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Multiple lamp detection primers, detection kits and detection methods for six foodborne pathogenic bacteria in fruits and vegetables

A technology for detecting food-borne pathogenic bacteria and primers, which is applied in the direction of microorganism-based methods, biochemical equipment and methods, recombinant DNA technology, etc., and can solve problems such as the difficulty of analyzing primer detection results

Inactive Publication Date: 2021-01-05
INST OF QUALITY STANDARDS & TESTING TECH FOR AGRO PROD OF SHANDONG ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the difficulty and complexity of the mutual interference between primers, the optimization of multiple LAMP detection conditions, and the analysis of detection results, there has been no report so far that can simultaneously detect the multiplex of the six foodborne pathogens in the fruits and vegetables. LAMP method

Method used

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  • Multiple lamp detection primers, detection kits and detection methods for six foodborne pathogenic bacteria in fruits and vegetables
  • Multiple lamp detection primers, detection kits and detection methods for six foodborne pathogenic bacteria in fruits and vegetables
  • Multiple lamp detection primers, detection kits and detection methods for six foodborne pathogenic bacteria in fruits and vegetables

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0080] Embodiment 1: the preparation of kit

[0081] The kit includes several LAMP reaction tubes filled with reaction solution, wherein each 25 μL reaction solution contains: 2.5 μL of Thermoplol buffer, 2 μL of 25 mM magnesium chloride solution (MgCl 2 ), 2.5 μL of 10 M betaine Betaine, 1.0 μL of 8 U / μL Bst DNA polymerase, 0.8 μL of 1 U / μL UNG enzyme, 0.5 μL of 10 μM forward outer primer (F3 Sequence such as SEQ No.1, SEQ No.5, SEQ No.9, SEQ No.13, SEQNo.17, SEQ No.21), 0.5 μL concentration of 10 μM reverse outer primer (B3 sequence such as SEQ No. 2. SEQ No.6, SEQ No.10, SEQ No.14, SEQ No.18, SEQ No.22), 2 μL of forward internal primers with a concentration of 10 μM (FIP sequence such as SEQ No.3, SEQ No. 7. SEQ No.11, SEQ No.15, SEQ No.19, SEQ No.23), 2 μL reverse internal primer (BIP sequence such as SEQ No.4, SEQ No.8, SEQ No. 12. SEQ No.16, SEQ No.20, SEQ No.24), 2.5 μL of deoxyribonucleoside triphosphate (dNTP) with a concentration of 10 mM, 2.5 μL of the DNA templat...

Embodiment 2

[0082] Embodiment 2: specificity experiment

[0083] (1) Take 6 vegetable samples and add cultured suspensions of Listeria monocytogenes, Enterobacter sakazakii, Shigella, Staphylococcus aureus, Salmonella and Escherichia coli O157:H7 respectively, After mixing, samples were taken to enrich the bacteria for testing.

[0084] (2) Take the bacterial genomic DNA template to be detected, and perform gradient dilution with sterile water to obtain each dilution.

[0085] (3) Using the dilution obtained in step 2 as a template, the kits prepared in Example 1 were used to perform loop-mediated isothermal amplification. Reaction conditions: constant temperature at 65°C for 60min.

[0086] The sensitivity of the primer set to detect the target gene was 1.2×10 2 CFU / 25g, the test results are shown in Table 1:

[0087] Table 1 Specificity test results

[0088]

[0089] From the above table, it can be obtained that the Staphylococcus aureus LAMP method only detects Staphylococcus a...

Embodiment 3

[0090] Embodiment 3: Sensitivity experiment

[0091] (1) Remove the soil on the surface of the vegetable and fruit samples to be tested, and weigh four portions each weighing 25g, three of which correspond to the addition of cultured Listeria monocytogenes, Enterobacter sakazakii, Shigella, golden Low (L), medium (M) and high (H) concentrations of Staphylococcus aureus, Salmonella and Escherichia coli O157:H7 bacterial liquid, then add 225mL of combined enrichment liquid, and incubate at 36°C for 18-24 hours; The combined enrichment solution was used as a control.

[0092] (2) Extract the genomic DNA template of the bacteria to be tested: Take 1 mL of the cultured bacterial culture to be tested and add it to a 1.5 mL sterile centrifuge tube, centrifuge at 14000 r / min for 2 min, discard the supernatant, add 80 μL of the DNA extraction solution, and mix well Afterwards, incubate at 95°C for 10 minutes; centrifuge at 14,000r / min for 2 minutes, and the supernatant is the nucleic ...

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Abstract

The invention discloses a multiplex LAMP detection primer, kit and method for six food-borne pathogenic bacteria in fruits and vegetables and belongs to the technical field of bacterial gene detection. A rapid detection primer set for the six pathogenic bacteria including listeria monocytogenes, enterobacter sakazakii, shigella spp, staphylococcus aureus, salmonella spp and escherichia coli O157:H7 is designed, and multiplex LAMP reaction is performed on the genome DNA of the bacteria extracted from a sample to be detected in the same reaction system by use of the detection kit including the primer set to determine whether the sample contains the six food-borne pathogenic bacteria or not. The multiplex LAMP detection primer is high in specificity and sensitivity and can accurately detect the genome DNA of the six food-borne pathogenic bacteria in the same reaction system, can realize simple and convenient, quick and accurate detection, is suitable for on-site rapid detection and has significance on improving the pathogenic bacterium analysis and detection technology and the fruit and vegetable edible quality security.

Description

technical field [0001] The invention relates to the technical field of bacterial gene detection, in particular to a six-species food product containing Listeria monocytogenes, Enterobacter sakazakii, Shigella, Staphylococcus aureus, Salmonella and Escherichia coli O157:H7 in fruits and vegetables. A detection primer for multiple LAMP detection of pathogenic bacteria, a detection kit and a detection method thereof. Background technique [0002] As a global problem, foodborne diseases widely threaten public safety, and the phenomenon of diseases caused by pathogenic microorganisms carried by fruits and vegetables is also increasing year by year. In recent years, with the development of the national economy, the demand for fresh agricultural products is increasing. However, fresh fruits and vegetables, especially green leafy vegetables, are easy to spread pathogenic bacteria and viruses, and it is difficult to clean them by conventional methods. Therefore, it is of great signi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/689C12Q1/6844C12Q1/14C12Q1/10C12Q1/04C12N15/11C12R1/445C12R1/42C12R1/19C12R1/01
CPCC12Q1/6844C12Q1/689C12Q2600/16C12Q2531/119C12Q2537/143
Inventor 王文博刘宾苑学霞李瑞菊
Owner INST OF QUALITY STANDARDS & TESTING TECH FOR AGRO PROD OF SHANDONG ACADEMY OF AGRI SCI
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