Real-time fluorescence nucleic acid isothermal amplification detection kit for Cronobacter spp

A technology of Enterobacter zosteri and a kit is applied in the field of biological detection of pathogenic microorganisms, which can solve the problems of false negative detection cost, low sensitivity and false positive experimental results.

Inactive Publication Date: 2018-07-10
SHANGHAI RENDU BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In order to solve the problems of low sensitivity of the existing Cronobacter sakazakii (Cronobacter spp) detection method, long detection cycle, easy to cause contamination of amplified products, resulting in false positive or false negative results and high detection cost, The invention provides a real-time fluorescent nucleic acid constant temperature amplification detection technology for Cronobacter sakazakii (Cronobacter spp) with short detection period, high sensitivity, high specificity, low pollution, stable reaction, low detection cost and easy popularization and application , including special primers, probes, kits and their use

Method used

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  • Real-time fluorescence nucleic acid isothermal amplification detection kit for Cronobacter spp
  • Real-time fluorescence nucleic acid isothermal amplification detection kit for Cronobacter spp
  • Real-time fluorescence nucleic acid isothermal amplification detection kit for Cronobacter spp

Examples

Experimental program
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Effect test

Embodiment 1

[0100] Example 1. Design of special primers and probes for real-time fluorescent nucleic acid constant temperature amplification detection of Cronobacter sakazakii (Cronobacter spp)

[0101] Based on personal experience and reading a large number of documents and patents, the present invention selects 23s-ITS2-5S Rrna, 16s-ITS-23S Rrna in the Cronobacter spp gene, 16s rRNA has no secondary structure and is highly conserved The segment is used as the amplified target sequence region. According to the principle of primer probe design, use DNAStar, DNAMAN software and artificially design special primers and probes for real-time fluorescent nucleic acid constant temperature amplification to detect Cronobacter sakazakii (Cronobacter spp) Sequence, the following specific sequence is obtained:

[0102] Experimental group 1:

[0103] nT7: 5'-TTAACCTTACAACGCCAA-3'

[0104] T7: 5'-AATTTATACGACTCACTATAGGGAGACCGCTTCCGCCATGA-3'

[0105] Probe: 5'-CCGCCAGUCUGGCGUGUGGCGG-3'

[0106] Expe...

Embodiment 2

[0125] Example 2. Specific primers and probes for the detection of Cronobacter sakazakii Enterobacter sakazakii (Cronobacter spp) by real-time fluorescent nucleic acid constant temperature amplification

[0126] The primers and probes selected last in Example 1 were selected, and the sequences were put into NCBI BLAST, and the results indicated that this region could detect all subtypes of Enterobacter sakazakii of the genus Cronobacter, namely Cronobacter dublinenesis and Cronobacter dublinenesis subsp lausannensis , Cronobacter malonaticus, Cronobacter muytjensii, Cronobacter sakazakii, Cronobacter turicensis, Cronobacter universalis, Cronobacter genomospecies, Cronobacter condiment.

Embodiment 3

[0127] Example 3. Preparation of a real-time fluorescent nucleic acid constant temperature amplification detection kit for Cronobacter sakazakii (Cronobacter spp)

[0128] Using the special primers and probes provided in Example 1, the real-time fluorescent nucleic acid constant temperature amplification detection kit for Cronobacter sakazakii (Cronobacter spp) of the present invention was obtained. The kit contains components such as capture probe (TCO, Target Capture Oligo), T7 primer, nT7 primer, detection probe, internal standard detection probe, internal standard, M-MLV reverse transcriptase and T7 RNA polymerase .

[0129] The capture probe exists in the nucleic acid extraction solution, the T7 primer, nT7 primer, detection probe, and internal standard detection probe exist in the detection solution, and the M-MLV reverse transcriptase and T7 RNA polymerase exist In the SAT enzyme solution, specifically, the kit is divided into A box (specimen processing unit) stored at...

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Abstract

The invention discloses a real-time fluorescence nucleic acid isothermal amplification detection kit for Cronobacter spp. The kit comprises a capture probe, a detection primer T7, an nT 7 primers, a detection probe, M-MLV reverse transcriptase, T7 RNA polymerase and other reagents. The kit can detect the RNA of Cronobacter spp in food and has the advantages of being high in specificity and sensitivity (which can reach 1,000 CFU / ml), low in contamination (wherein amplified product RNA is easily degraded under the natural environment) and capable of achieving rapid detection (which is completedwithin 60 min conventionally), the kit can play an important role in food security, and the application prospect is wide.

Description

technical field [0001] The invention relates to the technical field of biological detection of pathogenic microorganisms, in particular to a method for detecting Cronobacter sakazakii (Cronobacterspp) by using magnetic bead-RNA enrichment technology to extract and purify target RNA and real-time fluorescent nucleic acid constant temperature amplification detection technology Reagent test kit. The detection of Enterobacter sakazakii in food can be realized by the kit of the invention. Background technique [0002] Enterobacter sakazakii (Cronobacter spp) is a foodborne opportunistic pathogen that can cause severe meningitis, necrotizing enterocolitis, and bacteremia in newborns. have a high fatality rate. In recent years, studies on the contamination rate of Enterobacter sakazakii in food, beverages, processed raw materials, and the environment have found that Enterobacter sakazakii is widely distributed in nature. [0003] Currently, culture and molecular biology methods ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6806C12Q1/6844C12Q1/689C12Q1/10
CPCC12Q1/6806C12Q1/6844C12Q1/689Y02A50/30
Inventor 许雪荷司康于明辉居金良
Owner SHANGHAI RENDU BIOTECH
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