LAMP detection reagent kit of food-borne yersinia enterocolitica and application of LAMP detection reagent kit

A technology of enterocolitis and detection kit, which is applied in the determination/inspection of microorganisms, recombinant DNA technology, DNA/RNA fragments, etc. It can solve the problems of long detection cycle and inaccurate test results, and achieve visible and convenient results. The effect of quick safety monitoring and accurate detection

Inactive Publication Date: 2020-01-31
SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the detection method of Yersinia enterocolitica is mainly based on the traditional physiological and biochemical detection method, which takes a long detection cycle and consumes a lot of manpower and material resources, and the overall detection result will be affected by other factors such as the environment, resulting in the overall detection of The result is not accurate

Method used

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  • LAMP detection reagent kit of food-borne yersinia enterocolitica and application of LAMP detection reagent kit
  • LAMP detection reagent kit of food-borne yersinia enterocolitica and application of LAMP detection reagent kit
  • LAMP detection reagent kit of food-borne yersinia enterocolitica and application of LAMP detection reagent kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1 Establishment of LAMP detection method for foodborne Yersinia enterocolitica

[0025] The ail gene encoding the deadhesive invasion site was selected as the target gene for detection (the complete gene sequence of ail of Yersinia enterocolitica is shown in SEQ ID NO.1), and two sets of detection primers were designed. The composition of FIP / BIP and outer primer F3 / B3, the primer sequence is shown in Table 1 below.

[0026] Table 1 Primer Sequence

[0027]

[0028] The DNA of Yersinia enterocolitica, a food-borne pathogen, was extracted as a template, and water was set up as a template as a negative control for LAMP amplification reaction, and the reaction product was tested for results.

[0029] The composition ratio of each component in the LAMP amplification is shown in Table 2 below.

[0030] Table 2 Composition of each component in the LAMP amplification reaction

[0031]

[0032] Add the above mixture into a PCR tube, and carry out isothermal amp...

Embodiment 2

[0036] Example 2 Specificity of LAMP in detecting foodborne Yersinia enterocolitica

[0037] Genomic DNA of Yersinia enterocolitica strain ATCC23715 was extracted, and tested as EHEC O157:H7ATCC43889, Salmonella SL7207, Staphylococcus aureus standard strain SA29213, Staphylococcus aureus isolate SAFX02, and Listeria Strain LMF2365, Listeria monocytogenes isolate FX01, Campylobacter jejuni HU-CJFX01, Enterotoxigenic E. coli ETEC-2, Enterotoxic E. coli ETEC-4, Enterotoxic E. 6 Genomic DNA of non-enterocolitica Yersinia ATCC23715, and water as negative control, were subjected to LAMP amplification reaction.

[0038] LAMP was amplified with the screened ail(1) primer set, and the reaction system was shown in Table 3 below.

[0039] Table 3 LAMP amplification reaction system

[0040]

[0041] Add the above mixture into a PCR tube, and carry out isothermal amplification in a water bath. The amplification steps are: constant temperature incubation at 65°C for 1 hour, and inactiv...

Embodiment 3

[0044] Example 3 Sensitivity of LAMP to detect foodborne Yersinia enterocolitica

[0045] Take the foodborne pathogen Yersinia enterocolitica ATCC23715 in the logarithmic growth phase, dilute it 10 times with normal saline, and count the colonies on the plate to calculate the concentration of the original bacterial solution. The concentration of the original bacterial liquid of Yersinia enterocolitica, a food-borne pathogen, was determined to be 2.8×10 by the plate colony counting method. 9 CFU / mL.

[0046] It was serially diluted 10-fold with Yersinia enterocolitica, a food-borne pathogenic bacterium whose concentration is known, respectively 2.8×10 9 CFU / mL, 2.8×10 8 CFU / mL, 2.8×10 7 CFU / mL, 2.8×10 6 CFU / mL, 2.8×10 5 CFU / mL, 2.8×10 4 CFU / mL, 2.8×10 3 CFU / mL, 2.8×10 2 CFU / mL, 2.8×10 1 CFU / mL and 2.8×10 0 CFU / mL, and the genomic DNA was extracted from the 10-fold gradient dilution bacterial solution, and the genomic DNA extracted from the diluted gradient bacterial s...

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Abstract

The invention discloses an LAMP detection reagent kit of food-borne yersinia enterocolitica. The reagent kit comprises a primer designed special for a food-borne yersinia enterocolitica ail gene and the primer comprises SEQID NO.2, SEQID NO.3, SEQID NO.4 and SEQID NO.5. The invention further discloses an application of the LAMP detection reagent kit to detection of food-borne yersinia enterocolitica of foods. For the LAMP detection reagent kit of the food-borne yersinia enterocolitica, the detection sensitivity can achieve 2.8 *10<2>CFU / mL, is 1000 times higher than common PCR, has the advantages of being good in specificity, visual in results and the like, and is suitable for real-time quick and accurate detection of the yersinia enterocolitica for food safety monitoring sites by grass-roots units.

Description

technical field [0001] The invention relates to the technical field of biological monitoring, in particular to a LAMP detection kit for foodborne Yersinia enterocolitica and its application. Background technique [0002] Yersinia enterocolitica, a Gram-negative bacillus, is found in a variety of environments. As a food-borne pathogen, Yersinia enterocolitica has a wide range of hosts, can be transmitted through contaminated food and water, and can cause gastroenteritis, arthritis, sepsis, and constipation in humans and other animals. Erythema and other diseases. Yersinia is the main factor leading to enterocolitis, and it is highly contagious and can be transmitted between humans and animals. It is a very important pathogen of zoonotic diseases. At present, the detection method of Yersinia enterocolitica is mainly based on the traditional physiological and biochemical detection method, which has a long detection cycle and consumes a lot of manpower and material resources, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/6844C12Q1/04C12N15/11
CPCC12Q1/689C12Q1/6844
Inventor 胡青海王西胡仲浩谭攀攀杜晓莉徐欣欣王权刘光清
Owner SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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