Nucleic acid mass spectrometry method for detecting 10 common pathogenic bacteria of clinical infection

A technology for detection of pathogenic bacteria and pathogenic bacteria, applied in the direction of microorganism-based methods, biochemical equipment and methods, and measurement/inspection of microorganisms, can solve problems such as inability to meet the clinical needs of various pathogenic microorganisms, and achieve a high degree of automation and high detection sensitivity High, easy and safe operation effect

Inactive Publication Date: 2018-04-27
GENERAL HOSPITAL OF PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the limitation of fluorescent detection channels (only 2 to 3 sites can be detected at a ti

Method used

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  • Nucleic acid mass spectrometry method for detecting 10 common pathogenic bacteria of clinical infection
  • Nucleic acid mass spectrometry method for detecting 10 common pathogenic bacteria of clinical infection
  • Nucleic acid mass spectrometry method for detecting 10 common pathogenic bacteria of clinical infection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0094] Example 1: Primer Design and Synthesis

[0095] Targeting 10 virulence factor genes related to pathogenic bacteria: Klebsiella pneumonia virulence factor khe gene, Acinetobacter baumannii virulence factor OXA gene, Enterococcus faecium virulence factor ddl gene, Streptococcus pneumonia virulence factor Lyt gene, Enterococcus faecalis virulence factor ddl gene, Escherichia coli virulence factor phoA gene, Staphylococcus aureus virulence factor nuc gene, Proteus mirabilis virulence factor ureR gene, Stagphylococcus epidermidis virulence factor nuc gene, Pseudomonas aeruginosa virulence factor ecfX gene related specific sites, design corresponding Specific PCR primer core sequence (SEQ ID No: 1 to SEQ ID No: 20) and specific extension primer core sequence (SEQ ID No: 21 to SEQ ID No: 30).

[0096] Among them, in order to prevent the PCR primers from entering the detection window of the mass spectrometer and interfering with the detection effect, a certain number of bases c...

Embodiment 2

[0099] Embodiment 2: sample DNA extraction

[0100] Collect samples from patients with clinical pathogen infection (hot (hot stained samples or low temperature hot stained samples with chills, leukocytosis (count greater than 10.0) 9 / L, especially when there is "nuclear shift to the left", skin and mucous membrane hemorrhage, coma, multiple organ failure, lower blood pressure, higher C-reactive protein and faster breathing, neutropenia and thrombocytopenia in patients with blood diseases, etc., or at the same time With the above signs) blood samples from 6 cases. Among them, DNA extraction and so on are in accordance with the requirements of the instructions, and human venous blood is collected when the patient has chills, fever, and body temperature reaches the highest point, and is collected with EDTA anticoagulant tubes. According to the instructions, the collected blood should not be stored at 2-8°C for more than one week, and at -20°C for no more than one month. Try to ...

Embodiment 3

[0101] Embodiment three: Biological experiment

[0102] The gene loci related to the virulence factor genes of 10 common pathogenic bacteria in clinical infection were tested using the PCR instrument of Yixin Bochuang Company.

[0103] The components used in the detection system for PCR, PCR product purification and single base extension are:

[0104] serial number

component name

main ingredient

1

PCR mix

dNTPs, MgCl2

2

PCR Primer Mix

PCR primers

3

PCR enzyme

Taq enzyme

4

SAP Enzyme Mix

SAP enzyme

5

Extension Primer Mix

extension primer

6

elongase mix

iPLEX enzymes, ddNTPs

7

positive control

Genomic DNA of 10 kinds of bacteria (10ng / ul)

[0105] According to the manual, the specific operation method is as follows:

[0106] 1. PCR amplification

[0107] 1.1 In the PCR preparation area, prepare 200ul PCR reaction tubes according to the number of samples to be...

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Abstract

The invention discloses a primer system for detecting 10 common pathogenic bacteria of clinical infection. The detection system is used for detecting blood or body fluid (pleural fluid, ascitic fluid,drainage fluid, synovial fluid and cerebrospinal fluid) infected samples of patients infected by the pathogenic bacteria; detection results are combined with other clinical indicators, reference canbe provided for early diagnosis for clinicians and rational use of antibiotics, and treatment delaying and nonstandard use of the antibiotics are avoided. Virulence factor gene loci of 10 differentpathogenic bacteria can be simultaneously detected in two reaction systems; compared with sequencing, real-time fluorescence quantitative PCR and other technologies, the cost is lower, the operation is more convenient, and the accuracy and sensitivity are improved.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a detection method and product for determining specific gene loci of 10 kinds of common pathogenic bacteria infecting genus. Specifically, multiplex PCR technology, single base extension technology and mass spectrometry technology are used. A method for detecting specific gene loci of 10 genera associated with common pathogenic bacteria in clinical infection and a corresponding kit. Background technique [0002] Infectious diseases caused by pathogenic microorganisms are a serious threat to human health, among which bacterial diseases are the most common, and bacterial clinical infections have received widespread attention due to their acute onset, severe illness, and high fatality rate. The prerequisite for improving the treatment rate is to identify the pathogenic bacteria and select sensitive drugs for targeted treatment. At present, the diagnosis of clinical infection mainly relie...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/6858C12Q1/14C12Q1/10C12Q1/04C12N15/11C12R1/22C12R1/46C12R1/19C12R1/445C12R1/45C12R1/385C12R1/01
CPCC12Q1/6858C12Q1/689C12Q2600/156C12Q2600/16C12Q2531/113C12Q2537/143C12Q2533/101C12Q2565/627
Inventor 王成彬何赏陈琛张可昕
Owner GENERAL HOSPITAL OF PLA
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