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Real-time fluorescence quantitative PCR detection method of campylobacter jejuni and kit

A real-time fluorescence quantitative, Campylobacter jejuni technology, applied in the determination/inspection of microorganisms, fluorescence/phosphorescence, biochemical equipment and methods, etc., to achieve the effects of improved detection efficiency, convenient sampling and high sensitivity

Active Publication Date: 2010-11-17
NAT INST FOR FOOD & DRUG CONTROL
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0011] However, whether and how to use real-time fluorescent quantitative PCR technology in practical applications still has technical problems to be overcome, such as probe design, detection sensitivity, etc., and there is still no real-time fluorescent quantitative PCR technology using probe method. Quantitative detection of Campylobacter jejuni in stool samples from clinical diarrhea patients, monkey and dog stool samples, and ileocecal content samples of miniature pigs

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  • Real-time fluorescence quantitative PCR detection method of campylobacter jejuni and kit
  • Real-time fluorescence quantitative PCR detection method of campylobacter jejuni and kit

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Embodiment 1

[0039] Example 1. Design of primers and TaqMan probes for real-time fluorescent quantitative PCR detection of Campylobacter jejuni (CJ)

[0040] Referring to the complete genome sequence of Campylobacter jejuni subsp.jejuni NCTC 11168 strain (GeneBank: NC_002163) included in GenBank, the sequences of Campylobacter coli and Campylobacter fetalis, which are very similar to the genome structure of Campylobacter jejuni, were compared respectively, and flagellin was selected For the conserved region of the A(flaA) gene, TaqMan probes and primers were designed and synthesized using ABI Primer Express 3.0 real-time fluorescent quantitative PCR primer design software. The fluorescent labeling of the probe selects FAM (5' end) as the reporter luminescent group, and NFQ (3' end) as the quenching group. The sequence is as follows:

[0041] Upstream primer (CJ-1): 5'-AACCTGAGCCTGCAGAAACATTAT-3' (SEQ ID NO: 1 in the sequence listing);

[0042] Downstream primer (CJ-2): 5'-CCACAGGTTTTGCAA...

Embodiment 2

[0044] Embodiment 2, real-time fluorescent quantitative PCR detection of Campylobacter jejuni

[0045] 1. Establishment of standard curve

[0046] 1. Construction of pMD-CJ-flaA recombinant plasmid

[0047] 1.1 Cloning of the target gene-Campylobacter jejuni flagellin A (flaA) gene

[0048] 1.1.1 Extraction of Genomic DNA from Campylobacter jejuni

[0049] The following operations must be carried out in the P2 laboratory negative pressure biological safety cabinet in strict accordance with the requirements.

[0050] (1) Under aseptic conditions, take 100 mg of feces samples from patients with Campylobacter jejuni infectious diarrhea (or 100 mg of monkey or dog feces, or 100 mg of ileocecal contents of minipigs), put them into a 5 mL sterile centrifuge tube, and add 3 mL of sterile PBS (pH7.6), mix well, and let stand at room temperature for 5 minutes; draw the supernatant with a sterile syringe, filter through a microporous membrane (0.45 μm) to remove large bacteria and re...

Embodiment 3

[0085] Embodiment 3, Campylobacter jejuni probe method real-time fluorescent quantitative PCR detection kit

[0086] CJ TaqMan assay mix 50 μL (TaqMan probe concentration 250 nM, upstream and downstream primer concentrations 900 nM each), CJ TaqMan mix 500 μL, CJ positive quality control (pMD-CJ- flaA recombinant plasmid) 50 μL, CJ negative quality control product 50 μL and RNase-free water 500 μL were packaged together to obtain a real-time fluorescent quantitative PCR detection kit for Campylobacter jejuni.

[0087]

[0088]

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Abstract

The invention discloses a real-time fluorescence quantitative PCR detection method of campylobacter jejuni and a kit. In the invention, a primer and a Taqman probe are designed according to a conserved region of a campylobacter jejuni flagellin A (flaA) gene for qualitatively detecting the copy number of nucleic acid of the campylobacter jejuni in a fecal sample of a clinical patient, the fecal samples of a monkey and a dog, and the sample of ileocecal contents of a miniature swine, an upstream primer sequence SEQ ID NO:1, a downstream primer sequence SEQ ID NO:2 and a Taqman probe sequence SEQ ID NO:3. The invention has a great practical significance on quality control of relevant people and animal derived biological products and the inspection and quarantine field of import and export animals, and can ensure the quality of the relevant biological products. The detection method and the kit of the invention can be applied to diagnosis of campylobacter diseases and verification of the campylobacter jejuni in the biological products, thus having wide application prospect.

Description

technical field [0001] The invention relates to a molecular biology detection method of bacteria in the field of biotechnology, in particular to a real-time fluorescence quantitative PCR detection method and a kit for campylobacter jejuni. Background technique [0002] Campylobacteriosis is a zoonotic infectious disease caused by Campylobacter. There are 3 kinds of pathogenic Campylobacter, namely Campylobacter jejuni, Campylobacter coli and Campylobacter fetus. Campylobacter jejuni is the most harmful. Since Dekeyser et al first isolated Campylobacter jejuni from the stool of patients with diarrhea in 1972, it has been confirmed that Campylobacter jejuni is one of the most important pathogens of human acute gastroenteritis (Butzler JP, Dereume JP, Barbier P, et al . Digestive origin of Campylobacter septicemias. Nouv Presse Med. 1977, 6(12): 1033-1035.). In some developed countries, diarrhea caused by Campylobacter jejuni has surpassed that of Salmonella and Shigella. C...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/06C12N15/11G01N21/64
CPCY02A50/30
Inventor 高正琴
Owner NAT INST FOR FOOD & DRUG CONTROL
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