Method and kit for directly extracting DNA of three pathogenic bacteria from milk

Abstract

The invention discloses a kit for directly extracting DNA of three pathogenic bacteria from milk. The kit mainly comprises a solution A, wherein the solution A contains EDTA-2Na, NaOH, SDS and NaCl. The kit is simple in reagent component; lysozyme and proteinase k are not used; the high-toxicity organic solvents, such as phenol, chloroform and ethyl alcohol, are not used; the cost is low and the operation is simple and convenient. Based on the well-designed extracting solution and a microwave method, the invention also discloses a method for directly extracting DNA of three pathogenic bacteria from milk. The three pathogenic bacteria comprise escherichia coli, staphylococcus aureus and streptococcus agalactiae. According to the method, the cell splitting effect is improved, the conventional extracting steps are simplified, only one EP tube is required during the milk processing process, the whole extracting process is shortened within 30min, an acquired template can be directly used for PCR reaction, the PCR amplification product is detected through agarose gel electrophoresis, and then the detection of the sample is finished within short time.

Description

technical field [0001] The invention belongs to the technical field of DNA extraction in molecular biology, and in particular relates to a method and a kit for directly extracting the DNA of three pathogenic bacteria in milk. Background technique [0002] Mastitis is one of the most common diseases in dairy farms and causes the most serious economic losses. The disease reduces milk production and quality, greatly increases treatment costs, and prematurely eliminates dairy cows, which restricts the healthy development of dairy farming and dairy processing industries. How to effectively prevent and control the disease has become the focus of attention at home and abroad. [0003] Pathogenic bacterial infection is the most important cause of the disease, and Escherichia coli (Escherichiacoli), Staphylococcus aureus (Staphylococcus aureus), and Streptococcus agalactiae (S. agalactiae) are the main pathogenic bacteria. The detection of pathogenic bacteria is a prerequisite for t...

AI Technical Summary

Problems solved by technology

The detection of pathogenic bacteria is a prerequisite for the prevention and control of mastitis. The current method for diagnosing the pathogenic bacteria of cow mastitis is based on the culture of microorganisms in milk and bacterial biochemical tests. On the one hand, the workload is heavy, and on the other hand, it takes a long time

Due to the limitations of the bacterial culture method, the PCR method has been widely studied and applied, which greatly improves the efficiency of diagnosing the pathogenic bacteria of dairy cow mastitis, and the preparation of the DNA template directly affects the detection speed and sensitivity of the PCR method. Usually, it is very difficult to directly isolate the DNA of target pathogenic bacteria with high purity from milk, and generally requires a selective enrichment culture process, which improves the sensitivity of detection, but greatly reduces the speed of detection

[0004] The methods for directly extracting bacterial DNA from milk reported in recent years have the disadvantages of complex steps, time-consuming, expensive reagents and toxicity, which limit the promotion and application of these methods.

Therefore, there is an urgent need to establish a convenient new method for extracting the DNA of mastitis-causing bacteria from milk, so as to cooperate with early diagnosis, gain valuable time for mastitis diagnosis, and avoid further economic losses caused by time-consuming diagnosis

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