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Constant-temperature detection method and kit for instantly detecting listeria monocytogenes

A Listeria, detection method technology, applied in the field of constant temperature amplification technology that does not depend on high-energy energy molecules, can solve the problems of long detection time, insufficient sensitivity, and high technical requirements for detection personnel, and achieve simple composition, sensitivity and accuracy High, fast amplification and stable results

Inactive Publication Date: 2017-06-13
GUANGZHOU HEAS BIOTECH CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Commonly used detection methods for Listeria monocytogenes include the immunogold standard test strip method and the polymerase chain reaction method. Among them, the immunogold standard test strip method is simple to operate, and the detection results are fast and intuitive, but there are insufficient sensitivity. Disadvantages; while the polymerase chain reaction method has advantages in terms of sensitivity and specificity, but there are disadvantages such as requiring a long time for detection and relatively high technical requirements for detection personnel.

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  • Constant-temperature detection method and kit for instantly detecting listeria monocytogenes
  • Constant-temperature detection method and kit for instantly detecting listeria monocytogenes
  • Constant-temperature detection method and kit for instantly detecting listeria monocytogenes

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Effect test

Embodiment 1

[0063] Embodiment 1 (sensitivity experiment):

[0064] In this embodiment, the Listeria monocytogenes nucleic acid is diluted 10 times, 100 times, 1000 times, and 10000 times, and the diluted nucleic acid is sequentially labeled as sample 1, sample 2, sample 3, and sample 4, and the concentrations are respectively 10ng / ul, 1ng / ul, 0.1ng / ul, 0.01ng / ul, use sterilized pure water as negative control. Take five 200ul reaction tubes and mark them as reaction tubes 1~5 in turn, add 20ul reaction solution and 5ul reaction enzyme system to reaction tubes 1~5 respectively, add 2ul sample 1 to reaction tube 1, and add 2ul sample 2, and so on, add 2ul sterilized pure water to reaction tube 5, and finally make up the system to 45ul with sterilized pure water, mix well and then add 5ul starter solution respectively.

[0065]Select a constant temperature water bath to provide the constant temperature environment required for the reaction, set the temperature of the constant temperature wat...

Embodiment 2

[0067] Embodiment 2 (specificity experiment):

[0068] In this embodiment, Listeria monocytogenes nucleic acid, Salmonella nucleic acid, Escherichia coli nucleic acid, Shigella nucleic acid, and Staphylococcus aureus nucleic acid are sequentially labeled as sample 1, sample 2, sample 3, sample 4, and sample 5. The concentration is 10ng / ul. Take five 200ul reaction tubes and mark them as reaction tubes 1~5 in turn, add 20ul reaction solution and 5ul reaction enzyme system to reaction tubes 1~5 respectively, add 2ul sample 1 to reaction tube 1, and add 2ul sample 2, and so on, and finally make up the system to 45ul with sterilized pure water, mix well, and then add 5ul starter solution respectively.

[0069] Select a constant temperature water bath to provide the constant temperature environment required for the reaction, set the temperature of the constant temperature water bath to 37°C, and the reaction time to 30min.

[0070] After the reaction, 10ul products were taken fro...

Embodiment 3

[0071] Embodiment 3 (mixed infection experiment):

[0072] In this embodiment, the Salmonella-Listeria monocytogenes nucleic acid and the Escherichia coli-Listeria monocytogenes nucleic acid were labeled as sample 1 and sample 2 in sequence, and the concentrations were both 10 ng / ul. Take three 200ul reaction tubes and mark them as reaction tubes 1~3 in turn, add 20ul reaction solution and 5ul reaction enzyme system to reaction tubes 1~3 respectively, add 2ul sample 1 to reaction tube 1, and add Add 2ul of sterilized pure water to 2ul of sample 2 and reaction tube 3, and finally make up the system to 45ul with sterilized pure water, mix well and then add 5ul of starter solution respectively.

[0073] Select a constant temperature water bath to provide the constant temperature environment required for the reaction, set the temperature of the constant temperature water bath to 37°C, and the reaction time to 30min.

[0074] After the reaction, 10ul products were taken from react...

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Abstract

The invention discloses a constant-temperature detection method and kit for instantly detecting listeria monocytogenes. The constant-temperature detection kit does not depend on high-energy molecules such as ATP and phosphocreatine. Compared with the existing constant-temperature amplification system, the constant-temperature detection kit is relatively simple in components, relatively low in cost and relatively convenient to use. Key components comprise dNTPs, polymerase Bsu, single-strand binding protein and recombinase (E.coli Rect). The constant-temperature detection kit can be matched with a fluorescently-labeled probe based on a constant-temperature amplification reaction reagent to carry out instant fluorescence detection. The constant-temperature amplification system is applied to amplification of complex templates; amplification is rapid and stable; the amplification reaction can be completed in 30 minutes; amplification is high in sensitivity and accuracy; the possibility of mismatching is low; the amplification effect is excellent.

Description

technical field [0001] The invention relates to a constant temperature detection method and kit for instant detection of Listeria monocytogenes, in particular to a constant temperature amplification technology independent of high-energy energy molecules. Background technique [0002] Listeria monocytogenes exists widely in nature, is not easy to be frozen and thawed, and can withstand high osmotic pressure. It can be found in soil, surface water, sewage, waste water, plants, silage feed, and rotten vegetables. Therefore, animals can easily ingest the bacteria and spread it through the oral-fecal route. According to reports, the carrying rate of Listeria monocytogenes in the feces of healthy people is 0.6-16%, 70% of people can carry the bacteria for a short time, 4-8% of aquatic products, 5-10% of milk and its products, More than 30% of meat products and more than 15% of poultry are contaminated by this bacteria. Humans do this primarily by ingesting soft cheeses, underhea...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04
Inventor 刘淑园陈华云肖湘文
Owner GUANGZHOU HEAS BIOTECH CO LTD
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