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Primer, kit and method for detecting bacillus coli shiga toxin through PSR (Polymerase Spiral Reaction) isothermal amplification reaction detection

A technology of Escherichia coli and Shiga toxin, which is applied in the biological field, can solve the problems of complex operation process, heavy workload, and high cost, and achieve the effect of simple and fast operation, low detection cost, and guaranteed reliability

Pending Publication Date: 2018-11-13
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The workload of conventional detection and identification methods is relatively large, and the test results usually take 4 to 5 days to get the results, which takes a long time. Judging the identification results based on experimental phenomena is difficult to meet the needs of rapid identification
The gene chip detection and PCR amplification methods developed in recent years have strong specificity, rapidity and sensitivity, but the cost is high
Immunological detection methods, such as ELISA, immunochromatography, etc., have high sensitivity, but the operation process is complicated and the cost is high, so it is not suitable for large-scale detection of samples
At present, the most widely used constant temperature amplification technology-LAMP also has its limitations, such as complex primer design, high false positive rate, high reagent price, and due to the protection of intellectual property rights in Japan, my country has strong limitations in transformation and application

Method used

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  • Primer, kit and method for detecting bacillus coli shiga toxin through PSR (Polymerase Spiral Reaction) isothermal amplification reaction detection
  • Primer, kit and method for detecting bacillus coli shiga toxin through PSR (Polymerase Spiral Reaction) isothermal amplification reaction detection
  • Primer, kit and method for detecting bacillus coli shiga toxin through PSR (Polymerase Spiral Reaction) isothermal amplification reaction detection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 Based on the polymerase helical reaction isothermal amplification technology to detect the microbial method of E.coli O157:H7

[0039]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:

[0040] a. The detection primer Ft aqueous solution and the Bt aqueous solution primer sequences with a concentration of 50 μM are as follows (5'-3'):

[0041] Detection primer Ft: CTCTTCAGCCAGTCGTCGTG-CAACAGCGACATCATCCG (SEQ ID NO.1);

[0042] Detection primer Bt: GTGCTGCTGACCGACTTCTC-ATTCCTTCCCGTAACAACT (SEQ ID NO.2);

[0043] b.2 × reaction stock solution: Tris-HCl with a concentration of 40.0mM, ammonium sulfate 20.0mM, potassium chloride 20.0mM, magnesium sulfate 16.0mM, 0.2% (v / v) Tween 20, betaine 1.4 M, dNTPs (each) 10.0mM mixture composition;

[0044] c. Bst DNA polymerase (large fragment, NEB company) aqu...

Embodiment 2

[0052] Example 2 Polymerase helical reaction detection Shiga toxin specificity test

[0053] Escherichia coli containing Shiga toxin (Escherichia coli E019 [1] , E. coli E020 [1] , E. coli E043 [1] , E. coli E044 [1] , Escherichia coli ATCC43895) and other strains that do not contain Shiga toxin (Salmonella ATCC29629, Salmonella ATCC19585, Salmonella ATCC14028, Salmonella ATCC 13076, Listeria monocytogenes ATCC19116, Listeria monocytogenes ATCC19114, Listeria monocytogenes ATCC19115, Pseudomonas aeruginosa Genomic DNA of Monomonas ATCC27853, Pseudomonas aeruginosa ATCC9027, Staphylococcus aureus ATCC23235, Staphylococcus aureus ATCC6358, and Vibrio parahaemolyticus ATCC17802 was established according to the above reaction system and conditions. Test.Set the genome containing Shiga toxin escherichia coli as positive control, and ultrapure water as negative control (negative control and figure 1 The results of the blank control in the same), the results are as follows figu...

Embodiment 3

[0054] Example 3PSR detects the sensitivity test of Escherichia coli Shiga toxin

[0055] The genome of Escherichia coli O157 was serially diluted 10 times to 68ng / μl, 6.8ng / μl, 680pg / μl, 68pg / μl, 6.8pg / μl, 680fg / μl, and a negative control (deionized water) was set at the same time According to the reaction system in the above-mentioned embodiment 1, the polymerase helical reaction amplification method is constructed to determine the sensitivity of the detection method, and the results are as follows image 3 shown. The results showed that the established Escherichia coli Shiga toxin polymerase helical reaction method could detect 680pg / μL of Escherichia coli DNA in the sample.

[0056] Conclusion: From the above experimental results, it can be seen that the polymerase helical reaction amplification method has the following advantages over conventional PCR and fluorescent PCR:

[0057] The operation and identification are simple and fast: the whole process of conventional PC...

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Abstract

The invention discloses a primer, a kit and a method for detecting bacillus coli shiga toxin through PSR (Polymerase Spiral Reaction) isothermal amplification reaction detection. The primer comprisesa detection primer Ft and a detection primer Bt, wherein a nucleotide sequence of the detection primer Ft is shown in SEQ ID NO.1; a nucleotide sequence of the detection primer Bt is shown in SEQ ID NO.2. The invention also provides a kit for detecting the bacillus coli shiga toxin through the PSR isothermal amplification reaction. The kit comprises the primers, BstDNA polymerase and a mixed solution of calcein and manganese chloride; detection of polymerase spiral reaction on the bacillus coli shiga toxin can be carried out, time loss and short time consumption cannot be caused by the changeof a temperature; a reaction process is simple and convenient, a detection period is short, the specificity is strong, and detection results can be observed by naked eyes. According to the primer, thekit and the method for detecting the bacillus coli shiga toxin through the PSR isothermal amplification reaction detection disclosed by the invention, significance on amplification development of a novel constant-temperature amplification technology and detection on site of microorganism can be achieved.

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 Shiga toxin by PSR isothermal amplification reaction. Background technique [0002] Shiga toxin is mainly produced by Escherichia coli, and Shiga toxin-producing Escheria coli (STEC) is one of the main food pathogens. The main virulence factors of STEC are located on the stx1 and stx2 genes on the phage, and these two genes encode Shiga toxins Stx1 and Stx2 respectively. Shiga toxins Stx1 and Stx2 cause infection in patients at very low doses, which can cause severe abdominal pain and diarrhea in patients, and acute renal failure and death in severe cases. [0003] At present, the detection methods of Shiga toxin mainly include the traditional biochemical identification method, the PCR method of molecular biology technology, the immunology-based ELISA kit and the detection method using various biochemical automatic identifi...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/6844C12Q1/10C12N15/11C12R1/19
CPCC12Q1/6844C12Q1/689
Inventor 刘君彦徐振波徐行勇徐瑞瑞苏健裕刘丽艳李冰李琳
Owner SOUTH CHINA UNIV OF TECH
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