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Primer, probe and kit for multiplex PCR detection of five pathogenic bacteria

A technology for pathogenic bacteria and kits, applied in the field of biological detection, can solve the problems of increased mortality and decreased survival chances, and achieve the effects of high sensitivity, reduced pollution possibility, and high sensitivity

Pending Publication Date: 2021-11-09
PILOT GENE TECH HANGZHOU CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, if a patient is initially treated with an inappropriate antibiotic, their chances of survival may drop fivefold
However, early antifungal treatment is often ignored, and antifungal treatment is not started until 48 hours after blood culture is positive. Delay in treatment is an independent risk factor for death in the hospital, and the fatality rate increases significantly by 1.5 to 2 times.

Method used

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  • Primer, probe and kit for multiplex PCR detection of five pathogenic bacteria
  • Primer, probe and kit for multiplex PCR detection of five pathogenic bacteria
  • Primer, probe and kit for multiplex PCR detection of five pathogenic bacteria

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Embodiment 1 Primer probe of the present invention

[0041] The sequences of the primer combinations involved in this example are shown in Table 1.

[0042] Table 1 Primer Probe Combination Sequence

[0043]

Embodiment 2

[0044] Embodiment 2 detection method of the present invention

[0045] The experimental procedure is as follows:

[0046] Plasma sample nucleic acid extraction→configuration of digital PCR reaction solution→droplet chip generation→amplification→reading→result analysis and report output.

[0047] It is recommended to use the plasma cell-free DNA nucleic acid extraction kit produced by Linghang Gene Technology (Hangzhou) Co., Ltd. for the nucleic acid extraction experiment kit, and perform nucleic acid extraction according to the instructions.

[0048] 1. Target gene detection program

[0049] Digital PCR detection process (configuration of digital PCR reaction solution, droplet chip generation and amplification process).

[0050] 1. Prepare a 20 μl droplet PCR detection system. The specific system formulations are shown in Table 2.

[0051] Table 2

[0052] components Volume (μl) 5x Taq Mix 4 Forward Primer (100μM) 0.06 Reverse Primer (100μM) 0....

Embodiment 3

[0061] Embodiment 3 kit of the present invention

[0062] 1. Nucleic acid extraction kit (preferably Tiangen DP710 kit)

[0063] Preferably, the commercially available Tiangen DP710 kit is used for DNA extraction according to the instructions, as follows:

[0064] Add the lysate GHH to the separated sample at a ratio of 1:1.5 to the plasma, then add proteinase K, magnetic beads and carrierRNA in sequence, vortex and oscillate to mix, and incubate at room temperature for about 20 minutes, shaking and shaking twice during the incubation;

[0065] Place the centrifuge tube on the magnetic stand for 2 minutes, and discard the waste liquid when the magnetic beads are completely absorbed;

[0066] Add 750ul buffer GDF, mix upside down for 30s to fully suspend the magnetic beads, place the centrifuge tube on the magnetic stand for 2min, and discard the waste solution when the magnetic beads are completely absorbed;

[0067] Add 750ul rinse solution PWG, mix upside down for 30s to f...

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Abstract

The invention relates to the technical field of biological detection, in particular to a primer, probe and kit for multiplex PCR detection of five pathogenic bacteria. The primer and the probe disclosed by the invention support high-throughput detection, can quickly realize joint detection of five pathogenic bacteria including staphylococcus aureus, coagulase negative staphylococcus, enterococcus faecium, enterococcus faecalis and candida, and are high in accuracy and good in specificity. According to the quantitative / semi-quantitative detection result of the target pathogen, the change of the specific pathogen in the body of a blood flow infection patient can be rapidly monitored in real time, the condition of the patient can be evaluated in time, the pathogen can be effectively and accurately diagnosed, early discovery, early prevention, early treatment and early control are achieved, auxiliary treatment suggestions are provided for clinicians, for example, antibacterial drugs are reasonably selected and used, so that the hospital infection opportunity is reduced, and the primer and the probe have extremely high application value in the scientific research field and clinics.

Description

technical field [0001] The invention relates to the technical field of biological detection, in particular to primers, probes and kits for multiplex PCR detection of five pathogenic bacteria. Background technique [0002] Blood stream infection (BSI) refers to various pathogenic microorganisms invading the blood circulation, multiplying in the blood, releasing toxins and metabolites, and inducing the release of cytokines, causing systemic inflammatory response syndrome, further leading to blood pressure drop, infection Sexual shock, changes in blood coagulation and fibrinolytic systems, and eventually cause multiple organ dysfunction throughout the body, which is a serious systemic infection. Pathogens that invade blood are mainly bacteria, fungi, and molds. Bacteria (including G - Negative bacteria and G + positive bacteria) are still the main pathogenic microorganisms. According to CHINET 2020 data, the main bacteria isolated from clinical blood samples are Escherichia ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12Q1/689C12Q1/6851C12Q1/14C12Q1/06C12N15/11C12R1/445C12R1/44C12R1/01C12R1/72
CPCC12Q1/6895C12Q1/689C12Q1/6851C12Q2600/16C12Q2531/113C12Q2537/143C12Q2563/159
Inventor 宋小慧夏江
Owner PILOT GENE TECH HANGZHOU CO LTD
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