High-sensitivity rapid detection kit for campylobacter jejuni

A technology of Campylobacter jejuni and a detection kit, which is applied in the determination/testing of microorganisms, methods based on microorganisms, microorganisms, etc., and can solve the urgent problems of rapid detection

Inactive Publication Date: 2012-07-04
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In addition, how to use the lack of testing conditions for rapid testing at the epidemic site is also an urgent problem

Method used

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  • High-sensitivity rapid detection kit for campylobacter jejuni
  • High-sensitivity rapid detection kit for campylobacter jejuni
  • High-sensitivity rapid detection kit for campylobacter jejuni

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1: Design and screening of highly specific primers for loop-mediated isothermal amplification for the detection of Campylobacter jejuni

[0034] (1) Obtain the nucleotide sequence of the gene flhA related to flagella synthesis of Campylobacter jejuni NCTC11168 from NCBI (National Center for Biotechnology Information, USA) (GeneBank gene accession number is 905174);

[0035] (2) For the nucleotide sequence of flhA, use the PrimerExploerV4 software to design the internal primers and external primers for LAMP amplification. Through bioinformatics analysis, 2 sets of primers were preliminarily screened, numbered ID-8 and ID-90:

[0036] Primer ID-8:

[0037]

[0038] Primer D-90:

[0039]

[0040] (3) Use the primers ID-8 and ID-90 obtained from the primary screening to perform loop-mediated isothermal amplification (the template DNA is extracted from Campylobacter jejuni ATCC33291, 25uLLAMP reaction system, 63°C water bath for 60 minutes), and take 4uL of th...

Embodiment 2

[0041] Example 2: Comparative analysis of the sensitivity of detecting Campylobacter jejuni

[0042] (1) The genome of Campylobacter jejuni ATCC33291 was extracted and used as template DNA.

[0043] (2) Perform PCR amplification with flhA-F3 and flhA-B3 as primers, and perform LAMP amplification with flhA-FIP, flhA-BIP, flhA-F3, and flhA-B3 as primers. The LAMP reaction system and reaction conditions are the same as above, The 25uLPCR reaction system is as follows:

[0044] PCR reaction system (total volume 25uL):

[0045]

[0046] PCR reaction program: pre-denaturation at 94°C for 5 minutes, and then enter the following cycle: denaturation at 94°C for 30 seconds, annealing at 52°C for 30 seconds, extension at 72°C for 30 seconds, after 35 cycles, extension at 72°C for 10 minutes, and incubation at 4°C .

[0047] (3) analyze with 1% and 2% agarose gel electrophoresis respectively, observe LAMP amplification and PCR amplification product under ultraviolet light, the result ...

Embodiment 3

[0048] Example 3: The kit and detection method provided by the present invention can detect Campylobacter jejuni with high specificity

[0049] (1) Genomic DNA of Campylobacter jejuni ATCC33291, Bacillus sakazakii ATCCBAA-894, Staphylococcus aureus ATCC25923, Escherichia coli and Listeria monocytogenes were extracted respectively.

[0050] (2) Use the above-mentioned genomic DNA as a template, use the LAMP amplification primers designed in the present invention, the established LAMP reaction system and reaction conditions to simultaneously perform product amplification to test the specificity of the reaction.

[0051] (3) The results of ultraviolet observation on 2% agarose gel electrophoresis show that only Campylobacter jejuni has a LAMP amplification band, and Escherichia coli, Listeria monocytogenes, Staphylococcus aureus, and Bacillus sakazakii are all negative, indicating that the present invention The provided kit and detection method can detect Campylobacter jejuni wit...

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Abstract

The invention provides a high-sensitivity rapid molecule detection kit for campylobacter jejuni being one of food-borne pathogens; and the high-sensitivity rapid molecule detection kit comprises four high-specificity primers (two internal primers flhA-FIP/flhA-BIP and two external primers flhA-F3/flhA-B3) and a high-efficiency reaction system, wherein the four high-specificity primers aim at the gene flhA of a specificity detection target and can be applied to a loop-mediated isothermal amplification target sequence, and the high-efficiency reaction system can carry out the loop-mediated isothermal amplification target sequence. Simultaneously, the invention also discloses a method for detecting the campylobacter jejuni by using the kit. Compared with the detection sensitivity of a PCR (Polymerase Chain Reaction) detection method, the detection sensitivity of the kit and method provided by the invention for detecting the campylobacter jejuni is increased by more than 100 times, and the kit and method have high specificity on the detection of the campylobacter jejuni. The kit and the detection method provided by the invention have the advantages of high sensitivity, strong specificity, simple and convenient operation, short time consumption and low cost, thereby a high-efficiency reliable convenient and practical means is provided for detecting the campylobacter jejuni in food.

Description

technical field [0001] The invention relates to a highly sensitive and rapid detection kit for Campylobacter jejuni, in particular to a method for highly sensitive and specific detection of food-borne pathogen Campylobacter jejuni at the DNA molecular level by using a loop-mediated isothermal amplification technique. Background technique [0002] Campylobacter jejuni (Campylobacter jejuni) is the main foodborne pathogen that can cause acute diarrhea and gastroenteritis in humans. pathogenic bacteria. Strengthening the screening and detection of Campylobacter jejuni is the fundamental measure to effectively prevent and control the contamination, transmission and pathogenicity of Campylobacter jejuni, and the key to monitoring and preventing Campylobacter jejuni in food and environment is to establish a highly sensitive, fast and simple Easy detection system. [0003] At present, many countries including my country still mainly use traditional detection methods (isolation cu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12R1/01
Inventor 李烨王小元史锋徐洋
Owner JIANGNAN UNIV
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