CN-119979330-B - Cracking method of food-borne pathogenic bacteria and application thereof

CN119979330BCN 119979330 BCN119979330 BCN 119979330BCN-119979330-B

Abstract

The invention relates to the technical field of food safety, in particular to a low-power water bath ultrasonic method for cracking food-borne pathogenic bacteria and application thereof. According to the invention, a low-power water bath ultrasonic machine is used, ABS pellets with the size of about 1 mm are added into a solution sample, high-frequency vibration is generated on the ABS pellets by utilizing ultrasonic waves, bacteria are rapidly cracked by a local high-sound pressure field and a high-density cavitation field generated by vibration, and meanwhile, the movement of the pellets can accelerate the solution at different positions in a pipe to perform rapid material exchange, so that a1 mL bacteria sample to be tested can be cracked within 3 minutes. The method provided by the invention has the advantages of high cracking efficiency and short time consumption. When the method is applied to food-borne pathogenic bacteria detection, nucleic acid purification and enrichment of lysate are not needed, the time and labor cost required by nucleic acid detection can be greatly shortened, and a novel efficient, economical and practical bacterial lysis and nucleic acid extraction method is provided for the field of food safety detection.

Inventors

LIN JIANHAN
Jia kaiyuan
WANG LEI

Assignees

中国农业大学

Dates

Publication Date: 20260512
Application Date: 20250122

Claims (9)

1. The method for cracking food-borne pathogenic bacteria is characterized by comprising the steps of adding ABS pellets into a solution sample of the food-borne pathogenic bacteria, and utilizing ultrasound to cause high-frequency vibration of the ABS pellets so as to crack the bacteria, wherein the particle size of the ABS pellets is 1.0+/-0.2 mm, and the ultrasound parameters are 0.12+/-0.01W/mL and 40+/-3 kHz; the bacterial content in the solution sample of the food-borne pathogenic bacteria is 6.8-13.6 CFU/mL, 13.6-13600 CFU/mL or >13600CFU/mL.
2. The method of claim 1, wherein the ultrasonic treatment time is 2.5.+ -. 0.1min.
3. The method of claim 1, wherein 0.2.+ -. 0.01 g of ABS pellets are added to each 1mL of the solution sample of the food-borne pathogenic bacteria.
4. A method of lysing food-borne pathogenic bacteria according to any of claims 1 to 3 wherein the low power water bath sonicator is used to sonicate the sample of the solution of food-borne pathogenic bacteria in a centrifuge tube at an angle of 90±5° to the ultrasound transducer during sonication.
5. The method of claim 1, wherein the solution sample of food-borne pathogenic bacteria is a cultured or non-cultured food sample to be tested.
6. The method of claim 5, wherein the food sample is at least one of chicken viscera, milk, coconut milk, chicken leg, chicken counter waste, orange juice, and apple juice.
7. The method of claim 1, wherein the food-borne pathogenic bacteria comprise gram-positive bacteria and/or gram-negative bacteria.
8. The method for lysing food-borne pathogenic bacteria according to claim 7, wherein the gram-positive bacteria comprise Bacillus cereus and/or the gram-negative bacteria comprise Escherichia coli (ESCHERICHIA COLI) or Salmonella typhimurium (Salmonella Typhimurium).
9. Use of the method for the lysis of food-borne pathogenic bacteria according to any of claims 1 to 8 for the detection of food-borne pathogenic bacteria for non-disease diagnosis purposes.

Description

Cracking method of food-borne pathogenic bacteria and application thereof Technical Field The invention relates to the technical field of food safety, in particular to a low-power water bath ultrasonic method for cracking food-borne pathogenic bacteria and application thereof. Background In the field of food safety, rapid and accurate detection of food-borne pathogenic bacteria is an important link for guaranteeing public health. According to the national standard GB 4789.4-2016, etc., the sampling stage of detecting food-borne pathogenic bacteria is usually subjected to pre-enrichment and enrichment treatment to ensure that the bacterial quantity in the sample reaches the detection limit of the detection method, and the process needs at least 8-18 hours. However, the conventional detection methods such as a culture method and a PCR gel electrophoresis method have the problems of long time consumption, complex operation, high cost and the like although the detection result is accurate, and the requirements of the modern food safety detection on the rapidity and the high efficiency are difficult to meet. In recent years, with the rapid development of molecular biology technology, qPCR (real-time fluorescent quantitative nucleic acid amplification detection technology) has been widely used in detecting food-borne pathogenic bacteria due to its advantages of high sensitivity, high specificity, rapidity, and the like. The qPCR method realizes the rapid quantitative detection of pathogenic bacteria by directly detecting bacterial nucleic acid in a sample. Currently, qPCR detection methods are already the "gold standard" method among a variety of pathogen detection methods. However, to perform qPCR assays, high quality, high concentration nucleic acids are first extracted from the sample, and bacterial lysis is a key step in nucleic acid extraction, whose efficiency and cost directly impact the performance of the overall assay procedure. Traditional bacterial lysis methods include chemical lysis, thermal lysis, sonication, and the like. Although the chemical lysis method has high efficiency, the experimental process is complex, additional nucleic acid purification steps are needed, the reagent cost is high, and professional laboratory conditions and professional experiment operators are needed, so that qPCR detection needs additional laboratory cost, time cost and labor cost, the thermal lysis method has relatively low efficiency and is easy to cause nucleic acid aerosol pollution, and ultrasonic bacteria disruption depends on ultrasonic treatment equipment with higher power, so that the related pathogen nucleic acid extraction method is lacking, but expensive ultrasonic lysis instruments and longer lysis time are generally needed, and the detection cost is increased. In view of this, the present invention has been made. Disclosure of Invention In order to solve the technical problems, the invention provides a low-power water bath ultrasonic method for cracking food-borne pathogenic bacteria and application thereof. Specifically, the technical scheme of the invention is as follows: In the first aspect, the invention provides a method for cracking food-borne pathogenic bacteria, which comprises the steps of adding ABS pellets into a solution sample of the food-borne pathogenic bacteria, and utilizing ultrasound to cause high-frequency vibration of the ABS pellets so as to crack the bacteria, wherein the particle size of the ABS pellets is 1.0+/-0.2 mm, and the ultrasound parameters are 0.12+/-0.01W/mL and 40+/-3 KHz. Preferably, the treatment time of the ultrasound is 2.5.+ -. 0.1min. Preferably, 0.2.+ -. 0.01 g ABS pellets are added to each 1mL sample of the solution of food-borne pathogenic bacteria. Preferably, the solution sample of the food-borne pathogenic bacteria is placed in a centrifuge tube, and the included angle between the centrifuge tube and an ultrasonic transducer in the ultrasonic process is 90+/-5 degrees. Preferably, the solution sample of food-borne pathogenic bacteria is a test food sample, either cultured or not. Preferably, the source of the food sample to be tested is at least one of chicken viscera, milk, coconut juice, drumstick, chicken counter sewage, orange juice and apple juice. Preferably, the bacteria content in the solution sample of the food-borne pathogenic bacteria is <6.8CFU/mL, 6.8-13.6 CFU/mL, 13.6-13600 CFU/mL or >13600CFU/mL. Preferably, the food-borne pathogenic bacteria comprise gram-positive bacteria and/or gram-negative bacteria. Preferably, the gram positive bacteria comprise Bacillus cereus ATCC 14579 and/or the gram negative bacteria comprise E.coli O157: H7 ATCC 43888 or Salmonella typhimurium ATCC 14028. In a second aspect, the invention provides the use of the method for the lysis of food-borne pathogenic bacteria in the detection of food-borne pathogenic bacteria for non-disease diagnosis purposes. The beneficial effects are that: The invention provides a low-po