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Loop-mediated isothermal amplification kit detecting campylobacter jejuni and detection method

A loop-mediated isothermal, Campylobacter jejuni technology, applied in the field of biotechnology detection, can solve the problems of harsh culture conditions, low specificity, and complicated operation, and achieve the effects of short detection time, strong specificity, and simple operation.

Active Publication Date: 2017-02-01
YANGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The national standard method used to detect Campylobacter jejuni is the traditional culture method, but due to the harsh culture conditions of Campylobacter jejuni, the traditional culture method has disadvantages such as cumbersome operation, low specificity, and time-consuming

Method used

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  • Loop-mediated isothermal amplification kit detecting campylobacter jejuni and detection method
  • Loop-mediated isothermal amplification kit detecting campylobacter jejuni and detection method
  • Loop-mediated isothermal amplification kit detecting campylobacter jejuni and detection method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] The preparation of embodiment 1 kit

[0042] Primer design and synthesis:

[0043] cjaA is a conserved gene of Campylobacter jejuni. Its C-terminal amino acid is bound to the inner membrane, and some amino acids are exposed to the outer membrane. It is one of the components of the ABC-type cysteine ​​transport system. By reviewing the literature and performing BLAST comparison on NCBI, it was found that the cjaA gene has good specificity, high conservation and high homology, and is a reliable target gene that can be used for the detection of Campylobacter jejuni.

[0044] Primers were designed with reference to the cjaA gene sequence of Campylobacter jejuni NCTC11168 in GenBank, and the primers included: FIP, BIP, F3, B3, LF and LB. The PrimerExplorer V4 software of Japan Eiken Chemical Co., Ltd. was used to design according to the default settings (http: / / primerexplorer.jp / elamp4.0.0 / index.html), and was synthesized by GenScript according to the PAGE purity level.

[0...

Embodiment 2

[0051] Example 2 Kit Performance Verification in Example 1

[0052] 1. When using the primers in the kit described in Example 1, the establishment of the loop-mediated isothermal amplification reaction system and reaction program

[0053] Loop-mediated isothermal amplification reaction system: F3 and B3 each 0.2μM, LF and LB each 0.8μM, FIP and BIP each 1.6μM, 0.8mM dNTPs, magnesium chloride 4mM, betaine 0.8M, Bst DNA polymerase large fragment 8U , 10×ThermoPol Buffer2.5μl, 2μl sample genomic DNA, and finally add ultrapure water to 25μl.

[0054] Loop-mediated isothermal amplification reaction procedure: constant temperature water bath reaction, that is, after 30 minutes at 65°C, stop the reaction at 80°C for 3 minutes.

[0055] The result shows: through the agarose (1.5% w / v) electrophoresis analysis of the LAMP amplification product of Campylobacter jejuni standard strain NCTC 11168, gradient band (such as figure 1 shown).

[0056] Two, the specific identification of kit ...

Embodiment 3

[0075] Example 3 The kit in Example 1 detects Campylobacter jejuni in food

[0076] 1. Sample Collection and DNA Extraction

[0077] Collect 200 chicken surface wipe samples (wetted with PBS) from the farm, centrifuge at 8000×g for 5 minutes, and suspend and wash the pellet with PBS. Then centrifuge at 8000×g to obtain the pellet and resuspend the bacteria in 70 μl ultrapure water. Bacteria were lysed using the boiling method, that is, the bacterial suspension was boiled at 100° C. for 10 minutes, centrifuged at 12,000×g for 3 minutes, and the supernatant was taken as a template (sample genomic DNA).

[0078] 2. Detection of Campylobacter jejuni in food samples

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Abstract

The invention belongs to the field of biotechnology detection, and particularly relates to a loop-mediated isothermal amplification kit detecting campylobacter jejuni and a detection method. The kit comprises a cjaA gene detection primer. The loop-mediated isothermal amplification kit is easy to operate and short in detection time, detected sample DNA is detected, and the detection result can be obtained only in about 1 hour; the specificity is high, and the sensitivity is high; the application range is wide, the kit can be used for detecting the campylobacter jejuni in food samples such as chicken and pork, and the lowest detection limit is campylobacter-jejuni 20 CFU / ml; the detection method can replace a detection method of the campylobacter jejuni in traditional food.

Description

technical field [0001] The invention belongs to the field of biotechnology detection, and in particular relates to a loop-mediated isothermal amplification kit and a detection method for detecting campylobacter jejuni. Background technique [0002] Campylobacter jejuni (Campylobacter jejuni) is an important zoonotic pathogen that widely exists in the intestinal tract of poultry animals. Humans are mainly infected by ingesting poultry meat, food, dairy products and water contaminated by it. Human clinical campylobacteriosis, with acute, self-limiting gastroenteritis as the typical clinical symptom, is listed by the World Health Organization (WHO) as the most common foodborne disease. Campylobacter jejuni, as an important indicator of pathogenic bacteria detection, is a necessary detection item in public health, food safety, animal husbandry and veterinary medicine, and entry-exit inspection and quarantine, and has important social and economic significance. The national sta...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/10
CPCC12Q1/6844C12Q1/689C12Q2531/119C12Q2561/113C12Q2563/107Y02A50/30
Inventor 焦新安臧筱琦黄金林孙林
Owner YANGZHOU UNIV
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