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Method for jointly detecting microorganisms through combination of nucleic acid isothermal amplification and CRISPR/Cas13a, and application of method

An isothermal amplification and combined detection technology, applied in the biological field of pathogenic microorganism and food-borne pathogen detection, can solve the problems of expensive instruments and cannot be used in basic laboratories, and achieve the effect of high sensitivity, good specificity and simple operation.

Pending Publication Date: 2021-10-01
ZHEJIANG UNIV
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  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to: overcome the deficiency that the current microbial detection technology requires expensive instruments and cannot be applied in the basic laboratory, and provides a combination of nucleic acid isothermal amplification (Recombinase Aided Amplification, RAA) based on recombinase mediated T7 transcription and CRISPR / Cas13a The method and application of the simple and rapid detection of microorganisms, the method uses two steps of RAA and combined T7 transcription-CRISPR / Cas13a detection, and the specific detection of microbial (such as Salmonella) DNA samples can be completed in 30 minutes, and only ordinary metal baths and Portable fluorescence detector, no need for fluorescent quantitative PCR instrument

Method used

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  • Method for jointly detecting microorganisms through combination of nucleic acid isothermal amplification and CRISPR/Cas13a, and application of method
  • Method for jointly detecting microorganisms through combination of nucleic acid isothermal amplification and CRISPR/Cas13a, and application of method
  • Method for jointly detecting microorganisms through combination of nucleic acid isothermal amplification and CRISPR/Cas13a, and application of method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1. Establishment of a system based on RAA isothermal amplification and CRISPR / Cas13a detection of Salmonella

[0052] 1. Design and synthesis of RAA primers

[0053] 1. Design principles

[0054] Different from PCR primers, RAA primers are in the range of 30-35nt in length, and there is no palindromic sequence, continuous single-base repeat sequence and internal secondary structure in the sequence, so it is usually not necessary to consider the Tm value of the primer. Specificity and conservation need to be confirmed by NCBI Blast screening.

[0055] 2. Design RAA Primers

[0056] Preferably, the well-conserved invA gene of Salmonella is selected. According to the above principles, after NCBI Blast screening, the RAA primer sequence was determined to be:

[0057]

[0058] (Note: the underlined sequence is the T7 promoter.)

[0059] 2. Design and synthesize crRNA

[0060] 1. Design principles

[0061] The function of crRNA is to specifically recognize t...

Embodiment 2

[0079] Embodiment 2. Feasibility analysis is carried out to the system

[0080] Cultivate fresh Salmonella and Escherichia coli, and detect according to the steps described in Example 1.

[0081] The test results are attached figure 1 As shown, when the template is Salmonella, the reaction tube will produce a strong fluorescent signal, while Escherichia coli has no fluorescent signal, which proves that the detection system is feasible and can specifically detect Salmonella.

Embodiment 3

[0082] Embodiment 3. Sensitivity detection is carried out to the system

[0083] Take fresh Salmonella, adjust OD 620 to the range of 0.05-0.09 (about 10 8 CFU / mL), using PBS for ten-fold serial dilution, take 1mL 10 5 -10 0 The CFU / mL bacterial liquid was detected according to the steps described in Example 1 (at the same time, a suitable gradient bacterial liquid was taken for plate counting), and DEPC water was set up as a negative control, and the high-concentration Salmonella genome was used as a positive control.

[0084] The test results are attached figure 2 As shown, the detection limit of the proof system is 10 1 CFU / mL, with strong sensitivity.

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Abstract

The invention relates to a method for detecting microorganisms through combination of recombinase-mediated nucleic acid isothermal amplification and CRISPR / Cas13a, and application of the method. The method comprises the following steps: carrying out RAA isothermal amplification on microbial genome DNA to obtain a DNA product with a T7 promoter (referred as T7-DNA), and then jointly carry out T7 transcription and CRISPR / Cas13a detection. When a Cas13a-crRNA compound recognizes and specifically bind to RNA obtained by transcription of the T7-DNA, Cas13a randomly cuts surrounding irrelevant single-chain RNA fluorescent reporter molecules, and a fluorescent signal is generated. The method disclosed by the invention can specifically detect 10<1> CFU / mL of microorganisms only in 30 minutes, is short in time, high in sensitivity and good in specificity, only needs a common metal bath and a portable fluorescence detector, and is suitable for field detection of food-borne pathogens, medicine and veterinary pathogenic microorganisms or suitable for use in primary laboratories.

Description

technical field [0001] The invention belongs to the technical field of detection of pathogenic microorganisms and food-borne pathogens, and is a method and application for rapid and specific detection of microorganisms based on the combined application of recombinase-mediated nucleic acid isothermal amplification technology (RAA) and CRISPR / Cas13a technology. Background technique [0002] Kary Mullis invented the polymerase chain reaction (PCR) in 1983, making amplification and cloning of DNA a routine procedure. Therefore, in vitro DNA amplification technology has developed rapidly, and technologies such as real-time quantitative PCR and immune PCR have come out one after another. However, due to the need for thermal cycling, these techniques rely on relatively expensive PCR machines, which limits their application in basic laboratories or on-site. In order to solve the above shortcomings, isothermal nucleic acid amplification technology has quietly emerged. Recombinase A...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/6844C12Q1/04C12R1/42
CPCC12Q1/689C12Q1/6844C12Q2521/507C12Q2522/101C12Q2521/327C12Q2563/107
Inventor 方维焕付豪李肖梁
Owner ZHEJIANG UNIV
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