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Method for detecting microorganism based on CRISPR-Cas13a system and application

A technology to be detected for microorganisms, which is applied in the field of detecting Staphylococcus aureus, can solve problems such as undiscovered patent publications, and achieve the effects of broadening applications, accurate copy number, and wide detection range

Pending Publication Date: 2020-06-23
TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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[0005] Through the search, no patent publications related to the patent application of the present invention have been found

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  • Method for detecting microorganism based on CRISPR-Cas13a system and application
  • Method for detecting microorganism based on CRISPR-Cas13a system and application
  • Method for detecting microorganism based on CRISPR-Cas13a system and application

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Embodiment Construction

[0069] The embodiments of the present invention will be described in detail below. It should be noted that the embodiments are illustrative, not restrictive, and cannot limit the protection scope of the present invention.

[0070] The raw materials used in the present invention, unless otherwise specified, are conventional commercially available products; the methods used in the present invention, unless otherwise specified, are conventional methods in the art.

[0071] A method for detecting microorganisms based on the CRISPR-Cas13a system, the method obtains RNA through two-step amplification, that is, PCR amplification, and performing transcription and amplification of PCR products, which is called targetRNA, that is, target RNA, and the nucleic acid sequence of crRNA is designed, Detection of targetRNA using Cas13a-crRNA complex;

[0072] The specific steps are: in the presence of targetRNA, it can stimulate the accessory cutting ability triggered by the nucleic acid-speci...

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Abstract

The invention relates to a method for detecting a microorganism based on a CRISPR-Cas13a system. Firstly, a bacterium to be detected is pretreated, a genome of the bacterium to be detected is extracted, RNA, called target RNA, is obtained through two steps of PCR amplification and in-vitro transcription. When the target RNA exists, the attached cleavage ability of a Cas13a-crRNA compound can be motivated, namely single-stranded RNA is cleaved. A fluorophore is modified at one end of the RNA, and a quencher is modified at the other end of the RNA. When the Cas13a-crRNA compound detects the corresponding Target RNA, a fluorescent probe can be cleaved, the increase of a fluorescence value is caused, the increase of the fluorescence value is in a linear correspondence relationship with the bacterium to be detected, and the microorganism is detected. The method has the characteristics of excellent reliability, extremely high sensitivity, high specificity, easiness in implementation and short detection time, and has great potential for detection of the microorganism.

Description

technical field [0001] The invention belongs to the field of food safety detection biotechnology, in particular to a sensitive and specific detection method and application of Staphylococcus aureus based on CRISPR-Cas13a system. Background technique [0002] Since entering the 21st century, foodborne diseases have become an important factor affecting public health. According to the statistics of the World Health Organization (WHO), nearly 1.5 billion people in the world are infected with foodborne diseases every year, of which 70% are caused by pathogenic microorganisms in food. caused by pollution. The "China Food Safety Development Report (2018)" issued by the Jiangnan University Food Safety Risk Governance Research Institute and several universities pointed out that there are still four major risks in my country's food safety, among which microbial contamination is the most serious, accounting for the unqualified 32.74% of the total samples, an increase of 4.84% compared ...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/686C12Q1/14C12R1/445
CPCC12Q1/689C12Q1/686C12Q2521/327C12Q2563/107Y02A50/30
Inventor 马龙满淑丽
Owner TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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