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Primer combination and kit for simultaneously detecting cronobacter and salmonella and application of primer combination and kit

A Cronobacter and primer combination technology, applied in the direction of microorganisms, recombinant DNA technology, microorganism-based methods, etc., can solve the problem of long detection time, inability to meet the needs of rapid detection of food-borne pathogens, cumbersome detection steps, etc. problem, to achieve the effect of simple production method, good storage stability and good specificity

Pending Publication Date: 2021-11-26
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Traditional microbiological methods require extremely long detection time and extremely cumbersome detection steps, which are not suitable for the rapid pace of contemporary life and production. Therefore, this method cannot meet the needs of rapid detection of foodborne pathogens, so there is an urgent need for a A simple and rapid detection technique

Method used

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  • Primer combination and kit for simultaneously detecting cronobacter and salmonella and application of primer combination and kit
  • Primer combination and kit for simultaneously detecting cronobacter and salmonella and application of primer combination and kit
  • Primer combination and kit for simultaneously detecting cronobacter and salmonella and application of primer combination and kit

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Embodiment 1. The establishment of double LAMP reaction system

[0054] (1) Design of primers for LAMP amplification of different pathogenic bacteria

[0055] Primers were designed and screened for the siiA gene of Salmonella and the ITS sequence of Cronobacter as target sequences.

[0056] ①Salmonella

[0057] LAMP primers were designed for the conserved sequence siiA gene of Salmonella (STM4257, the sequence number in NCBI is: NC_003197), and 6 sets of primers were designed by using the online software PrimerExplorer V4 (Table 1-6). Finally, the screened specific primers were used as templates for LAMP amplification, and a set of primers with the best amplification efficiency was used for subsequent experiments. The result is as figure 1 , primer siiA-1 had the best amplification effect.

[0058] Table 1 LAMP detection Salmonella primer siiA-1

[0059]

[0060] Table 2 LAMP detection Salmonella primer siiA-2

[0061]

[0062] Table 3 LAMP detection Salmone...

Embodiment 2

[0101] Example 2. Double test strip construction

[0102] (1) Determination of coating antibody conditions

[0103] T1 line coated with anti-streptavidin antibody, T2 line coated with anti-digoxigenin antibody: the optimization of the antibody coating in the NC membrane determines the detection effect of the double LFD, and the antibody pH value and antibody concentration are optimized respectively. The pH value of the anti-digoxigenin antibody was optimized, and the experimental results were as follows Figure 10 shown. The results showed that no matter what the pH value of the anti-digoxigenin antibody was, there were weak false positives in the negative results, and when the pH value was 9, the detection signal of the T2 line in the positive results was the strongest. Carry out the optimization of the second antibody concentration according to table 11, the result is as follows Figure 11 shown. When the dilution concentration of T1 line antibody is 0.5 mg / mL, the detec...

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Abstract

The invention discloses a primer combination and a kit for simultaneously detecting cronobacter and salmonella and application of the primer combination and the kit and belongs to the technical field of food safety. In order to solve the problems existing in detection on the salmonella and the cronobacter by a traditional microbiological method, the invention provides a dual test strip based on a LAMP reaction and a primer combination as shown in SEQ ID NO: 1-8, and the dual test strip comprises the following steps: extracting DNAs (deoxyribonucleic acids) from the salmonella and the cronobacter, and amplifying a large number of target products through the LAMP reaction; and directly interpreting a result by naked eyes through the dual test strip constructed on the basis of preparation of stable colloidal gold, a gold-labeled antibody, a redissolution solution and a diluent. The dual test strip obtained by the invention has the advantages of simplicity in manufacturing, low cost, sensitivity in detection and the like, and can be used for simultaneously detecting salmonella and cronobacter pollution in foods and food processing processes, especially pathogenic bacterium pollution in infant formula milk powder.

Description

technical field [0001] The invention belongs to the technical field of food safety, and specifically relates to a primer combination, a kit and an application thereof for simultaneously detecting Cronobacter and Salmonella. Background technique [0002] In recent years, with the outbreak of a series of milk powder recalls and major infection incidents involving foodborne pathogenic bacteria, the contamination of milk with pathogenic bacteria has attracted worldwide attention. Foodborne pathogens in milk powder include Cronobacter, Escherichia coli, Salmonella, Staphylococcus aureus, Citrobacter, Enterobacter cloacae and Listeria monocytogenes, etc., which seriously threaten infants The types of health and life safety are mainly Cronobacter and Salmonella. [0003] Salmonella, one of the most common food-borne pathogens worldwide, belongs to the Enterobacteriaceae family, a motile, non-spore facultative anaerobic Gram-negative bacteria, when the human body is infected with S...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/6844C12Q1/10C12N15/11C12R1/42C12R1/01
Inventor 满朝新姜毓君秦雪付世骞杨鑫焱
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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