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Complete set reagent for detecting staphylococcus haemolyticus

A staphylococcus and reagent technology, applied in the field of complete sets of reagents for the detection of hemolytic staphylococcus, to achieve high sensitivity, good repeatability, amplification efficiency, and high specificity

Inactive Publication Date: 2018-05-04
MICROBE EPIDEMIC DISEASE INST OF PLA MILITARY MEDICAL ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Whether the TaqMan MGB probe method can realize the direct detection of staphylococcus hemolyticus pathogens in whole blood samples infected by staphylococcus hemolyticus is whether a set of primers and probes with high sensitivity and specificity can be designed. At present, domestic and foreign There are no related MGB probes reported in the literature

Method used

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  • Complete set reagent for detecting staphylococcus haemolyticus
  • Complete set reagent for detecting staphylococcus haemolyticus
  • Complete set reagent for detecting staphylococcus haemolyticus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Embodiment 1, the preparation of the complete set of reagents that detects staphylococcus hemolyticus

[0054] The complete set of reagents for detecting hemolytic staphylococcus consists of primer pairs and probe P for detecting or assisting the detection of hemolytic staphylococcus;

[0055] The primer pair is composed of single-stranded DNA named F and R respectively; F is the single-stranded DNA molecule shown in sequence 1 of the sequence listing; R is the single-stranded DNA molecule shown in sequence 2 of the sequence listing;

[0056] The sequence of the probe P is the sequence 3 in the sequence list, the 5' end of the probe is marked with a FAM fluorescent group, and the 3' end is marked with a non-fluorescence quenching group NFQ-MGB.

[0057] The first position of sequence 1-sequence 3 is the 5' terminal nucleotide of the corresponding sequence.

[0058] Each single-stranded DNA and probe of the complete set of reagents for detecting hemolytic staphylococcus a...

Embodiment 2

[0059] Embodiment 2, detect the specificity of the complete set of reagents of staphylococcus hemolyticus

[0060] 1. Strains to be tested

[0061] The strains used are the following 23 strains:

[0062] Ten species of Gram-positive bacteria (Streptococcus agalactiae, Staphylococcus hemolyticus (Shu Xiaoli; Wu Yidong; Shang Shiqiang, Establishment of a real-time fluorescence quantitative and typing method for bacterial 16SrRNA gene. Chinese Journal of Contemporary Pediatrics, 2008.10(6): p. 732-736.), Staphylococcus aureus (Li, L., et al., Phenol-soluble modulin alpha4 mediates Staphylococcus aureus-associated vascular leakage by stimulating heparin-binding protein release from neutrophils. Sci Rep, 2016.6: p.29373. ), Streptococcus pyogenes (Wang, J., et al., Identification and cluster analysis of Streptococcuspyogenes by MALDI-TOF mass spectrometry.PLoS One, 2012.7(11): p.e47152.), Streptococcus pneumoniae (Shu Xiaoli; Wu Yidong ; Shang Shiqiang, Establishment of a real-ti...

Embodiment 3

[0071] Embodiment 3, detect the sensitivity of the complete set of reagents of staphylococcus hemolyticus

[0072] After the hemolytic staphylococcus was quantified, a 10-fold serial dilution was performed to obtain a concentration of 10 3 cfu / mL, 10 2 cfu / mL, 10 1 cfu / mL and 10 0 For each cfu / mL strain suspension, 1 mL of each strain suspension was used to extract genomic DNA using the same method, and eluted with 80 μL elution buffer to collect the genome, and 5 μL was used as a template.

[0073] According to the reaction system and reaction procedure of Example 2, the sensitivity of the kit of reagents in Example 1 was tested using the above genome solutions as templates, and three replicates were set for each concentration of genome solutions.

[0074] result( figure 2 ) shows that 10 3 cfu / mL, 10 2 cfu / mL, 10 1 cfu / mL and 10 0 There are amplification curves in the reaction system of cfu / mL, show that, the complete set of reagents of embodiment 1 can detect 10 0...

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Abstract

The invention discloses a complete set reagent for detecting staphylococcus haemolyticus. The complete set reagent for detecting the staphylococcus haemolyticus disclosed by the invention is composedof a primer pair for detecting staphylococcus haemolyticus and a probe, wherein the primer pair consists of single-chain DNA with names of F and R; F is a single-chain DNA molecule shown in sequence 1of a sequence table; R is a single-chain DNA molecule as shown in sequence 2 of the sequence table; and a sequence of the probe is sequence 3 of the sequence table. According to the experiment, the complete set reagent for detecting staphylococcus haemolyticus has high specificity, high sensitivity and very good repeatability and amplification efficiency while detecting staphylococcus haemolyticus, is simple and convenient, quick and accurate in detecting staphylococcus haemolyticus, and is a reagent used for detecting staphylococcus haemolyticus and preparing staphylococcus haemolyticus.

Description

technical field [0001] The invention relates to a set of reagents for detecting hemolytic staphylococcus in the field of biotechnology. Background technique [0002] Haemolytic Staphylococci are Gram-positive cocci. The bacterium is an opportunistic pathogen. Normally, humans have a certain natural immunity to pathogenic staphylococci. When the skin and mucous membranes are traumatized, or when the balance of the human immune system is lost, it may lead to serious infections in patients, such as bloodstream infections. [0003] Bloodstream infections (BSI) refer to the systemic inflammatory response syndrome (SIRS) caused by one or more pathogenic microorganisms invading the blood circulation of the body, releasing toxins in the blood, growing and multiplying, and releasing metabolites. ). [0004] With the development of genomics and proteomics, molecular biology techniques, such as polymerase chain reaction, mass spectrometry, gene chips, and high-throughput sequencing,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/14C12R1/44
CPCC12Q1/689
Inventor 张超江华姜永强郑玉玲郑新刘鹏孔德聪律清宇
Owner MICROBE EPIDEMIC DISEASE INST OF PLA MILITARY MEDICAL ACAD OF SCI
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