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CPA detection primer, detection kit and detection method for salmonella

A Salmonella and detection primer technology, applied in the biological field, can solve the problem of high difficulty in the design of reaction primers, and achieve the effects of ensuring reliability, shortening cycle, and short time-consuming

Pending Publication Date: 2021-10-01
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the design of primers for this reaction is very difficult, and it is necessary to design five primers in a limited product length, and to avoid non-specific amplification of the five primers themselves and affect the results, so the design of primers is particularly important

Method used

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  • CPA detection primer, detection kit and detection method for salmonella
  • CPA detection primer, detection kit and detection method for salmonella
  • CPA detection primer, detection kit and detection method for salmonella

Examples

Experimental program
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Effect test

Embodiment 1

[0039] The primer screening test for detection of Salmonella by cross constant temperature amplification reaction comprises the following steps:

[0040] According to the sequence of the invA gene, after designing with NCBI Primer-Blast, three sets of detection primers with higher scores were selected for comprehensive comparison, and the 4s and 5a primers of each set of primers were used as upstream detection primers and downstream detection primers for PCR amplification. To determine whether the region corresponding to the primer can be amplified and the specificity of the 4s and 5a stripping primers. When the reaction is single and consistent with the size of the corresponding product, that is, the upstream and downstream stripping primers have good specificity. If the stripping primers have non-specific amplification, a ladder-like band will appear, which may lead to subsequent false positive results;

[0041] Three sets of detection primers were designed as follows:

[004...

Embodiment 2

[0064] The method for detecting Salmonella based on cross-primer constant temperature amplification reaction technology comprises the following steps:

[0065] 1. The method for detecting pathogenic microorganisms based on the cross-primer constant temperature amplification reaction technology, the present embodiment takes Salmonella as an example, and the reagents used are as follows:

[0066] a. The stripping primers 4s and 5a, the cross-amplification primers 2a1s, and the specific primers 2a and 3a each at a concentration of 10 μM, the primer sequences are as shown in the preceding SEQ ID NO.1-SEQ ID NO.5;

[0067] b.2×Reaction stock solution: Tris-HCl with concentration of 40.0mM, ammonium sulfate of 20.0mM, potassium chloride of 20.0mM, magnesium sulfate of 16.0mM, Tween 20 of 0.2% (v / v), 1.4M Betaine, 10.0mM dNTPs (each) composition;

[0068] c. Bst DNA polymerase (large fragment, NEB company) aqueous solution with a concentration of 8U / μL;

[0069] d. Mixed solution o...

Embodiment 3

[0080] The cross constant temperature amplification reaction detection Salmonella specificity test comprises the following steps:

[0081] The genomic DNA of Salmonella and non-Salmonella was established according to the reaction system and conditions in Example 1. The cross constant temperature amplification reaction detection method was carried out, and the specificity test was carried out; wherein, the non-Salmonella was: Escherichia coli O157:H7ATCC43895; Escherichia coli O157:H7ATCC43894; Escherichia coli O157:H7E019; Escherichia coli O157:H7E043; Escherichia coli O157:H7E044; Listeria monocytogenes ATCC19116; Listeria monocytogenes ATCC19114, Listeria monocytogenes ATCC19115; Listeria monocytogenes ATCC15313; Listeria monocytogenes Bacteria ATCC19113; Pseudomonas aeruginosa ATCC27853; Staphylococcus aureus ATCC27664; Methicillin-resistant Staphylococcus aureus NCTC10442; Methicillin-resistant Staphylococcus aureus N315; Staphylococcus aureus CA05; Vibrio parahaemolyticus...

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Abstract

The invention discloses a CPA detection primer, a detection kit and a detection method of salmonella. The CPA primer designed aiming at a target invA comprises stripping primers 4s and 5a, a cross amplification primer 2a1s and specific primers 2a and 3a; the nucleotide sequences of the primers are respectively as shown in SEQ ID NO.1 to SEQ ID NO.5. The cross primer isothermal amplification reaction detection and identification system designed for the salmonella specific target sequence invA provided by the invention overcomes the defects of long period, low sensitivity, high cost, difficulty in field application and the like of the method in the prior art. By selecting a conserved region of a specific sequence invA of a target strain, a pair of stripping primers, a cross primer and a specific primer are designed to construct a cross primer isothermal amplification reaction system, and a detection result can be obtained within about 60 minutes so as to shorten the traditional salmonella detection period.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a CPA detection primer for Salmonella, a detection kit and a detection method. Background technique [0002] Salmonella is a common food-borne zoonotic pathogen, which can infect humans and animals through contaminated food, feed or feces, and can survive in the intestinal tract of humans and animals, clinically manifested as sepsis, gastroenteritis and local inflammation and food poisoning of other tissues, causing great harm to human and animal life and health. The main medium of transmission of Salmonella is through poultry, eggs, milk, meat and other nutritious poultry and livestock products, which easily pollute water sources and food. According to statistics, among the types of bacterial food poisoning in various countries in the world, food poisoning caused by Salmonella often ranks first. Salmonella is also the first in my country's inland areas. In our country...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/6844C12Q1/10C12N15/11
CPCC12Q1/689C12Q1/6844C12Q2531/119C12Q2537/1376C12Q2563/107Y02A50/30
Inventor 徐振波林欣骆玉婷刘君彦陈玲叶燕锐李晓玺洪玮彭芳付欣户帅锋苏健裕
Owner SOUTH CHINA UNIV OF TECH
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