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Method for rapidly detecting listeria monocytogenes in food

A Listeria mononuclear technology, applied in the field of microbial detection, can solve the problems of low specificity, high labor intensity, low detection sensitivity, etc., and achieve the effect of simple result judgment, easy operation, and low detection cost

Inactive Publication Date: 2012-10-10
NANCHANG UNIV
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Problems solved by technology

For example (Yang Ruijun, Analysis of the detection results of pathogenic bacteria in raw meat food [J] China Health Inspection Journal. 2009:19 (9) 2122-2125), the traditional method has the disadvantages of high labor intensity, time-consuming, and high cost
In addition, the detection sensitivity of traditional methods is relatively low and the specificity is not strong, especially for strains of similar origin, it is difficult to distinguish them through physiological and biochemical methods

Method used

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  • Method for rapidly detecting listeria monocytogenes in food
  • Method for rapidly detecting listeria monocytogenes in food
  • Method for rapidly detecting listeria monocytogenes in food

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Experimental program
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Embodiment

[0030] 1. Food sample pretreatment

[0031] Weigh 1 g of food sample, add 9 ml of phosphate buffered saline (PBS), grind to make a homogenate, centrifuge at 900 g for 5 min, remove large food residues, and take the supernatant (this process must be performed aseptically).

[0032] 2. PMA treatment

[0033] PMA was dissolved in dimethyl sulfoxide (DMSO), prepared into a 0.5 mg / mL PMA solution, and stored at -20°C in the dark. Take 500 μL of the prepared bacterial suspension and place it in a 1.5 mL microcentrifuge tube, add 3 μL of 0.5 mg / mL PMA solution to make the final mass concentration of PMA 3 μg / mL; Incubate in the dark at room temperature for 5 min, and expose to a 500 W halogen lamp for 5 min. When cross-linking with light, place the sample on ice (to avoid overheating), and place the cross-linked suspension at 10,000 g at a distance of 20 cm from the light source. After centrifugation for 5 min, the resulting pellet was used for DNA extraction.

[0034] 3. Extracti...

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Abstract

The invention discloses a method for detecting listeria monocytogenes in food by loop-mediated isothermal amplification (LAMP). A set of specificity detecting primers are designed and screened for the listeria monocytogenes, a food sample to be detected is detected by LAMP, whether specific gene segments of the listeria monocytogenes exist or not is confirmed, and whether the food sample to be detected is polluted by the listeria monocytogenes or not is further determined. In addition, before the LAMP detection, the food sample is treated with azide propidium bromide to remove the disturbance of dead bacteria, and only the detection signals of live bacteria can exist in the follow-up LAMP detection. The detection method disclosed by the invention has high sensitivity, strong specificity and short detection time, an expensive experimental instrument is not needed, the operation process is simple and can not be disturbed by the residual DNA or the dead bacteria in the food sample, and the method is especially suitable for self detection in the open air, and in grass-roots detecting organizations and food enterprises.

Description

technical field [0001] The invention belongs to the field of microorganism detection, in particular to a method for rapidly detecting Listeria monocytogenes in food. technical background [0002] Currently, the genus Listeria contains 8 species, and the vast majority of Listeria that infect humans are related to Listeria monocytogenes ( L. monocytogenes )related. In non-pregnant individuals, Listeria monocytogenes infection causes meningitis and meningoencephalitis; in pregnant women, it primarily infects the unborn child. Listeria monocytogenes can spread from one cell to other nearby cells during infection. Listeria monocytogenes is commonly found in decaying plant matter and soil, but it also enters livestock through drinking water and food and colonizes their gastrointestinal tracts. Humans can become sick from eating the meat or milk of infected livestock. Eating food contaminated with Listeria monocytogenes, such as cheese, sausage, milk, etc., is the main r...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12R1/01
Inventor 魏华徐锋杨友均郭亮许恒毅赖卫华熊勇华
Owner NANCHANG UNIV
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