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Toehold structure for rapid detection of food-borne pathogenic microorganisms and application of Toehold structure

A rapid technology for pathogenic microorganisms, applied in the field of molecular biology, can solve problems such as the inability to distinguish dead bacteria from live bacteria, insufficient sensitivity, and limitations in primer design, and achieve rapid and efficient detection, a wide range of detection, and low prices.

Active Publication Date: 2020-02-21
ZHEJIANG GONGSHANG UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But at the same time, the disadvantages are also obvious, such as high cost, limitations in primer design, insufficient sensitivity, inability to distinguish dead bacteria from live bacteria, professional operation is required, and specificity is not strong, etc.

Method used

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  • Toehold structure for rapid detection of food-borne pathogenic microorganisms and application of Toehold structure
  • Toehold structure for rapid detection of food-borne pathogenic microorganisms and application of Toehold structure
  • Toehold structure for rapid detection of food-borne pathogenic microorganisms and application of Toehold structure

Examples

Experimental program
Comparison scheme
Effect test

Embodiment example 1

[0025] Implementation Case 1: Selection of Pathogenic Bacteria and Detection Objects

[0026] With reference to the most important pathogens in food contamination, the present invention selects 10 common food-borne pathogens as the main objects for developing the Toehold detection method. The principle of Toehold detection is based on the interaction between RNAs, that is, complementary base pairing. Therefore, the selection of detection objects is based on the mRNA sequence of the highly conserved bacterial species or the characteristic protein-coding gene, which is the trigger. Among them, the conserved region in the 16s rRNA of Staphylococcus aureus and Vibrio parahaemolyticus, the Shiga toxin coding gene stx1 of Escherichia coli O157:H7, and the stx1 gene of Salmonella enterica Secreted toxin protein coding gene invA, Enterobacter aerogenes (Enterobacter aerogenes) type I pili coding gene fimH, Pseudomonas aeruginosa (Pseudomonas aeruginosa) exotoxin A coding gene toxA, Kl...

Embodiment example 2

[0027] Implementation case 2: Toehold structure and sequence design

[0028] Toehold structure includes toehold sequence, RBS, AUG and other parts, and the downstream is directly composed of linker and reporter gene fluorescent protein coding sequence (mCherry) ( figure 1 ). The design process is mainly as follows: search the coding gene sequence of the detection object from the NCBI database, analyze the two-segment structure of the RNA level by software, and select the specific chain partial sequence. High sequence specificity means that the sequence does not exist in other bacterial species or the part with a low matching degree is used as a trigger candidate sequence (this sequence is a trigger for in vitro testing). The Toehold structure and sequence were designed by software, and 6 Toehold structure sequences were obtained for each type of bacteria for testing. A total of 60 Toehold structure sequences were obtained for 10 microorganisms. See SEQ ID No. 1-60 for sequen...

Embodiment example 3

[0029] Implementation Case 3: In Vitro Testing the Feasibility and Efficiency of the Designed Toehold Structure

[0030] Using the whole gene synthesis method, synthesize Toehold 1 structural sequence, including T7 promoter, Toehold and reporter gene, T7 terminator and other parts, such as figure 2 shown. Using the synthesized pUCT1 as a template, primers corresponding to the Toehold sequence were used to amplify and prepare another 59 Toehold structure transcription templates. See SEQ ID Nos. 121-238 for sequence information. The materials used for plasmid construction, including PCR system, restriction endonuclease, ligase, plasmid extraction kit, DNA fragment purification kit, and competent cell production kit, were all purchased from Treasure Bioengineering (Dalian) Co., Ltd.; Completed by Sangon Bioengineering (Shanghai) Co., Ltd.

[0031] Toehold RNA and trigger RNA were synthesized in vitro. According to the sequence of the trigger, design forward and reverse primers...

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Abstract

The invention provides a Toehold structure for rapid detection of food-borne pathogenic microorganisms and an application of the Toehold structure, and belongs to the technical field of molecular biology. The invention discloses the Toehold structure for the rapid detection of ten food-borne pathogenic microorganisms, and a sequence of the Toehold structure is shown as SEQ ID No. 1-60, and the invention discloses the application of the Toehold structure for the rapid detection of the ten food-borne pathogenic microorganisms. Compared with a current method, a method provided by the invention has the advantages of simple operation, high detection accuracy, accurate results, a low price, low detection limit (fM level), a large detection sample size (simultaneous detection of hundreds of samples), and short time (the whole process is 2-3 h).

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to a Toehold structure for rapid detection of food-borne pathogenic microorganisms and an application thereof. Background technique [0002] Food safety hazards are mainly caused by pathogenic microorganisms. Common foodborne pathogenic microorganisms such as Salmonella, Escherichia coli O157:H7, Staphylococcus aureus, Les Listeria monocytogenes, Vibrio parahaemolyticus, etc. Solving food safety problems In addition to controlling possible contamination routes during the production process, an effective detection method is more important for ensuring food safety and preventing the spread of diseases. With the development of molecular biology techniques, different technical means have been continuously developed for the detection of pathogenic microorganisms. Common immunological detection techniques such as enzyme-linked immunosorbent assay (ELISA), immunomag...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/6865C12Q1/14C12Q1/10C12Q1/04C12N15/11
CPCC12Q1/689C12Q1/6865C12Q2563/107Y02A50/30
Inventor 梁新乐夏凯
Owner ZHEJIANG GONGSHANG UNIVERSITY