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Fluorescent quantitative PCR detection kit and method for predicting sepsis infection

A detection kit and fluorescence quantitative technology, applied in the field of biomedicine, can solve problems such as difficult diagnosis of sepsis, and achieve good clinical application prospects

Active Publication Date: 2021-07-09
FLASHDX SHENZHEN INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The present invention aims at the technical problem of difficult diagnosis of sepsis in the prior art, and aims to provide a fluorescent quantitative PCR detection kit for pre-judging the infection of sepsis

Method used

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  • Fluorescent quantitative PCR detection kit and method for predicting sepsis infection
  • Fluorescent quantitative PCR detection kit and method for predicting sepsis infection
  • Fluorescent quantitative PCR detection kit and method for predicting sepsis infection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Step S1 Use the QIAamp Fast DNAStool Mini Kit (51604) kit to extract the stool genome sample

[0029] Weigh 200 mg of fresh or frozen (stored at -80°C) stool sample in a 2 mL centrifuge tube and place it on ice, add 1 mL of InhibitEx Buffer, and shake for 1 min. The above suspension was heated in a metal bath at 70° C. for 5 minutes, and centrifuged at 8000 rpm for 1 minute at room temperature to obtain a supernatant. Add 15uL proteinase K to the bottom of a new 1.5mL centrifuge tube, add 200μL supernatant; add 200μL Buffer AL, shake for 15s; heat in a metal bath at 70°C for 10min, and centrifuge briefly to remove the liquid along the inner edge of the tube cap. Add 200 μL of ethanol (concentration: 96% to 100%) into the above centrifuge tube, shake for 15 seconds and centrifuge for a short time. Carefully add the above mixture into the QIAamp spin column, put the spin column into a 2mL collection tube, close the cap of the centrifuge tube, centrifuge at 8000rpm for 1m...

Embodiment 2

[0047] Quantitative analysis of the level of intestinal flora was carried out on the stool samples of patients diagnosed with sepsis and normal controls, and the experimental procedures and conditions were the same as those in Example 1. The plasmid stock solution of each gate (that is, the pure plasmid stock solution sample obtained by TA cloning) was equally diluted with distilled water, and the dilution factor was 10, and a total of 8 concentration gradients were diluted, respectively starting from 10 -1 diluted to 10 -8 , as the standard curve points in the respective gate qPCR, the results of the standard curve are shown in Figure 1. The qPCR reaction was performed using the Bio-Rad CFX Connect instrument, and the cycle conditions are shown in Table 2 above. The output of the sample to be tested is the final concentration of the target product of each gate, and then the logarithm (log10) is taken to obtain the relative abundance. Compare the obtained flora structure wit...

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PUM

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Abstract

The invention discloses a fluorescent quantitative PCR detection kit and a method thereof for predicting sepsis infection. The kit includes: Bacteroidetes forward primer and reverse primer, Firmicutes forward primer and reverse primer, Proteobacteria forward primer and reverse primer, Actinobacteria forward primer and reverse primer Primers, PCR master mixes and reagents for fluorescent quantitative PCR detection methods. The invention provides reliable evidence of infection and the etiological type of infection for the diagnosis of sepsis by detecting the classification level structure of human intestinal flora, can be used for auxiliary diagnosis of clinical sepsis, and provides relevant antibiotic treatment for patients. Effective basis, good prospects for clinical application.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a fluorescent quantitative PCR detection kit and a method thereof for predicting sepsis infection. Background technique [0002] Sepsis is a clinical syndrome of fatal organ dysfunction caused by host response imbalance caused by infection. Its morbidity and mortality have been high and it is a prominent problem faced by the global medical community. Although the international guidelines for sepsis have been continuously updated and improved, clinicians still face many difficulties when diagnosing sepsis. Among them, whether the patient's condition is infection, or whether the organ dysfunction is caused by infection, can not be well explained according to the existing technology. Currently, as many as 40% of ICU patients diagnosed with sepsis do not in fact have an infection and thus are not truly sepsis. Therefore, how to effectively judge the infection of sep...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6851C12Q1/689C12Q1/06C12N15/11
CPCC12Q1/6851C12Q1/689C12Q2531/113C12Q2563/107C12Q2545/114
Inventor 杜白露沈楠莫茜曹清李璧如王莹钱娟宁铂涛陶悦干驰赵瑞珂胡金凤
Owner FLASHDX SHENZHEN INC
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