Detection kit for Yersinia rohdei causing enterocolitis and detection method

A technology for enterocolitis and Yersinia, which is applied in the field of bacterial detection, can solve the problems of complex amplification and high cost, and achieve the effects of strong specificity, short time consumption and good application prospects

Inactive Publication Date: 2015-11-25
SOUTHWEST UNIVERSITY FOR NATIONALITIES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] The patent with the notification number 103468811B discloses a kit for detecting Yersinia enterocolitica, which needs to be ampli

Method used

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  • Detection kit for Yersinia rohdei causing enterocolitis and detection method
  • Detection kit for Yersinia rohdei causing enterocolitis and detection method
  • Detection kit for Yersinia rohdei causing enterocolitis and detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 Primer Design

[0026] 1. Experimental method

[0027] 1. PCR primer design and synthesis

[0028] The upstream and downstream primers for the ail gene (SEQ ID NO.1~2): ail-F: 5ˊ-taatgtgtacgctgcgag-3ˊ, ail-R: 5ˊ-gacgtcttacttgcactg-3ˊ, the expected fragment size is 351bp; the inv gene upstream and downstream primers (SEQIDNO.3~ 4): intB-Forword: 5ˊ-tgcgccatgcggtccatc-3ˊ, intB-Reverse: 5ˊ-ggtgcataagattctcgg-3ˊ, the expected fragment size is 520bp; intB gene upstream and downstream primers (SEQ ID NO.5~6): intB-F: 5ˊ-tgcgccatgcggtccatc-3ˊ , intB-R: 5ˊ-ggtgcataagattctcgg-3ˊ, expected fragment size 722bp. The primers were synthesized by Shanghai Yingwei Jieji Trading Co., Ltd.

[0029] 2. Template preparation

[0030] Yersinia enterocolitica was quickly revived at 37°C, and then inoculated in LB broth medium for 18 hours at 28°C, and the bacterial genomic DNA was extracted according to the operation steps of the Tiangen Bacteria Genomic DNA Extraction Kit as a ...

Embodiment 2

[0044] Embodiment 2 specificity test

[0045] 1. Test method

[0046] 1. PCR primers

[0047] Same as the primer pair in Example 1 (the primer pair shown in SEQ ID NO.1-2, SEQ ID NO.3-4, SEQ ID NO.5-6).

[0048] 2. Template preparation:

[0049]Put Yersinia enterocolitica and Aeromonas vibrio, Vibrio harveii, Pseudomonas, Vibrio minimal, Aeromonas somniform, Citrobacter, Aeromonas intermedius at 37 After rapid recovery under the condition of ℃, they were inoculated in LB broth medium and cultured at 28 ℃ for 18 hours, and the bacterial genomic DNA was extracted according to the operation steps of the Tiangen Bacteria Genomic DNA Extraction Kit, which was used as a template for PCR amplification.

[0050] With embodiment 1.

[0051] 3. PCR amplification

[0052] Multiplex PCR amplification with embodiment 1.

[0053] 4. Result detection

[0054] With embodiment 1.

[0055] 2. Results

[0056] The result is as figure 2 As shown, only Yersinia enterocolitica can amplif...

Embodiment 3

[0058] Embodiment 3 sensitivity test

[0059] 1. Test method

[0060] 1. PCR primers

[0061] Same as the primer pair in Example 1 (the primer pair shown in SEQ ID NO.1-2, SEQ ID NO.3-4, SEQ ID NO.5-6).

[0062] 2. Template preparation:

[0063] Yersinia enterocolitica was quickly revived at 37°C, and then inoculated in LB broth medium for 18 hours at 28°C, and the bacterial genomic DNA was extracted according to the operation steps of the Tiangen Bacteria Genomic DNA Extraction Kit.

[0064] Determine the concentration of the DNA sample, do 10 times, 20 times, 50 times, 10 times -2 、10 -3 、10 -4 、10 -5 、10 -6 、10 -7 Make a dilution. After determination, the concentration of the DNA sample was 170.4ng / μL, and after dilution, the concentration was 17.04ng / μL, 8.52ng / μL, 3.41ng / μL, 17.04×10 -1 ng / μL, 17.04×10 -2 ng / μL, 17.04×10 -3 ng / μL, 17.04×10 -4 ng / μL, 17.04×10 -5 ng / μL, 17.04×10 -6 ng / μL DNA sample solution.

[0065] 3. PCR amplification

[0066] Multiplex ...

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PUM

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Abstract

The invention discloses a detection kit for Yersinia rohdei causing enterocolitis. The detection kit comprises primer pairs shown as SEQ ID NO: (1 to 2), SEQ ID NO: (3 to 4) and SEQ ID NO: (5 to 6). The invention further provides a detection method of Yersinia rohdei, the three primer pairs, and application of the primer pairs. The detection kit and the detection method has the advantages that Yersinia rohdei can be detected accurately and effectively; the specificity and the sensitivity are high; the time consumption is low; the detection is fast; fast detection and forecast of water sources and foods can be realized; diseases can be prevented; the economic benefit is improved.

Description

technical field [0001] The invention relates to a detection technique for bacteria, in particular to a detection kit and a detection method for Yersinia enterocolitica. Background technique [0002] Yersinia enterocolitica (Y.entrocolitica) is a Gram-negative bacillus or coccus belonging to the Enterobacteriaceae Yersinia genus. Another bacteria after bacteria. As early as the 1980s, the bacterium has attracted the attention of the international community. The pathogenic bacterium has been detected from the environment, water body, crowd, animals and food, and can pass through human-human, human-animal, animal-animal, food, Water sources and other means of transmission. The clinical symptoms caused by the bacteria are mainly manifested as acute abdominal pain, diarrhea, vomiting and other gastrointestinal phenomena and complications such as erythema nodosum, arthritis, osteomyelitis, hepatitis and sepsis. Based on the above characteristics, Yersinia enterocolitica has bec...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12Q1/68C12Q1/04
Inventor 王利苟小兰段荟芹
Owner SOUTHWEST UNIVERSITY FOR NATIONALITIES
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