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A kind of primer, kit and method for detecting Escherichia coli type I Shiga toxin by PSR

A technology of Escherichia coli and Shiga toxin, which is applied in biochemical equipment and methods, microbial measurement/testing, DNA/RNA fragments, etc., can solve problems such as high technical cost, high reagent price, and many influencing factors, and achieve operational Simple and quick, shortened detection cycle, low detection cost

Active Publication Date: 2021-09-21
SOUTH CHINA UNIV OF TECH
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  • Description
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AI Technical Summary

Problems solved by technology

Immunological detection methods such as ELISA method are to adsorb immunologically active antigens or antibodies on the surface of a solid phase carrier, add an active enzyme-labeled antibody or antigen, and then add an enzyme chromogenic substrate, and the active enzyme acts on the substrate. Judging by producing color, this method has high sensitivity and strong specificity, but its operation process is complicated, there are many influencing factors, and the cost is high, so it is not suitable for mass detection
The gene chip detection method developed in recent years has the characteristics of high throughput, rapidity, and high degree of automation. However, due to the high cost of this technology, it cannot be widely used in the field of routine detection.
Although the most widely used constant temperature amplification technology-LAMP has the advantages of high sensitivity and specificity, it also has disadvantages such as complex primer design, high false positive rate, and high reagent prices.

Method used

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  • A kind of primer, kit and method for detecting Escherichia coli type I Shiga toxin by PSR
  • A kind of primer, kit and method for detecting Escherichia coli type I Shiga toxin by PSR
  • A kind of primer, kit and method for detecting Escherichia coli type I Shiga toxin by PSR

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 Polymerase helical reaction detection stx1 primer screening

[0044] 1. Design primers

[0045] According to the principle of PSR amplification reaction, three sets of primers as shown in Table 1 were designed for the stx1 target using Primer Premier software.

[0046] Table 1 Primer Information

[0047] Primer name Sequence 5'-3' Ft-stx1-1 CCTCTGTATTTGCCGAAAAC-GCAAGAGCGATGTTACGG Bt-stx1-1 CAAAAGCCGTTTATGTCTCC-AGGCAGGACACTACTCAACC IF-stx1-1 GCTTCAGCTGTCACAGT IB-stx1-1 TCTTACATTGAACTGGGGA Ft-stx1-2 CAGTCATTACATAAGAACGCC-TTCGGCAAATACAGAGGG Bt-stx1-2 CCGCAAGAATACATTACTGAC-AGGCAGGACACTACTCAACC IF-stx1-2 GATCATCCAGTGTTGTA IB-stx1-2 TACATTGAACTGGGGA Ft-stx1-3 CAGTGTTGTACGAAATCCCC-GAGCGATGTTACGGTTTG Bt-stx1-3 CCCCTAAAGCATGTTGTGAC-AGGCAGGACACTACTCAA IF-stx1-3 CTGTATTTGCCGAAAAC IB-stx1-3 ACATTGAACTGGGGAAG

[0048] 2. Establish a polymerase helical reaction detection met...

Embodiment 2

[0058] Embodiment 2 detects the microbiological method of E.coli O157:H7 based on polymerase helical reaction isothermal amplification technology

[0059] 1. The method for detecting pathogenic microorganisms based on polymerase helical isothermal amplification technology. In this embodiment, E.coliO157:H7 is used as an example, and the reagents used are as follows:

[0060] a. The detection primer Ft aqueous solution, the Bt aqueous solution, the accelerated primer IF aqueous solution, and the IB primer aqueous solution with a concentration of 50 μM each. The primer sequences are as follows (5'-3'):

[0061] Detection primer Ft:

[0062] 5'-CAGTGTTGTACGAAATCCCCGAGCGATGTTACGGTTTG-3' (SEQ ID NO.1);

[0063] Detection primer Bt:

[0064] 5'-CCCCTAAAGCATGTTGTGACAGGCAGGACACTACTCAA-3' (SEQ ID NO. 2);

[0065] Accelerated Primer IF:

[0066] 5'-CTGTATTTGCCGAAAAC-3' (SEQ ID NO.3);

[0067] Accelerated Primer IB:

[0068] 5'-ACATTGAACTGGGGAAG-3' (SEQ ID NO. 4).

[0069] b.2×Rea...

Embodiment 3

[0076] Example 3 Polymerase helical reaction detection type I Shiga toxin specificity test

[0077] Escherichia coli containing type I Shiga toxin (Escherichia coli O157:H7ATCC43895, Escherichia coli E019, Escherichia coli E020, Escherichia coli E043, Escherichia coli E044) and other strains without type I Shiga toxin (Staphylococcus aureus ATCC23235, Staphylococcus aureus ATCC6358, Staphylococcus aureus ATCC12600, Staphylococcus aureus ATCC25923, Staphylococcus aureus ATCC19095, Salmonella ATCC29629, Salmonella ATCC19585, Salmonella ATCC14028, Salmonella ATCC13076, Salmonella ATCC700155, Salmonella ATCC91 Listeria monocytogenes 9 Listeria monocytogenes ATCC19116, Listeria monocytogenes ATCC19114, Listeria monocytogenes ATCC19115, Listeria monocytogenes ATCC15313, vibrio parahaemolyticus ATCC27969, vibrio parahaemolyticus ATCC17802) Genomic DNA according to above-mentioned embodiment 1 Reaction system and conditions Establish the detection method of polymerase helical reaction...

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Abstract

The invention discloses a primer, a kit and a method for detecting Escherichia coli type I Shiga toxin by PSR. The present invention designs primers for detecting Escherichia coli type I Shiga toxin for the heterosexual target sequence stx1 of type I Shiga toxin, and the primers include detection primer Ft and detection primer Bt, acceleration primer IF and acceleration primer IB, and its nucleotide The sequences are shown as SEQ ID NO.1-SEQ ID NO.4. The present invention also provides a kit for detecting Escherichia coli type I Shiga toxin by PSR, comprising the above-mentioned primers, Bst DNA polymerase, and a mixed solution of calcein and manganese chloride, capable of detecting Escherichia coli type I Shiga toxin For the detection of polymerase helical reaction, the result can be judged by the naked eye by directly using fluorescent dyes to develop color. The operation is simple and fast, and the detection cost is low, which is suitable for on-site detection applications.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a primer, a kit and a method for detecting Escherichia coli type I Shiga toxin by PSR. Background technique [0002] Shiga toxin is mainly produced by Escherichia coli, and Shiga toxin-producing Escheriachia coli (STEC) is the main pathogenic factor of human food poisoning in the world, and it is also the main cause of large-scale foodborne food poisoning. pathogenic bacteria. There are more than 100 serotypes of pathogenic Escherichia coli in STEC that can cause disease, and can cause non-hemorrhagic diarrhea, hemorrhagic colitis, hemolytic uremic syndrome and other diseases. The pathogenic characteristic virulence factors are Shiga toxin type I (stx1) and Shiga toxin type II (stx2), encoded by stx1 and stx2 respectively, and diseases caused by Stx1 are often reported. [0003] Currently, Shiga toxin detection methods mainly include conventional detection methods, PCR detection me...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/689C12Q1/6844C12Q1/10C12N15/11
CPCC12Q1/6844C12Q1/689C12Q2531/119C12Q2563/107
Inventor 徐振波徐瑞瑞徐行勇刘君彦陈玲苏健裕李冰李琳李晓玺张霞
Owner SOUTH CHINA UNIV OF TECH
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