Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Specific primer, probe and real-time fluorescence quantitative PCR (Polymerase Chain Reaction) kit for detecting cronobacter sakazakii

A technology of real-time fluorescence quantification and Enterobacter sakazakii, applied in the field of molecular biology and in vitro diagnostic reagents, can solve problems such as unfavorable, finding pathogens, and a large amount of time

Inactive Publication Date: 2018-06-19
SUZHOU BAIYUAN GENT CO LTD
View PDF6 Cites 2 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Currently, microbiologists do not know the source of E. sakazakii contamination, but many case reports indicate that infant formula is the main source of infection found so far
The traditional method of diagnosing Enterobacter sakazakii is mainly based on the classification of bacterial physiological and biochemical characteristics, which requires a lot of time to determine the results of physiological and biochemical reactions, which is not conducive to finding the pathogen in time and diagnosing the cause

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Specific primer, probe and real-time fluorescence quantitative PCR (Polymerase Chain Reaction) kit for detecting cronobacter sakazakii
  • Specific primer, probe and real-time fluorescence quantitative PCR (Polymerase Chain Reaction) kit for detecting cronobacter sakazakii
  • Specific primer, probe and real-time fluorescence quantitative PCR (Polymerase Chain Reaction) kit for detecting cronobacter sakazakii

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 116

[0029] The preparation of embodiment 1 16SrDNA gene standard

[0030] To establish a real-time fluorescent quantitative PCR method, the external standard required by the method must first be prepared. The standard should contain highly conserved and specific sequences, and high specificity of the reaction must be ensured. The 16S rDNA gene widely exists in Enterobacter sakazakii and has high conservation. The present invention uses Enterobacter sakazakii 16SrDNA gene as the target sequence. This example mainly uses PCR technology to amplify the 16SrDNA gene of Enterobacter sakazakii, and uses gene recombination technology to connect it to the plasmid vector pMD18-T to construct the recombinant plasmid pMD18-T-16S, and carry out corresponding PCR identification and sequencing identification , and finally quantified as a standard for the method to be established, laying the foundation for the next method and evaluation.

[0031] 1. Preparation of template DNA

[0032] Genomic...

Embodiment 2

[0095] Embodiment 2 real-time fluorescent quantitative PCR kit

[0096] 1. Design and synthesis of specific primers and probes

[0097] The present invention performs bioinformatics comparison and analysis on the complete sequence of Enterobacter sakazakii 16SrDNA gene in the NCBI database, selects the conserved fragment sequence suitable for designing primers and probes as the target (see Example 1), and further applies Primer express 3 Software, Primer Premier 5 software and Oligo 7 software, designed multiple sets of real-time fluorescent quantitative PCR primers and probes, after preliminary screening of the test, finally determined a set of fluorescent quantitative PCR primers and probes for the detection of Enterobacter sakazakii.

[0098] As the core of the present invention, a group of primers and probe nucleotide sequences for real-time fluorescent PCR detection of Enterobacter sakazakii are as follows:

[0099] Upstream primer: 16SrDNA-304F 5'-GAACTGACCGATGACGTA-3' ...

Embodiment 3

[0119] Example 3 The quantitative detection method of the sample to be tested by the real-time fluorescent quantitative PCR kit

[0120] Respectively with the 16SrDNA gene series concentration standard substance (series concentration standard substance serial concentration of sample DNA to be tested and embodiment 1 preparation is: 1.00 * 10 10 copies / ml, 1.00×10 9 copies / ml, 1.00×10 8 copies / ml, 1.00×10 7 copies / ml, 1.00×10 6 copies / ml, 1.00×10 5 copies / ml, 1.00×10 4 copies / ml, 1.00×10 3 copies / ml, 1.00×10 2 copies / ml) as a template, use the primers and probes in the kit to perform fluorescent quantitative PCR amplification, and set up positive and negative controls at the same time.

[0121] The PCR reaction system is as follows:

[0122]

[0123] Reaction conditions: 94°C pre-denaturation for 2 minutes, 94°C for 10 seconds, 60°C for 30s and collecting fluorescence signals, 40 cycles. Draw a standard curve, and perform rapid quantitative detection through the sta...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention provides a specific primer and a probe for detecting cronobacter sakazakii. A sequence of the probe is 5'-CGTTCAGGAGAGCCACCG-3'; a sequence of the specific primer is the following sequences or the complementary chain sequences of the following sequences: an upstream primer sequence is 5'-GAACTGACCGATGACGTA-3'; a downstream primer sequence is 5'-GTCTCATTAAACGGCTCG-3'. The invention further provides a corresponding real-time fluorescence quantitative PCR (Polymerase Chain Reaction) kit. The invention provides the specific primer and the probe, can fulfill the aim of accurately andquantitatively detecting content of cronobacter sakazakii in a to-be-detected specimen by extracting genome DNA (deoxyribonucleic acid) of the cronobacter sakazakii and combining a real-time fluorescence quantitative PCR detection technology, has high sensitivity and high specificity, and has important significance on judging the content of active bacteria of the cronobacter sakazakii and even performing subsequent research on urinary tract infection.

Description

technical field [0001] The invention belongs to the technical field of molecular biology and in vitro diagnostic reagents, in particular to specific primers and probes for detecting Enterobacter sakazakii and a real-time fluorescent quantitative PCR kit. Background technique [0002] Enterobacter sakazakii (Cronobacter sakazakii) is a kind of Enterobacteriaceae. It is a Gram-negative, rod-shaped bacterium that does not form spores and is most suitable for growth between 37°C and 43°C. Enterobacter sakazakii can cause severe neonatal meningitis, enterocolitis and bacteremia, with a mortality rate as high as 50%. Currently, microbiologists do not know the source of E. sakazakii contamination, but many case reports indicate that infant formula is the main source of infection found so far. The traditional method of diagnosing Enterobacter sakazakii is mainly based on the classification of bacterial physiological and biochemical characteristics, which requires a lot of time to d...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/686C12N15/11C12R1/01
CPCC12Q1/686C12Q1/689C12Q2561/113C12Q2563/107C12Q2545/114
Inventor 徐红车团结陈游高恺李亚鹏
Owner SUZHOU BAIYUAN GENT CO LTD
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com