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

Primer probe combination for identification of staphylococcus aureus and drug resistance of staphylococcus aureus

A Staphylococcus, primer probe technology, applied in the biological field, can solve problems such as unreasonable primer probe involvement, and achieve good specificity, good accuracy, and cost-saving effects

Active Publication Date: 2020-01-10
AUTOBIO DIAGNOSTICS CO LTD
View PDF4 Cites 2 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, at present, there are unreasonable phenomena in the selection of gene fragments identified for SA and MRSA and the involvement of primers and probes, resulting in the inability of most detection reagents to achieve accurate, rapid and sensitive detection

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
  • Primer probe combination for identification of staphylococcus aureus and drug resistance of staphylococcus aureus
  • Primer probe combination for identification of staphylococcus aureus and drug resistance of staphylococcus aureus
  • Primer probe combination for identification of staphylococcus aureus and drug resistance of staphylococcus aureus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Embodiment 1 Preparation of Staphylococcus aureus and methicillin-resistant Staphylococcus aureus dual nucleic acid detection kit

[0046] The primer and probe sequences in the kit are shown in Table 1 below:

[0047] Table 1 Primer, probe sequence

[0048] name Nucleotide sequence SA upstream primer SEQ ID NO: 1 AGAAAAATTGAAGTCGAGTTTGACAAAG SA downstream primer SEQ ID NO: 2 TAAACATAAGCAACTTTAGCCAAAGCC SA probe SEQ ID NO: 3 TGGACGTGGCTTAGCGTATATTTTATGCTG MRSA upstream primer SEQ ID NO: 4 CAGTTATTGGATATTCGTGTCGTCAT MRSA downstream primer SEQ ID NO: 5 ACATTCAAATACGATAGACATTGTTGT MRSA probe SEQ ID NO: 6 CCAGCCAATGATCCAGCTTTATTTGAATCT internal standard upstream primer SEQ ID NO: 7 TCTTATTCTTCCTCCCACAGCTCC internal standard downstream primer SEQ ID NO: 8 GTGGGGTGAATTCTTTGCCA internal standard probe SEQ ID NO: 9 CGTGCTGGTCTGTGTGCTGGCC

[0049] The kit also includes: 10mM dNTPs, 2...

Embodiment 2

[0050] The detection method of embodiment 2 kit of the present invention

[0051]The detection method of the present invention is Real time RT-PCR, and the reaction process of Real Time RT-PCR is (1) pre-denaturation, and the time and length depend on the length and base composition of the target nucleotide, and the temperature of pre-denaturation is generally 90°C- 105°C, the time is generally 2-10min, the purpose of pre-denaturation is to completely separate the double-stranded nucleotide sequence into single strands; (2) Denaturation, the temperature is generally 91°C-105°C, and the time is generally 10s-35s; ( 3) Annealing, annealing each primer to the target sequence of human Staphylococcus aureus and methicillin-resistant Staphylococcus aureus or internal standard quality control nucleic acid. The annealing temperature is usually 40°C to 60°C, and the annealing time can be 10s to 60s. It is 10s~1min.

[0052] The composition of each detection system is shown in Table 2...

Embodiment 3

[0064] The feasibility test of embodiment 3 kit of the present invention

[0065] 1. Lower limit of detection (LOD) test

[0066] (1) Preparation of dual nucleic acid detection reagents for Staphylococcus aureus and methicillin-resistant Staphylococcus aureus: the dual nucleic acid detection reagent for Staphylococcus aureus and methicillin-resistant Staphylococcus aureus was prepared by adopting the method in Example 1.

[0067] (2) Bacterial sample extraction

[0068] Mix the 5 different concentrations of Staphylococcus aureus in the tube with the eluate from human throat swab of methicillin-resistant Staphylococcus aureus with a pipette, take out 100 μl in a new centrifuge tube, centrifuge at 12000rpm for 5min, carefully Discard the supernatant; add 200 μl of bacterial lysate (from Beijing Biolab Technology Co., Ltd.) to the pellet, mix well, bathe in water at 100°C for 5 minutes, and centrifuge at 10,000 rpm for 5 minutes for later use.

[0069] (3) Sample testing

[00...

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 relates to the technical field of biotechnology, in particular to a primer probe combination for identification of staphylococcus aureus and drug resistance of staphylococcus aureus. Theprimer probe combination provided by the invention comprises general primer and probe targeting staphylococcus aureus and specific primer and probe targeting methicillin-resistant staphylococcus aureus.The primer and probe has good specificity and can achieve rapid, accurate and sensitive identification targeting SA and MRSA combined with real-time PCR detection method. Experiments show that minimum detection limit of the primer probe for detection of staphylococcus aureus is 3 CF / ml and minimum detection limit of the primer probe for detection of methicillin-resistant staphylococcus aureus is 5 CF / ml.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a combination of primers and probes for identifying Staphylococcus aureus and its drug resistance. Background technique [0002] Staphylococcus aureus (staphylococcus aureus, SA) is a Gram-positive coccus, an important human pathogen, belonging to the genus Staphylococcus (Staphylococcus), which can cause community and hospital infections. In recent years, with the widespread use of penicillin, some Staphylococcus aureus produce penicillinase, which can hydrolyze the β-lactam ring, showing resistance to penicillin, especially methicillin-resistant Staphylococcus aureus (methicillin-resistant staphylococcusaureus , MRSA) increased drug resistance has attracted more and more attention. [0003] There are various detection methods for Staphylococcus aureus. Bacterial isolation and culture is the "gold standard" for microbial detection. Although this method is reliable, it is time-consu...

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
IPC IPC(8): C12Q1/689C12Q1/686C12Q1/14C12N15/11C12R1/445
CPCC12Q1/689C12Q1/686C12Q2600/106C12Q2600/166C12Q2600/16C12Q2563/107C12Q2537/143Y02A50/30
Inventor 高歌高利飞李静静李振红付光宇吴学炜苗拥军
Owner AUTOBIO DIAGNOSTICS 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