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Method for rapid detection of Listeria monocytogenes viable bacteria in liquid milk

A technology of Listeria monocytogenes and liquid milk, which is applied in the field of detection of viable Listeria monocytogenes, can solve the problems of high detection cost, long detection time, inability to effectively distinguish dead bacteria and live bacteria, etc., and achieve high detection sensitivity , time-consuming, shorten the effect of detection time

Inactive Publication Date: 2011-12-14
黑龙江省乳品工业技术开发中心
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Problems solved by technology

[0005] The present invention aims to solve the problems that the existing LAMP detection method cannot effectively distinguish dead bacteria from live bacteria, and the traditional detection method of pathogenic bacteria in liquid milk has a long detection time and high detection cost, and provides a rapid detection method for single bacteria in liquid milk. Method for increasing live Listeria

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  • Method for rapid detection of Listeria monocytogenes viable bacteria in liquid milk
  • Method for rapid detection of Listeria monocytogenes viable bacteria in liquid milk
  • Method for rapid detection of Listeria monocytogenes viable bacteria in liquid milk

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specific Embodiment approach 1

[0011] Specific embodiment one: the method for quickly detecting the live bacteria of Listeria monocytogenes in liquid milk in this embodiment is carried out according to the following steps: 1. Pretreatment of the tested milk sample: add PMA to 1mL of the tested milk to make the PMA The concentration is 50μmol / L, then placed in a dark place for 5-30min, then placed 15-20cm away from a 650W halogen lamp, and exposed for 5-30min; Add 150 μL of sodium citrate with a concentration of 250 g / L and 100 μL of sodium hydroxide with a concentration of 1 mol / L, then centrifuge at 10,000 r / min for 2 minutes, discard the supernatant, and obtain a precipitate; 3. Wash with 0.85% normal saline Precipitate, then centrifuge at 10000r / min for 1min, discard the supernatant, and use the bacterial genome DNA extraction kit to extract DNA to obtain the extract; 4. Use the extract as a template for LAMP amplification to obtain the LAMP amplification product; 5. Identification : Take 5 μL of LAMP am...

specific Embodiment approach 2

[0014] Embodiment 2: This embodiment is different from Embodiment 1 in that: in step 1, place it in a dark place for 10-20 minutes. Others are the same as in the first embodiment.

specific Embodiment approach 3

[0015] Embodiment 3: This embodiment is different from Embodiment 1 in that: in step 1, place it in a dark place for 15 minutes. Others are the same as in the first embodiment.

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Abstract

A method for rapid detection of Listeria monocytogenes viable bacteria in liquid milk relates to a detection method of Listeria monocytogenes viable bacteria in liquid milk. The method solves problems of incapability of effective distinguishing dead bacteria from viable bacteria in a prior LAMP (loop-mediated isothermal amplification) detection method, and long detection time and high costs of a traditional detection method of pathogenic bacteria in liquid milk. The method comprises steps of: a pretreatment of milk sample to be detected; extraction of sample DNA; LAMP amplification; and identification. The invention has short time consuming and high detection sensitivity, and a detection time is substantially reduced to about 1.5h, so as to substantially reduce a storage time while waiting a detection result before raw material milk processing and before finished product leaving factory. A detection limit to Listeria monocytogenes viable bacteria in liquid milk is 6.37*10<1>cfu / mL. The method can be widely applied to the field of foodborne pathogenic bacteria detection.

Description

technical field [0001] The invention relates to a method for detecting live bacteria of Listeria monocytogenes. Background technique [0002] Listeria monocytogenes (Listeria monocytogenes), referred to as "Listeria monocytogenes", is one of the important food-borne pathogens, widely distributed, living environment plasticity, newborns, pregnant women and over 40 years old Adults and immunocompromised persons are susceptible to infection, and its fatality rate is very high. WHO has listed it as one of the four major pathogenic bacteria in food in the 1990s. Therefore, Listeria monocytogenes is the first choice for microbial detection in food in many countries. One of the must-check indicators. [0003] Loop-mediated isothermal amplification technology (Loop-mediated Isothermal Amplification, referred to as LAMP) is developing rapidly. It designs 4 specific primers (two outer primers and two inner primers) for six regions of the target gene, and uses a strand displacement D...

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04
Inventor 姜毓君满朝新杨秀茹卢雁单艺蒋亚男王旻谢鲲昊胡惠秩刘颖薛玉清
Owner 黑龙江省乳品工业技术开发中心
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