: adding 0.8 mL of a milk sample into 0.3 to 0.5 mL of an ES solution (EDTA with a pH value of 7.5 and concentration of 0.5 mol / L and SDS with concentration of 2%), carrying out uniform mixing and centrifugation and discarding supernatant; adding 0.8 to 1.2 mL of a PBS buffer solution, carrying out oscillation and shaking up, then carrying out centrifugation and discarding supernatant; adding 20 to 30 [mu]L of Staphylococcus lysozyme with a mass concentration of 0.02 mg / mL; adding 150 to 200 [mu]L of an aqueous Chelex-100 solution with a mass concentration of 10%, carrying out violent oscillation for 5 to 10 s and then carrying out heating in boiling water with a temperature of 100 DEG C for 8 to 12 min; and carrying out centrifugation and taking supernatant for a PCR reaction. The method provided by the invention employs a Chelex-100 process and can economically, rapidly, efficiently and directly extract bacterial DNA from the milk sample; and the bacterial DNA can be used for PCR detection of main pathogenic bacteria causing mastitis in cow.

Description

technical field [0001] The invention relates to the technical field of dairy cow molecular biology, in particular to a method for extracting microbial DNA in milk. Background technique [0002] Cow mastitis is one of the most important diseases affecting the development of the dairy industry in China and even the world. The infection of pathogenic bacteria such as Staphylococcus aureus, Streptococcus agalactiae, Mycoplasma bovis and Escherichia coli is the main cause of the disease. At present, there is still a lack of effective vaccines to prevent mastitis in dairy cows in China. The key factor in the prevention and treatment of this disease is to identify its causative bacteria. Once the pathogenic bacteria are identified, corresponding measures can be taken immediately. [0003] For the PCR detection of cow mastitis pathogenic bacteria, the preparation of bacterial DNA template in milk samples is one of the main factors affecting the detection speed and sensitivity. In ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
Inventor 郝永清
Owner CHINTEM TECH CONSULTING BEIJING CO LTD
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