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Kit for detecting salmonella in foods and application method thereof

A Salmonella and kit technology, applied in the direction of biochemical equipment and methods, microbe measurement/inspection, and resistance to vector-borne diseases, etc., can solve the problems of long time consumption and low sensitivity, and achieve simple reaction steps, low cost, and detection short-term effect

Inactive Publication Date: 2013-07-31
IPHASE BIOSCI BEIJING
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Traditional Salmonella biological detection methods have great disadvantages, such as time-consuming, low sensitivity, expensive equipment, etc.

Method used

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  • Kit for detecting salmonella in foods and application method thereof
  • Kit for detecting salmonella in foods and application method thereof
  • Kit for detecting salmonella in foods and application method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 The kit of the present invention

[0041] The kit: includes 24 reaction tubes, each containing 23μL of reaction mixture and 30μL of liquid paraffin; 1 tube of positive control (50μL); 2 tubes of DNA extraction reagent A (1300μL / tube); 1 tube of DNA extraction Reagent B (600μL). The reaction mixture contains: outer primer F3 with a concentration of 0.2 μM, the nucleotide sequence of which is shown in SEQ ID NO: 1; and outer primer B3 with a concentration of 0.2 μM, whose nucleotide sequence is as shown in SEQ ID NO: 2 The nucleotide sequence of the inner primer FIP with a concentration of 1.6 μM is shown in SEQ ID NO: 3; the nucleotide sequence of the inner primer BIP with a concentration of 1.6 μM is shown in SEQ ID NO: 4; The nucleotide sequence of the loop primer LoopF ​​of 0.8μM is shown in SEQ ID NO:5; the nucleotide sequence of the loop primer LoopB of 0.8μM is shown in SEQ ID NO:6; 20mM pH8.8 Tris buffer, 10mM KCl, 10mM NH 4 SO 4 , 0.1% by weight of Tween...

Embodiment 2

[0043] Example 2 Sensitivity test of the method of the present invention

[0044] Preparation of BPW medium: Weigh 4.5 g of CM201 buffered peptone water medium (purchased from Beijing Luqiao Technology Co., Ltd.), add water to prepare 225 mL of enrichment medium, which is called BPW medium. The pH value is 7.0±0.2. Sterilize at 121°C for 15 minutes, and then set aside.

[0045] Salmonella was inoculated into 4 mL of BPW medium and cultured overnight at 37°C. 40 μL of overnight culture broth was inoculated into another new 4 mL BPW medium and cultured at 35°C at 150 rpm for 4 hours to obtain metaphase cells. Then it was diluted with normal saline 10-fold gradient dilution method, and the treatment of each concentration was performed three times. Select 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 100 μL of solutions of 6 dilutions were spread on TSA plates (purchased from Beijing Luqiao Technology Co., Ltd.) and incubated at 37°C overnight. Colony count results are shown in Tabl...

Embodiment 3

[0053] Example 3 Specificity test of the method of the present invention

[0054] Pick 13 strains of Salmonella (Sal), 6 strains of Escherichia coli (E.coli), 8 strains of Vibrio parahaemolyticus (VP), 2 strains of Shigella (Shi), and 12 strains of Listeria monocytogenes. The bacteria (LM) and 6 strains of Staphylococcus aureus (SA) were added to 200μL of sterilized deionized water, heated at 98°C for 10min, centrifuged at 7000g for 2min, and the supernatant was taken as the LAMP detection DNA template solution.

[0055] Add 2 μL of the DNA template solution prepared above into a reaction tube containing 23 μL of the reaction solution described in Example 1 and 30 μL of liquid paraffin, and react at 64° C. for 60 min. The positive is green and the negative is orange.

[0056] The results showed that only the Salmonella test tubes were positive, and the test tubes for other strains were negative.

[0057]

[0058]

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Abstract

The invention relates to a kit for detecting salmonella in foods. The kit comprises reaction mixed liquor, a DNA extraction reagent A, a DNA extraction reagent B and a positive control, wherein the reaction mixed liquor contains an outer primer F3, an outer primer B3, an inner primer FIP, an inner primer BIP, an ring primer LoopF, a ring primer LoopB, 20mM of Tris buffer (pH being 8.8), 10mM of KCl, 10mM of nH4SO4, 0.1 wt% of polysorbate 20, 4-8mM of MgSO4, 0.8M of glycine betaine, 1.2mM of dNTP, 0.04-0.08mM of calcein, 0.4-1.6mM of MnCl2, 0.32U / mu L of Bst DNA polymerase, wherein the concentrations of the outer primer F3 and the outer primer B3 are both 0.2mu M, the concentrations of the inner primer FIP and the inner primer BIP are both 1.6mu M, and the concentrations of the ring primer LoopF and the ring primer LoopB are both 0.8mu M. The invention also relates to an application method of the kit. Compared with traditional detection methods, the method disclosed by the invention is short in detection time and low in cost, results can be directly determined, and no instrument is required.

Description

Technical field [0001] The present invention relates to the field of molecular biology detection. Specifically, the present invention relates to a kit for detecting Salmonella in food and a method of use thereof. Background technique [0002] Loop-mediated isothermal amplification (LAMP) is a new nucleic acid amplification method that uses a DNA polymerase (Bst DNA polymerase) with strand displacement activity to perform specific, sensitive, and Efficient nucleic acid amplification, LAMP reaction requires specific design of 2 inner primers (FIP and BIP) and 2 outer primers (F3 and B3). The target DNA is kept at a constant temperature of 60℃~65℃ (such as in a water bath). It takes about 60 minutes to complete the LAMP nucleic acid amplification reaction. The final product of LAMP includes a large number of stem-circular DNA mixtures. Because LAMP amplification produces a huge amount of DNA products and pyrophosphate ions, the reaction of pyrophosphate ions with magnesium ions in ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/10
CPCY02A50/30
Inventor 刘鸿君王倩李力
Owner IPHASE BIOSCI BEIJING
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