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Primer composition for detecting sepsis pathogen, nucleic acid detection kit and detection method thereof

A technology of primer composition and primer set, applied in the direction of microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve the problems of difficult high-throughput detection, poor stability of LAMP system, and large difference in constant temperature amplification looping efficiency To achieve the effect of improving detection sensitivity and specificity, improving detection sensitivity, and shortening the detection cycle

Pending Publication Date: 2022-07-29
SHANGHAI IGENETEC DIAGNOSTICS CO LTD +1
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  • Abstract
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0017] However, in recent years, the practical application of LAMP technology in the laboratory is relatively limited, and the progress is relatively slow. The reasons are: (1) the current LAMP system is difficult to achieve high-throughput detection in the true sense and needs to be further improved; (2) Although the LAMP system does not need to raise or lower the temperature during the detection process, it can greatly speed up the reaction speed and realize fast and efficient detection, but the stability of the LAMP system is poor, and the large number of primers in the LAMP amplification exacerbates the non-specific amplification of the source of the primers. Including non-specific amplification inside the primer (false positive in the blank control system) and non-specific binding amplification between primer and template, the problem of non-specific amplification seriously limits the practical application of this technology
[0018] The above-mentioned problems exist for the detection of a single pathogen, but for the simultaneous detection of multiple pathogens, the above-mentioned problems are more difficult to solve, whether it is the design of primer sets for different pathogens, or the simultaneous reaction of different primer sets under the same conditions to achieve ideal sensitivity and specificity. Challenging
This is due to the large difference in the constant temperature amplification looping efficiency of different primer sets, which can make the amplification efficiency of a variety of sepsis, especially more than 10 (for example, 12) pathogens, similar under the same system conditions. very difficult

Method used

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  • Primer composition for detecting sepsis pathogen, nucleic acid detection kit and detection method thereof
  • Primer composition for detecting sepsis pathogen, nucleic acid detection kit and detection method thereof
  • Primer composition for detecting sepsis pathogen, nucleic acid detection kit and detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0101] Example 1 Design and synthesis of target and primers

[0102] 1.1 Sequence acquisition:

[0103] By querying the conserved gene sequences of the above-mentioned bacteria in GeneBank, and comparing the gene sequences from various sources with Vector NTI software, it is found that the sequences are highly conserved and have good homology.

[0104] 1.2 Primer Design

[0105] (1) Import the conserved segments of each bacterial and internal standard gene into the primer design software, select F1c and B1c with similar Tm values, F3 / B3 / F2 / B2 with similar Tm values, and the Tm values ​​of F1c and B1c are higher than F3 / B3 / F2 / B2 has a Tm value greater than 5°C and a DNA sequence with an absolute value of 5'dG and 3'dG less than 4 as candidate primers.

[0106] (2) Primer synthesis: entrust the designed multiple sets of primer sequences to Shanghai Bailiger Company to synthesize them for future use.

[0107] (3) Primer confirmation: After dissolving the synthesized primers, ...

Embodiment 2

[0110] Example 2 Production of sepsis multi-target pathogen kit

[0111] Staphylococcus aureus, Klebsiella pneumoniae, Escherichia coli, Enterococcus faecalis, Acinetobacter baumannii, Pseudomonas aeruginosa, Stenotrophomonas maltophilia, Streptococcus pneumoniae, Standard strains of Neisseria meningitidis, Aspergillus, Enterobacter cloacae and Group B Streptococcus were obtained from Binabio.

[0112] 2.1 Vacuum-drying the premix (amplification system, loop-mediated isothermal amplification reagents)

[0113] The vacuum-dried premix contains 10mM dNTP (Shanghai Zhaowei), 5% BSA (sigma), 5MMgSO 4 (sigma), 100 μM fluorescent dye (Invitrogen), 5M betaine (sigma), 8U / μL Bst DNA polymerase (NEB), and target primer set (Shanghai Bailige) or internal standard primer set.

[0114] Specifically, in this example, the amplification system contains 10 mM dNTP, 5% BSA, 5 M MgSO 4 , 100 μM fluorescent dye, 5M betaine, 8U / μL Bst DNA polymerase and target / internal standard primer set, and...

Embodiment 3

[0132] Example 3 Preliminary results of detection using clinical samples

[0133] The sample types involved in the present invention mainly include bronchoalveolar lavage fluid and sputum. All samples need to be liquefied before use: put the sample at room temperature, take 200 μL of the sample, add 3 times the volume of 2M sodium hydroxide solution, vortex for 3 minutes, let it stand for 15 minutes, centrifuge at 12,000 rpm for 5 minutes, remove the supernatant, and add 1 mL of Wash with normal saline, centrifuge at 12,000 rpm for 5 min, remove the supernatant, and set aside.

[0134] For the detection of the sample, add 100 μL of the nucleic acid extraction reagent prepared in Example 2 to the centrifuge tube containing the liquefied sample, vortex for 15 s, centrifuge briefly, and then transfer it to the sample hole of the microfluidic chip. Stick the parafilm tightly, place it in the instrument, and react at 60-65°C for 30-45min.

[0135] In this embodiment, as figure ...

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Abstract

The invention discloses a primer composition for detecting pathogens of sepsis as well as a nucleic acid detection kit and a detection method of the primer composition. The invention discloses a primer composition for detecting pathogens of sepsis. The kit comprises a staphylococcus aureus primer group, a klebsiella pneumoniae primer group, an escherichia coli primer group, an enterococcus faecalis primer group, an acinetobacter baumannii primer group, a pseudomonas aeruginosa primer group, a stenotrophomonas maltophilia primer group, a streptococcus pneumoniae primer group, a neisseria meningitidis primer group and an aspergillus primer group. The primer group is at least one of an enterobacter cloacae primer group and a group B streptococcus primer group. According to the primer group for detecting the sepsis pathogens, the nucleic acid detection kit containing the primer group and the detection method provided by the invention, various sepsis pathogens can be rapidly and accurately detected, so that the defect that the existing sepsis pathogen detection technology is time-consuming and labor-consuming is overcome, the detection sensitivity and specificity are improved, the labor intensity is reduced, and the detection efficiency is improved. The detection period is shortened.

Description

technical field [0001] The present invention relates to the technical field of detection and diagnosis of pathogens, in particular to a primer composition for detecting sepsis pathogens, a nucleic acid detection kit and a detection method thereof. Background technique [0002] Sepsis can be caused by infection in any part. Clinically, it is common in pneumonia, peritonitis, cholangitis, urinary system infection, cellulitis, meningitis, abscess, etc. It refers to various bacteria or fungi entering the blood circulation, multiplying and producing Toxins induce systemic inflammatory response syndrome produced by the body. Sepsis is an important clinical problem faced by acute and critical care medicine. The number of patients with sepsis in the world exceeds 19 million every year, of which 6 million die, and the fatality rate exceeds 1 / 4. There are about 3 million surviving patients. cognitive dysfunction. Early recognition and appropriate management are critical to improving...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/6895C12Q1/6844C12N15/11C12R1/445C12R1/22C12R1/19C12R1/01C12R1/385C12R1/46C12R1/36C12R1/66C12R1/64
CPCC12Q1/689C12Q1/6895C12Q1/6844C12Q2531/119C12Q2537/1376C12Q2565/629
Inventor 方雪恩李杨李亚南刘闯孔继烈
Owner SHANGHAI IGENETEC DIAGNOSTICS CO LTD
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