Primer and/or probe composition for detecting bacillus for initiating blood flow infection and application of primer and/or probe composition

A composition and probe technology, applied in the field of molecular biology, can solve the problems of decreased patient survival rate, increased cost of bacterial drug resistance treatment, decreased patient survival rate, etc.

Pending Publication Date: 2020-01-07
宁波基内生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, for clinical treatment, if early diagnosis and reasonable antibacterial drug treatment are not carried out in the initial 24 hours of bloodstream infection, the survival rate of patients will be greatly reduced
The lack of accurate diagnosis r

Method used

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  • Primer and/or probe composition for detecting bacillus for initiating blood flow infection and application of primer and/or probe composition
  • Primer and/or probe composition for detecting bacillus for initiating blood flow infection and application of primer and/or probe composition
  • Primer and/or probe composition for detecting bacillus for initiating blood flow infection and application of primer and/or probe composition

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0107] This example provides the composition of exemplary kits.

[0108] Specifically, an exemplary kit includes reagent bottles or tubes respectively containing the following components, and a packaging box for collectively packaging these reagent bottles or tubes.

[0109] 1) Nucleic acid extraction solution: composed of 2% (M / V) Chelex-100, 1% (V / V) Tris-HCl, 1M, pH9.0;

[0110] 2) PCR reaction enzyme system: 5U / μL Taq DNA polymerase and 2U / μL uracil-N-glycosylase (UNG enzyme) mixed at a volume ratio of 3:1;

[0111] 3) Internal standard: a plasmid containing the internal standard int gene fragment;

[0112] 4) Negative control substance: double distilled water purified by Millipore water purifier;

[0113] 5) Positive controls: T vector plasmid carrying the target gene for detecting Mycobacterium tuberculosis (IS6110), T vector plasmid carrying the target gene for detecting Listeria monocytogenes (hlyA), carrying the target gene for detecting Klebsiella pneumoniae (phoE ...

Embodiment 2

[0125] This example provides a method for establishing an assay using the exemplary kit in Example 1.

[0126] Specifically, the method includes the following steps:

[0127] 1) Preparation of reference substances: Take 50 μL each of the positive control substance and negative control substance and place them in 1.5mL (or 0.5mL) centrifuge tubes (shake and mix for 10 seconds after the frozen reagent has melted), add 50 μL of nucleic acid extraction solution and mix well. Homogenize, incubate at 98°C for 10 min, then centrifuge at 12,000 rpm for 5 min, take 2 μL of supernatant for PCR reaction.

[0128] 2) Preparation of the reaction system mixture: 16 μL of each primer-probe mixture was mixed with 0.2 μL of the PCR reaction enzyme system, oscillated for several seconds, and centrifuged at 3000 rpm for several seconds.

[0129] 3) PCR amplification: Add 2 μL each of the treated supernatants of the negative control substance and the positive control substance into the PCR tubes...

Embodiment 3

[0135] This example provides specificity experiments performed on the kits and methods of the present application.

[0136] 1) Experimental materials

[0137] The experimental materials used in the following experiments and their sources are listed below:

[0138] Numbering Pathogen type source 1 Enterococcus faecalis Ningbo CDC, Zhejiang Province 2 Enterococcus faecium Ningbo CDC, Zhejiang Province 3 diphtheria Ningbo CDC, Zhejiang Province 4 Neisseria meningitidis Ningbo CDC, Zhejiang Province 5 Streptococcus sanguinis Ningbo CDC, Zhejiang Province 6 Candida albicans Ningbo CDC, Zhejiang Province 7 Klebsiella oxytoca Ningbo CDC, Zhejiang Province 8 Burkholderia cepacia Ningbo CDC, Zhejiang Province 9 Staphylococcus aureus Ningbo CDC, Zhejiang Province 10 Staphylococcus epidermidis Ningbo CDC, Zhejiang Province 11 positive control substance See Example 1 12 negative cont...

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Abstract

The application provides a primer and/or probe composition for detecting bacillus for initiating blood flow infection and an application of the primer and/or probe composition, and particularly relates to one or more primer and/or probe compositions or relevant reagents or reagent kits for detecting mycobacterium tuberculosis, listeria monocytogenes, klebsiella pneumoniae, pseudomonas aeruginosa,baumanii, escherichia coli, haemophilus influenzae and proteus mirabilis, so that the detection period of the bacillus can be greatly shortened, the efficiency and the detection flux are improved, andthe primer and/or probe composition can meet detection requirements. In addition, the method and relevant products thereof disclosed by the application have high specificity, sensitivity and accuracy.

Description

technical field [0001] The application belongs to the field of molecular biology, and in particular relates to a primer and / or probe composition and application thereof for detecting bacilli causing bloodstream infections, especially Gram-negative bacilli. Background technique [0002] Bloodstream infection (BSI) refers to the systemic inflammatory response, infection and poisoning caused by various pathogenic bacteria entering the blood circulation, and is a serious life-threatening infectious disease. Due to the insidious onset and rapid development of the disease, if it is not treated in time and effectively, it will endanger the life of the patient. However, the clinical manifestations of bloodstream infection lack specificity, and are easy to be missed and misdiagnosed. Therefore, early diagnosis of bloodstream infection has important clinical significance. [0003] Pathogens that cause bloodstream infections fall into three main categories: Gram-positive bacteria, Gra...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12N15/11C12Q1/04C12Q1/10C12R1/32C12R1/01C12R1/22C12R1/385C12R1/19C12R1/21C12R1/37
CPCC12Q1/689
Inventor 倪剑锋高华山史俊颖张博学杨实刘春燕童惠姗孙莎莎
Owner 宁波基内生物技术有限公司
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