Primer and/or probe composition for detecting cocci causing bloodstream infections 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 long cycle, increased cost of bacterial resistance treatment, and difficulty for clinicians to choose a treatment plan.

Pending Publication Date: 2020-01-10
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, this method requires a long cycle and is less sensitive to pathogenic bacteria that are difficult to grow or patients who have been treated with antibacterial drugs, so its role in the early diagnosis of bloodstream infections is very limited
The lack of accurate diagnosis results makes it difficult for clinicians to choose the best treatment plan, and the blind use of antibiotics may lead to adverse consequences such as reduced survival rate of patients, emergence of bacterial resistance, and increase in treatment costs

Method used

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  • Primer and/or probe composition for detecting cocci causing bloodstream infections and application of primer and/or probe composition
  • Primer and/or probe composition for detecting cocci causing bloodstream infections and application of primer and/or probe composition
  • Primer and/or probe composition for detecting cocci causing bloodstream infections and application of primer and/or probe composition

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0112] This example provides the composition of exemplary kits.

[0113] 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.

[0114] 1) Nucleic acid extraction solution: composed of 2% (M / V) Chelex-100, 1% (V / V) Tris-HCl, 1M, pH9.0;

[0115] 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;

[0116] 3) Internal standard: a plasmid containing the internal standard int gene fragment;

[0117] 4) Negative control substance: double distilled water purified by Millipore water purifier;

[0118] 5) Positive controls: T vector plasmid carrying the target gene for Streptococcus pneumoniae (lytA), T vector plasmid carrying the target gene for Enterococcus faecalis (Ef0027), T vector plasmid carrying the target gene for Enterococcus faecium (ddl), carrying T vect...

Embodiment 2

[0131] This example provides a method for establishing an assay using the exemplary kit in Example 1.

[0132] Specifically, the method includes the following steps:

[0133] 1) Preparation of reference substances: Take 50 μL each of the positive control substance and negative control substance, 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 to fully Mix well, incubate at 98°C for 10 min, then centrifuge at 12,000 rpm for 5 min, take 2 μL of supernatant for PCR reaction.

[0134] 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.

[0135] 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 of the re...

Embodiment 3

[0141] This example provides specificity experiments performed on the kits and methods of the present application.

[0142] 1) Experimental materials

[0143] The experimental materials used in the following experiments and their sources are listed below:

[0144]

[0145]

[0146] 2) Experimental method

[0147] Using the experimental method established in Example 2, the above-mentioned 12 kinds of pathogenic bacteria were detected respectively, so as to analyze and evaluate the specificity of the kit.

[0148] 3) Experimental results

[0149] Utilize the kit in the embodiment 1 to detect 12 kinds of common pathogenic bacteria (diphtheria, Haemophilus influenzae, Escherichia coli, Pseudomonas aeruginosa, Proteus, Proteus mirabilis, Enterobacter cloacae, Streptococcus sanguis, albicans, Klebsiella oxytoca, Burkholderia cepacia, Acinetobacter baumannii), the results were all negative (see Table 1).

[0150] Table 1 The specificity verification results of the kit of th...

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Abstract

The invention provides a primer and/or a probe composition for detecting cocci causing bloodstream infections and application of the primer and/or the probe composition. The invention specifically relates to the primer and/or the probe composition or a related reagent or a kit for detecting one or more of streptococcus pneumoniae, enterococcus faecalis, enterococcus faecium, streptococcus pyogenes, streptococcus agalactiae, neisseria meningitidis, coagulase negative staphylococcus and staphylococcus aureus. In this way, the detection cycle of the cocci is greatly shortened, the efficiency is improved, the detection flux is increased, and detection requirements are met; and in addition, a method and the related products 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 cocci causing bloodstream infection, especially Gram-positive cocci. 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 and 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, Gram...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/6851C12Q1/14C12Q1/04C12N15/11C12R1/44C12R1/445C12R1/01C12R1/46
CPCC12Q1/689C12Q1/6851C12Q2561/113C12Q2563/107C12Q2531/113
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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