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Primer probe for RNA isothermal amplification to detect cronobacter, kit and detection method

A technology for constant temperature amplification detection and Cronobacter, which is applied in the direction of microorganism-based methods, biochemical equipment and methods, DNA/RNA fragments, etc., can solve the problem of unable to monitor Enterobacter sakazakii in time, and cannot realize on-site detection and cultivation Higher condition requirements and other issues, to achieve the effect of low cost, reduced design and production costs, and easy pollution control

Active Publication Date: 2018-08-21
健垣科技(张家口)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The traditional detection method of Enterobacter sakazakii, due to the low concentration of bacteria and high requirements for culture conditions, takes a long time and has low inspection efficiency, which leads to the inability to monitor Enterobacter sakazakii in time and to realize on-site detection

Method used

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  • Primer probe for RNA isothermal amplification to detect cronobacter, kit and detection method
  • Primer probe for RNA isothermal amplification to detect cronobacter, kit and detection method
  • Primer probe for RNA isothermal amplification to detect cronobacter, kit and detection method

Examples

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Embodiment 1

[0080] Design of special primers and probes for detection of Cronobacter (CS) by constant temperature amplification of RNA

[0081] The present invention selects the highly conserved section of CS bacteria, SEQ ID NO: 1, as the amplified target sequence region, and uses DNAman software to artificially design the primer probe design principle for real-time fluorescent nucleic acid constant temperature amplification to detect Cronobacter (CS) The specific primers and probe sequences were obtained to obtain the following specific sequences:

[0082](1) A pair of CS detection primers for producing a DNA copy of the CS target nucleic acid (CS RNA) under the action of M-mLV reverse transcriptase, the CS detection primers are composed of upstream primer CS nT7 and downstream primer CS T7, the upstream primer The primer sequence of CS nT7 is 5'-CTTACCAGGT GTTGACATCC-3' (SEQ ID NO: 2), and the primer sequence of downstream primer CS T7 is 5'-AATTTAATACGACTCACTATAGGGAGACAACATCTCAGAACACG...

Embodiment 2

[0085] Preparation of Cronobacter (CS) RNA Constant Temperature Amplification Detection Kit

[0086] Using the special primers and probes provided in Example 1, the Cronobacter (CS) RNA constant temperature amplification detection kit of the present invention was obtained. The kit contains CS T7 primer, CS nT7 primer, CS detection probe, M-mLV reverse transcriptase and T7 RNA polymerase.

[0087] The capture probe exists in the nucleic acid extraction solution, the CS T7 primer, CS nT7 primer and CS detection probe exist in the CS detection solution, and the M-mLV reverse transcriptase and T7 RNA polymerase exist in the enzyme solution Medium; specifically, the main components of the kit are as follows:

[0088] (1) Lysis solution: 10% Triton X-100, 5mol / L guanidine isothiocyanate, 10mmol / L Tris HCI, pH 8.0;

[0089] (2) Nucleic acid extraction solution: 40-200mM EDTA, 50-500mg / L magnetic beads, 1-50μΜ capture probe;

[0090] (3) Washing solution: 2mmol / L NaCl, 5mol / L guani...

Embodiment 3

[0097] Sensitivity of RNA Isothermal Amplification Detection of Cronobacter

[0098] Use kit of the present invention (see embodiment 2 for composition) to detect Cronobacter genus (CS) in milk powder, and concrete method comprises the following steps:

[0099] ⑴ Bacterial solution dilution

[0100] The measured concentration is 1×10 8 CFU / mL of Cronobacter culture, 10-fold gradients were diluted to 10 7 CFU / mL, 10 6 CFU / mL, 10 5 CFU / mL, 10 4 CFU / mL, 10 3 CFU / mL, 10 2 CFU / mL, 10 1 CFU / mL, 10 0 CFU / mL.

[0101] ⑵ Nucleic acid extraction

[0102] Add 200 μL of lysate, 100 μL of nucleic acid extraction solution, and 200 μL of bacterial solution into a 1.5 mL EP tube, mix well, incubate at 60°C for 10 minutes, and place at room temperature for 10 minutes. Place the centrifuge tube on the magnetic bead separator and let it stand for 5 minutes. Keep the centrifuge tube on the magnetic bead separator, absorb the liquid, and keep the magnetic beads; add 1mL of washing sol...

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Abstract

The invention discloses a primer probe for RNA isothermal amplification to detect cronobacter, a kit and a detection method. The provided primer probe for detecting specificity, the detection kit containing the primer probe, and the detection method using the detection kit to carry out RNA isothermal amplification have the characteristics of high sensitivity, strong specificity, low pollution (amplification products RNA are easily degraded in nature), and rapidness; the requirements on instruments are low; the operation is simple; the primer probe and the kit are suitable for self detection offood enterprises and basic detection mechanisms and onsite detection, in particular, milk powder rapid detection, and the safety of milk powder is guaranteed.

Description

technical field [0001] The invention relates to the technical field of detection of pathogenic bacteria microorganisms, in particular to a rapid detection technology for detecting Cronobacter (CS) based on RNA constant temperature amplification, in particular to specific detection primer probes and kits for detecting Cronobacter and detection methods. Background technique [0002] Cronobacter (Cronobacter), formerly known as Enterobacter sakazakii, Enterobacter sakazakii is a Gram-negative non-bacillus parasitic in the intestinal tract of humans and animals, belonging to the family Enterobacteriaceae. It is a pathogenic bacterium newly discovered in dairy products that has attracted widespread attention in recent years. Cronobacter is highly virulent and can cause diseases such as neonatal meningitis, fatal enterocolitis, and bacteremia. Although it can be recovered with antibiotic treatment, it is accompanied by severe neurological sequelae and developmental disorders. Th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/6844C12Q1/10C12N15/11C12R1/01
CPCC12Q1/6844C12Q1/689C12Q2563/107C12Q2521/107C12Q2521/119C12Q2527/101
Inventor 刘喜富乌日琴于志鑫韩晓梅宋晓礁王罡
Owner 健垣科技(张家口)有限公司
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