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Novel method for rapidly detecting septicopyemia by using gram-negative bacterial infection

A technology for Gram-negative bacteria and sepsis, which is applied in the direction of measuring devices, instruments, and analysis materials, and can solve the problem of undetectable blood endotoxin

Active Publication Date: 2020-04-28
XIAMEN BIOENDO TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, in many cases of sepsis, especially during sepsis episodes, blood endotoxin levels are undetectable even with the most sensitive KT-TALA method

Method used

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  • Novel method for rapidly detecting septicopyemia by using gram-negative bacterial infection
  • Novel method for rapidly detecting septicopyemia by using gram-negative bacterial infection
  • Novel method for rapidly detecting septicopyemia by using gram-negative bacterial infection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Method for rapid detection of LPS in cultured blood of patients with suspected sepsis

[0037] A new step in the detection of LPS in blood suspected of GNB infection. Briefly, 3-5 ml of blood was added to the culture flask. After 2 hours, 0.5 ml samples were taken every hour for dynamic testing of LPS. Samples were diluted, heated, spun, and diluted again prior to LPS quantification with the KT-TAL system.

Embodiment 2

[0038] Example 2 Formulation of reference standards

[0039] Lyophilized endotoxin standard stock solutions were reconstituted in endotoxin-free water, diluted to final concentrations of 50, 10, 1, 0.1, 0.01 and 0 EU / ml, and three independent experiments were performed on microplates. After adding 100 μl of TAL reagent to each well, place the plate in a dynamic culture reader (BioTek TM ELx808IULALXH), and the reader immediately began to measure endotoxin levels using the kinetic assay procedure. Gel formation was recorded every 30 seconds for 30-60 minutes at a wavelength of 630 nm. For this set of studies, the resulting formula was Log(Y)=A*Log(X)+B, where Y=reaction time (onset time), A=Y intercept, X=endotoxin concentration, and B=regression curve The slope of. In this set of studies, A was -0.279, B was 5.95, and R 2 (correlation efficiency) was 0.994. If the standard curve is completed using the same batch of reagents and the same procedure, it can be stored in the...

Embodiment 3

[0040] Example 3 Effect of heating on LPS released from GNB samples and assay specificity

[0041]Based on the knowledge that (1) LPS has two forms, free LPS and LPS associated with intact cell walls (Jorgensen et al., 1973); and (2) heat can not only promote the release of LPS, but also denature / precipitate interfering substances , and contribute to more sensitive quantification of LPS, employing heat as a necessary step in sample preparation. As shown in Table 1, a series of cultured E. coli samples were collected at 4, 5, 6, 7, 8, and 9 hours, with or without heating, and the detected LPS levels were compared for concurrently prepared paired samples. At 4 hours, the LPS of the unheated sample was 0.144 EU / ml, while the LPS of the heated sample was 6.689 EU / ml. Similarly, unheated samples rose relatively slowly from 0.326, 0.999, 4.237 to 10.706 EU / ml at 5, 6, 7, 8 hours, and >14.125 EU / ml at 9 hours, while at 5 hours hour, the heating time reaches 10.854, which is equival...

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Abstract

Septicopyemia is a systemic infection endangering life and needs to be properly and quickly treated on the basis of quickly determining pathogens. Gram-negative bacteria (GNB) are the main pathogenicpathogens of endotoxin (LPS) as a characteristic substitute biomarker thereof. In the research, a new method is explored firstly, firstly, LPS is increased through GNB culture, and then the LPS of tachypleus amebocyte lysate (TAL) is detected through a dynamic turbidity measurement method (KT-TALA). Sample preparation procedures are optimized. Results show that under high GNB concentration, LPS can be detected 3 hours after culture, which is 6.5-7.5 hours ahead of time, and 9.5-10.5 hours is for a BD BACTEC positive report; under a low GNB load, a BD BACTEC system needs 22-26 hours to detect GNB, but the KT-TALA system only needs 9 hours to detect LPS of GNB, and the time is advanced by 13-17 hours. Compared with a traditional BD BACTEC system, the novel method for detecting GNB infected LPS is much faster, and especially in the early stage of the septicopyemia with a low bacterial load, and the method is helpful to make appropriate treatment decisions in earlier time.

Description

technical field [0001] The present invention generally relates to culturing blood suspected of sepsis and dynamically sampling endotoxin lipopolysaccharide (LPS) produced in blood by Gram-negative bacteria (GNB) using a dynamic turbidity TAL assay (KT-TALA) system, using for rapid detection of LPS. Background technique [0002] Sepsis is the most dangerous condition and requires immediate dedicated treatment, as each hour of delay increases mortality by 5-10%, resulting in up to ~30% of deaths. The high incidence of sepsis is mainly associated with trauma, infection, major organ dysfunction and the end stages of many diseases such as cancer, aging, etc. Rapid detection of system-invading pathogens is critical to saving lives. [0003] Currently, blood culture is still the gold standard for sepsis diagnosis, which utilizes metabolites accumulated to a certain level to trigger positive reporter genes with the BDBACTEC system ( http: / / www.bd.com / en-us / offerings / capabilities / ...

Claims

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Application Information

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IPC IPC(8): G01N33/569
CPCG01N33/56911G01N2400/50G01N33/569
Inventor 吴海苹吴尚毅周燕英
Owner XIAMEN BIOENDO TECH CO LTD
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