Primer, probe and kit for multiplex PCR detection of seven drug-resistant genes

A drug-resistant gene and multiple detection technology, applied in the field of probes and its kits, and primers for multiple PCR detection of drug-resistant genes, can solve public health threats, infectious disease prevention and control problems, and difficult to guide infectious diseases in time Treatment and other issues

Pending Publication Date: 2021-11-02
PILOT GENE TECH HANGZHOU CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These drug-resistant enzyme genes can be transmitted among different strains through plasmids, integrons, etc., resulting in a rapid increase in the rate of bacterial drug resistance, and the rapid increase in drug resistance, which brings great benefits to the prevention and control of infectious diseases in hospitals and communities. bothersome and a growing threat to public health
Since routine etiological diagnosis methods include bacterial culture, isolation and drug sensitivity in specimens, it takes a long time (3-5 days), and it is difficult to guide the treatment of infectious diseases in time

Method used

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  • Primer, probe and kit for multiplex PCR detection of seven drug-resistant genes
  • Primer, probe and kit for multiplex PCR detection of seven drug-resistant genes
  • Primer, probe and kit for multiplex PCR detection of seven drug-resistant genes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Embodiment 1 Primer probe of the present invention

[0039] The sequences of the primer combinations involved in this example are shown in Table 1.

[0040] Table 1 Primer Probe Combination Sequence

[0041]

[0042]

Embodiment 2

[0043] Embodiment 2 detection method of the present invention

[0044] The experimental procedure is as follows:

[0045] Nucleic acid extraction from plasma / anorectal test samples→configuration of digital PCR reaction solution→droplet chip generation→amplification→reading→result analysis and report output.

[0046] 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.

[0047] 1. Target gene detection program

[0048] 1. Digital PCR detection process (configuration of digital PCR reaction solution, droplet chip generation and amplification process) to prepare a 20 μl droplet PCR detection system, the specific system formulations are shown in Table 1;

[0049] Table 1

[0050] components Volume (μl) 5x Taq Mix 4 Forward Primer (100μM) 0.06 Reverse Prime...

Embodiment 3

[0059] Embodiment 3 kit of the present invention

[0060] 1. Nucleic acid extraction kit (preferably Tiangen DP710 kit)

[0061] Preferably, the commercially available Tiangen DP710 kit is used for DNA extraction according to the instructions, as follows:

[0062] 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;

[0063] Place the centrifuge tube on the magnetic stand for 2 minutes, and discard the waste liquid when the magnetic beads are completely absorbed;

[0064] 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;

[0065] 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 primers, probes and a kit for multiplex PCR detection of seven drug-resistant genes. The kit disclosed by the invention can be used for carrying out joint detection on carbapenem enzyme genes KPC, NDM, IMP, VIM and OXA48 genes in carbapenem drug-resistant enterobacteriaceae bacteria, methicillin-resistant staphylococcus mecA genes and vanA genes in vancomycin-resistant multi-drug-resistant enterococcus in a rapid, accurate and ultrahigh-sensitivity manner; the change of the specific drug-resistant gene in the body of a blood flow infection patient can be rapidly monitored in real time, the condition of the patient is evaluated in time, and auxiliary treatment suggestions are provided for clinicians. The kit disclosed by the invention can also be used for active screening of drug-resistant genes of seven bacteria in CRE, MRSA and VRE of critically ill patients and immunosuppressive people without symptom colonization, and has extremely high application value in the field of scientific research and clinical application.

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 seven drug-resistant genes. Background technique [0002] Antibacterial drugs are the most widely used drugs in clinical practice. However, the mortality rate of infectious diseases has increased rapidly in recent years. The reason is the overuse of antibiotics and the difficulty in drug use caused by drug-resistant bacteria. my country is one of the countries where the abuse of antibiotics is the most serious. However, with the increase in the use of antibiotics, the bacterial resistance is also becoming more and more serious. Currently, the monitoring / detection of bacterial resistance focuses on: 1. Carbapenem-resistant Enterobacteriaceae (CRE); 2. Methicillin-resistant Staphylococcus (MRSA); 3. Vancomycin-resistant multidrug-resistant enterococci (VRE). [0003] Carbapenem antibiotics have strong antibacter...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/6851C12N15/11
CPCC12Q1/689C12Q1/6851C12Q2600/106C12Q2600/16C12Q2531/113C12Q2537/143C12Q2563/159C12Q2563/107Y02A50/30
Inventor 宋小慧夏江
Owner PILOT GENE TECH HANGZHOU CO LTD
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