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Method and kit for detecting pathogenic bacterium of food source by test strip based on NASBA (Nucleic Acid Sequence Based Amplification)

A technology for food-borne pathogenic bacteria and test strips, which can be used in microorganism-based methods, microorganism determination/inspection, biochemical equipment and methods, etc.

Active Publication Date: 2013-06-12
SOUTH CHINA NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The operation of the culture identification method is cumbersome and takes a long time, while other methods have the disadvantages of low sensitivity, low specificity, long time-consuming, and expensive instruments.

Method used

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  • Method and kit for detecting pathogenic bacterium of food source by test strip based on NASBA (Nucleic Acid Sequence Based Amplification)
  • Method and kit for detecting pathogenic bacterium of food source by test strip based on NASBA (Nucleic Acid Sequence Based Amplification)
  • Method and kit for detecting pathogenic bacterium of food source by test strip based on NASBA (Nucleic Acid Sequence Based Amplification)

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Experimental program
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Effect test

Embodiment 1

[0075] (1) Extraction of total RNA from Listeria monocytogenes (strain CMCC54007, purchased from Guangzhou Institute of Microbiology):

[0076] ①Grinding with liquid nitrogen, take about 5mL of bacterial liquid in a mortar, add a small amount of liquid nitrogen, and grind quickly. During the grinding process, add liquid nitrogen continuously until the grinding is complete. After grinding, transfer the cell lysate into a 1.5mL EP tube, then add 0.8mL Trizol to the grinder, blow the residual lysate on the mortar, and pour it into the EP tube.

[0077] ②Cover the lid, invert and mix 10 times, and let stand at room temperature for 5 minutes.

[0078] ③Add 0.2mL chloroform to the EP tube, close the cap, shake vigorously for 15 seconds to mix the liquid thoroughly, let stand at room temperature for 5 minutes, and then centrifuge at 12,000 rpm for 15 minutes.

[0079] ④ Transfer the supernatant to a new 1.5mL EP tube (approximately 0.5mL), add 0.5mL isopropanol, shake vigorously for...

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Abstract

The invention discloses a method and a kit for detecting pathogenic bacterium of a food source by a test strip based on NASBA (Nucleic Acid Sequence Based Amplification) and belongs to the technical field of biological detection. The method comprises the following steps of: (1) extracting RNA of pathogenic bacterium of the food source; (2) designing an amplification primer; (3) performing NASBA reaction; (4) designing a catching probe; (5) preparing a nano-gold probe; (6) preparing a collaurum nucleic acid strip; and (7) detecting the samples. The kit using the method to detect Listeria monocytogenes comprises primers (as shown in SEQ ID NO. 1 and 2), enzyme, buffer solution, RNaseH, RNA enzyme inhibitor, dNTPs, NTPs, probes (as shown in SEQ ID NO. 4, 5 and 6) and collaurum nucleic acid strip, etc. By combining NASBA with detection of the test strip, the method disclosed by the invention can realize qualitative or quantitative detection, and has low cost, high detection speed, good specificity, high sensitivity and safety in use.

Description

technical field [0001] The invention belongs to the technical field of biological detection, and relates to a method and a kit for detecting food-borne pathogenic bacteria based on NASBA test strips. Background technique [0002] my country is the largest country in the world in terms of food consumption and production. Food safety is a major livelihood issue and a topic of close concern to the country and the people. However, in recent years, food safety accidents have occurred frequently in our country, and the problem of food safety has become a lingering nightmare in the hearts of Chinese people. According to the survey, disturbing food safety is the main reason for the low happiness index of the people. Foodborne diseases are the biggest problem in food safety, posing a serious threat to the health and safety of consumers. Foodborne pathogens are the main pathogenic factors causing foodborne diseases. In October 2011, Staphylococcus aureus was detected in the Synian ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12R1/42C12R1/19C12R1/445C12R1/63C12R1/085C12R1/01
Inventor 邢达詹芳芳周小明
Owner SOUTH CHINA NORMAL UNIVERSITY
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