CN113302494A

CN113302494A - Novel method for rapidly detecting sepsis by using gram-negative bacterial infection

Info

Publication number: CN113302494A
Application number: CN201980069612.7A
Authority: CN
Inventors: 吴海苹; 吴尚毅; 周燕英
Original assignee: Xiamen Bioendo Technology Co ltd
Current assignee: Xiamen Bioendo Technology Co ltd
Priority date: 2018-10-21
Filing date: 2019-10-21
Publication date: 2021-08-24
Also published as: CN111077307A; CN111077307B; WO2020083261A1

Abstract

A method for rapidly detecting sepsis using a gram-negative bacterial infection, comprising: blood culture, dynamic sampling, dilution, heating, rotation, further dilution, and determination with dynamic endotoxin assay system (K-LPS-A) and Limulus amebocyte lysate (TAL or LAL). The new method for detecting GNB infection is much faster (hours to tens of hours earlier) than the traditional BD BACTEC system, is particularly useful for detecting the early stages of sepsis with a low bacterial load, and helps to make appropriate treatment decisions at an earlier time.

Description

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

Technical Field

The present invention relates to the field of medicine, and more particularly to a method for rapidly detecting sepsis using gram-negative bacterial infection.

Background

Sepsis is the most dangerous situation and requires immediate dedicated treatment, since an hourly delay can increase mortality by 5-10%, resulting in up to-30% mortality. 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 systemic invading pathogens is critical to life saving.

Currently, blood culture is still the gold standard for sepsis diagnosis, using metabolites that accumulate to a certain level, triggering positive reporter genes with the BD BACTEC system. This takes about 16-24 hours for-50% of septic patients and > 24 hours for the rest of patients, which is too long for rapid treatment decision making. Furthermore, not all GNBs in blood can be detected by the BD BACTEC system.

Thomas et al reported that 12 samples were found to be endotoxin positive in culture without the corresponding GNB. In 7 of these 12 positive endotoxin tests, laboratory or clinical interpretation of these positive tests can be provided. This set of data indicates that a new assay is highly desirable to compensate for the "blind spot" of the BD BACTEC system in GNB infection.

However, in many cases of sepsis, especially during the onset of sepsis, it is difficult to detect blood endotoxins even with the most sensitive limulus reagents. It was suggested that endotoxin by GNB could only be determined if plasma LPS >0.5 EU/ml. However, this only occurs in patients with septic shock or severe sepsis, which is late for effective treatment.

Therefore, there is an urgent need in the art to develop sensitive, rapid methods of LPS detection to aid in the early diagnosis of GNB sepsis and to aid in monitoring therapeutic efficacy.

Disclosure of Invention

The object of the present invention is to provide a new method of sensitive LPS detection that facilitates not only the early diagnosis of GNB sepsis, but also the monitoring of the therapeutic effect.

In a first aspect of the present invention, there is provided a method for detecting endotoxin produced by Gram Negative Bacteria (GNB) in blood of a septic patient, the method comprising: blood culture, dynamic sampling, dilution, heating, spinning (spin), further dilution, and assay with a dynamic turbidity TAL assay (KT-TALA) system comprising:

(A) techniques and devices for blood culture, sampling, dilution, heating, spinning (spin), further dilution and testing in optimal conditions;

(B) special limulus amebocyte lysate reagent;

(C) reader for dynamic turbidity TAL assay (KT-TALA).

In another preferred embodiment, the method comprises blood culture, dynamic sampling, dilution, heating, rotation or centrifugation (spin), further dilution, and determination with A dynamic endotoxin assay system (K-LPS-A) comprising:

(A) techniques and devices for blood culture, sampling, dilution, heating, centrifugation, further dilution and testing in optimal conditions;

(B) special limulus amebocyte lysate (TAL or LAL) reagents;

(C) a dynamic incubation reader for dynamic turbidity, dynamic color or end-point color.

In another preferred embodiment, the limulus amebocyte lysate comprises a limulus reagent TAL or a limulus reagent LAL.

In another preferred embodiment, the system wherein component (C) is a dynamic incubation reader for dynamic turbidity, dynamic color or end-point color.

In another preferred embodiment, the subject is a human or an animal.

In another preferred embodiment, the test target is endotoxin Lipopolysaccharide (LPS) produced in blood by gram-negative bacteria (GNB).

In another preferred embodiment, the test agent is a limulus amoebocyte lysate, TAL.

In another preferred embodiment, the test agent is Limulus polyphemus amoebocyte lysate, LAL.

In another preferred embodiment, the method comprises dynamic sampling and dynamic reading.

In another preferred embodiment, the dynamic sampling is every 1-2 hours.

In another preferred example, the dynamic turbidity readings are used in the gelation process of LPS-targeted TAL.

In another preferred embodiment, the dynamic readout is a dynamic nephelometry based on the gelation process of LPS targeted limulus amoebocyte lysate (TAL or LAL).

In another preferred example, the dynamic readout is a dynamic color development method based on the color development reaction of a color development substrate added during the gelation process of LPS-targeted limulus amebocyte lysate (TAL or LAL).

In another preferred example, the dynamic readout is an end-point chromogenic method based on the chromogenic reaction of the LPS-targeted chromogenic substrate added during gelation of the limulus amoebocyte lysate (TAL or LAL).

In another preferred embodiment, the entire process for blood culture, sampling and testing is fully automated by the device.

In another preferred example, the method can be used in all fields related to LPS detection.

In a second aspect of the invention, there is provided a detection system comprising:

(a) techniques and devices for blood culture, sampling, dilution, heating, rotation, further dilution and testing in optimal conditions;

(b) special limulus amebocyte lysate (TAL or LAL) reagents;

(c) a reader (KT-TALA) for dynamic turbidity TAL assay; and

(d) limulus amoebocyte lysate (TAL).

In another preferred embodiment, the detection system comprises:

(a) techniques and devices for blood culture, sampling, dilution, heating, rotation (or centrifugation), further dilution and testing in optimal conditions;

(b) special limulus amebocyte lysate reagent; and

(c) dynamic endotoxin assay system (K-LPS-A).

In a third aspect of the invention, there is provided a use of the detection system according to the second aspect of the invention for preparing a reagent or a kit for detecting endotoxin (LPS) produced by gram-negative bacteria (GNB) in blood of a patient having sepsis.

In another preferred embodiment, the reagent or kit is also used for detecting lipopolysaccharide of endotoxin produced by gram-negative bacteria (GNB) in blood of a patient having sepsis.

In a fourth aspect of the invention, there is provided an in vitro method for detecting endotoxin produced by Gram Negative Bacteria (GNB) in the blood of a septic patient (or a patient suspected of having sepsis), the method comprising:

(a) providing a blood sample from a patient having sepsis or suspected of having sepsis;

(b) carrying out blood culture on the blood sample, and carrying out dynamic sampling and dynamic reading in the culture process; and

(c) determining whether Gram Negative Bacteria (GNB) in the blood produce endotoxin based on the dynamic readings.

In another preferred embodiment, in step (c), the determination comprises a qualitative determination (i.e., determining whether Gram Negative Bacteria (GNB) in the blood produce endotoxin), or a quantitative determination (giving a quantitative result of the production of endotoxin by Gram Negative Bacteria (GNB) in the blood).

In another preferred embodiment, in step (b), n dynamic sampling is performed, n is a positive integer of 4 to 50, preferably n is 5 to 30, more preferably n is 6 to 20.

In another preferred embodiment, the time interval between every two dynamic sampling is 0.25-3 hours, preferably 0.5-2 hours, such as every 20 minutes, 30 minutes, 45 minutes, or 1 hour.

In another preferred embodiment, in the step (b), in the dynamic sampling, the method includes: the withdrawn culture was diluted, heated, spun down (spin), and further diluted.

In another preferred embodiment, in step (b), in the dynamic reading, the detection and reading are performed by a dynamic endotoxin detection method.

In another preferred embodiment, the dynamic detection is selected from the group consisting of: dynamic turbidity methods, dynamic color development methods, endpoint color development methods, or combinations thereof.

In another preferred embodiment, the dilution is 2-50 fold dilution, preferably 5-20 fold dilution.

In another preferred embodiment, the heating is to a temperature of 50 ℃ to boiling temperature, preferably 60-100 ℃, more preferably 70-100 ℃.

In another preferred embodiment, the treatment time for heating is 1 to 25 minutes, preferably 2 to 20 minutes, more preferably 3 to 15 minutes.

In another preferred embodiment, centrifugation is centrifugation to remove the pellet (including the solid material resulting from the heat treatment) and to remove the supernatant.

In another preferred embodiment, the further dilution is a 2-10 fold dilution, preferably a 3-8 fold dilution.

In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

In another preferred embodiment, the method is an in vitro method.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

Figure 1 shows the new steps of LPS detection in blood suspected of being infected with GNB.

Figure 2 shows the results compared to LPS standard curves to determine if LPS increases in GNB in suspected blood over time.

Detailed Description

After extensive and intensive studies, the present inventors have unexpectedly found a novel method for detecting LPS in GNB-infected blood for the first time. Specifically, the present invention detects GNB in vitro using A limulus reagent-based kinetic assay system (e.g., K-LPS-A system). The methods and systems of the invention (e.g., the K-LPS-A system) can greatly reduce the detection time compared to the BD BACTEC system. Experiments have shown that in the present invention, GNB detection only takes 9 hours, which is several hours to more than ten and several hours earlier than the conventional BD BACTEC system. Furthermore, the method of the present invention has a very high sensitivity. On this basis, the present inventors have completed the present invention.

Term(s) for

As used herein, the terms "spin", "centrifugation" and "centrifugation" are used interchangeably to refer to the operation of centrifugation by rotation of a centrifuge.

Limulus amebocyte lysate

As used herein, the terms "specific limulus amebocyte lysate reagent", "limulus amebocyte lysate", "limulus reagent" are used interchangeably to refer to a reagent for detecting endotoxin from a limulus, representative examples include (but are not limited to): limulus reagent TAL or limulus reagent LAL. In the present invention, preferably, a limulus reagent having a sensitivity of 0.2 to 1.0Eu/ml, preferably 0.25 to 0.5Eu/ml can be used. Limulus reagent is the preferred reagent for detecting endotoxin.

As used herein, the term "limulus reagent TAL" or "TAL" is used interchangeably and refers to Tachypleus Amebocyte Lysate (TAL), which is a limulus reagent extracted from the blood of Tachypleus tridentatus (Chinese horseshoe crab).

As used herein, the terms "Limulus reagent LAL" or "LAL" are used interchangeably and refer to Limulus Amebocyte Lysate (LAL), which is a Limulus reagent extracted from the blue blood of North American horseshoe crab (Limulus polyphemus).

Detection method

In the present invention, A blood sample suspected of sepsis is cultured and endotoxin (LPS) produced in blood by gram-negative bacteriA (GNB) is dynamically measured using A dynamic endotoxin assay system (K-LPS-A), thereby performing A rapid and sensitive early diagnosis of GNB sepsis.

In the present invention, blood samples from GNB sepsis are incubated at a suitable temperature (e.g., 37 ± 2 ℃) for a short period of time (several hours) in a nutrient rich medium, and the amount of GNB rapidly doubles at intervals (e.g., about 20 minutes), thereby producing/releasing large amounts of endotoxin (e.g., LPS). The endotoxin produced was readily detectable by the K-LPS-A method and was much earlier than LPS + GNB + sepsis reported by blood culture of the BD BACTEC system.

It is known that a Limulus reagent prepared from amebocyte cells of a Limulus (Limulus polyphemus) is the most sensitive reagent for detecting LPS of GNB. The mechanism of action of limulus reagents is that endotoxin activates serine protease C, then serine protease B, and triggers a cascade of clotting enzymes and gel formation, which can be recorded kinetically with a dynamic incubation reader.

In the present invention, the sensitive detection method can be widely used for detecting endotoxin in different biological samples, such as blood, urine, ascites, pleural effusion, cerebrospinal fluid, bronchoalveolar lavage fluid, air and water. Compared with the traditional GNB culture test, the detection based on the limulus test is more sensitive and reliable.

In the present invention, representative limulus reagent-based dynamic endotoxin assay methods include (but are not limited to): dynamic turbidity methods, dynamic color development methods, endpoint color development methods, or combinations thereof.

The dynamic turbidity method, dynamic color method, and end-point color method are all methods for quantitatively detecting LPS. According to the detection principle, the dynamic turbidity method is a method for measuring the content of LPS by detecting the turbidity change in the reaction process of a limulus reagent and the LPS. Both the dynamic color development method and the end-point color development method belong to color development substrate methods. The chromogenic matrix method is a method for measuring the content of LPS by using the amount of a chromogen released by developing a specific substrate with a clotting enzyme produced during the reaction of a limulus reagent with LPS.

Typically, in the present invention, A limulus reagent-based dynamic endotoxin assay can be performed using A dynamic endotoxin assay system (K-LPS-A).

Preferably, the detection method based on the dynamic nephelometry can employ a dynamic turbidity TAL assay system (KT-TALA).

In the present invention, the detection value may be compared with a standard value or a standard curve. For example, one preferred method is to compare with a standard curve.

For example, when dynamic turbidity methods are employed, the formula for the standard curve may be formula Q1:

Log(Y)＝A*Log(X)+B (Q1)

wherein the content of the first and second substances,

reaction time (start time, e.g., s, min or hr),

the intercept of A is equal to the intercept of Y,

x ═ endotoxin concentration (e.g., EU/ml)

B is the slope of the regression curve.

For example, in one embodiment, A is-0.279, B is 5.95, and R²(associated 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 a reader or database as a reference for the following analysis. However, if the reagent or procedure changes, new criteria need to be created anew as a new reference. The results of the unknown samples were compared to the LPS standard curve to determine if LPS increases with time as GNB in the suspected blood increases.

The main advantages of the invention include:

(1) the method of the invention can greatly shorten the time for diagnosing the GNB and is convenient for early diagnosis of GNB sepsis.

(2) The method of the invention has very high sensitivity and prevents missing detection.

(3) Compared with the BD BACTEC system, the detection system adopted by the invention can greatly shorten the detection time, namely, the detection of GNB only needs 9 hours, and the detection of GNB sepsis is about 20 hours earlier than that of the traditional BD BACTEC system, thereby gaining valuable time for early treatment.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are percentages and parts by weight.

The materials and reagents used in the examples were all commercially available products unless otherwise specified.

Example 1 method for rapidly detecting LPS in cultured blood of suspected sepsis patient

In this example, a new procedure was used to detect LPS in blood suspected of having GNB infection. Briefly, 3-5ml of blood was added to the culture flask. After 2 hours, 0.5ml samples were taken every hour for dynamic testing of LPS. The samples were diluted, heated, spun (centrifuged) and diluted again before LPS quantification with A dynamic turbidity (KT-TALA) system or other dynamic endotoxin assay system (K-LPS-A).

EXAMPLE 2 reference Standard Generation

The lyophilized endotoxin standard stock solution was reconstituted with endotoxin-free water, diluted to final concentrations of 50, 10,1, 0.1, 0.01 and 0EU/ml and subjected to three independent experiments on microplates. After adding 100. mu.l TAL reagent to each well, the plate was placed in a dynamic incubation reader (BioTek)^TMELx808IULALXH) and the reader immediately begins to measure endotoxin levels using a 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 formula generated was log (Y) ═ a × log (X) + B, where Y ═ reaction time (onset time), a ═ Y intercept, X ═ endotoxin concentration, B ═ slope of the regression curve. In this study, A was-0.279, B was 5.95, and R was²(associated efficiency) was 0.994.

Example 3 Effect of heating on LPS Release from GNB samples and assay specificity

Based on the following knowledge: (1) LPS has two forms, free LPS and LPS associated with intact cell walls (Jorgensen et al, 1973); and (2) heat not only promotes the release of LPS but also denatures/precipitates interfering substances and facilitates more sensitive quantification of LPS, using heat as an essential 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 and compared to the detected LPS levels for pairs of samples prepared simultaneously, with or without heating. At 4 hours, the LPS of the unheated sample was 0.144EU/ml, while that of the heated sample was 6.689 EU/ml. Similarly, at hours 5, 6, 7, 8, the unheated sample rose relatively slowly from 0.326, 0.999, 4.237 to 10.706EU/ml, and at hours 9 > 14.125EU/ml, while at hour 5, the heating time reached 10.854, corresponding to the 8 th unheated sample. Later, at 6 hours, the heated sample reached > 14.125, corresponding to the unheated sample at 9 hours, indicating that the heating step can significantly increase LPS from an undetectable form to a detectable form and reduce assay interference factors, thereby significantly facilitating the detection of LPS in early GNB infections.

TABLE 1 Effect of heating on LPS released from GNB samples and assay specificity^#

^#Using water without LPS in a ratio of 1:4, heated at 100 ℃ for 10 minutes, spun at 450g for 3 minutes, and thenThe supernatant was further diluted at a ratio of 1:10 and LPS assay was performed using KT-TAL system. The experiment was repeated three times with the same trend, i.e. in the GNB E.coli group, the LPS of the heated sample was much higher than that of the unheated sample: (^*P<0.05) and there was no difference in LPS between the heated and unheated samples in the gram-positive s.

Example 4 Effect of centrifugation on LPS recovery

In measuring the formation of gel, LPS reacts with limulus reagent, and thus it is required that the sample be clean and transparent. However, after heating and denaturation, the blood sample becomes turbid, and turbid materials need to be removed by spin centrifugation.

Table 2 shows that the speed and time of centrifugation may affect the recovery of LPS. The turbid material was spun using a 1.5ml tube in an Allegra 21R centrifuge and it was found that LPS loss rate was 14.97% when spun at 10,000rpm for 3min and 8.75% at 3,000rpm for 3min, similar to 6.55% without spinning. This data indicates that LPS can co-precipitate during spinning. The high speed rotation pulls down more LPS than the low speed rotation. The optimum spin speed was 3,000rpm for 3 minutes. If the sample is clean and transparent, rotation may not be required. The speed and time of rotation is largely dependent on the transparency of the sample.

TABLE 2 Effect of centrifugation on LPS loss^*

^*The negative medium from the same BD BACTEC bottle cultured for 5 days was divided into several tubes, and standard 1EU/ml LPS was added to each tube, centrifuged at different speeds, LPS was measured and LPS loss rate was calculated as (1-measured LPS/1.045). times.100%.

Example 5 Effect of sample dilution on LPS assay results

BD BACTEC flasks with 3-10ml of whole blood contained a large number of components that could bind to the limulus reagent and prevent its gelation with LPS, and could produce negative results even if a large amount of LPS was present. Although the heating/centrifugation process can remove some of the interfering substances with large molecular weight, optimized dilution prior to the assay will help reduce interfering substances with small molecular weight.

To determine the optimized dilution factor, 4 samples (1 normal blood, designated M1; 3 patient blood, designated CN1,2,3) were cultured in BD BACTEC bottles, each divided into two tubes: one as background and the other with the addition of standard 1EU LPS. Paired samples were heated, spun at 3000rpm for 3 minutes and diluted at different ratios (1: 10, 1:20, 1:40 or 1: 100) prior to KT-TALA assay.

The results (Table 3) show that LPS was not detected (< 0.007EU/ml) in all 4 pairs of samples in the 1:10 dilution group, indicating that high levels of interferents block LPS-TAL gelation. In the 1:20 dilution group, 4 spiked solutions also showed very low levels of LPS (0.01-0.025 EU/ml). In the ratios 1:40 and 1: in the 100 dilution group, all the spiked solutions added to the samples showed LPS levels close to 1EU with high recovery of 97.66% -118.99%. The data support that the limulus reagent TAL starts to react with LPS and form a gel only after the interfering substance is diluted to a certain low level. With this new method, the optimal dilution factor is 1:40 to 1: 100.

TABLE 3 optimization of sample dilution for spiked sample recovery test^#

#: 1EU/ml LPS was added to all tubes labelled with-S and the LPS detected should be-1, < 1EU/ml, indicating the presence of some strong factor inhibiting the assay system.

M1 ^*: media supplemented with 3ml of blood was used as background control.

M1-S ^*: to M1^*To this was added standard LPS (1EU/ml) to perform LPS recovery (%) at different dilutions.

CN1 ^*，CN2 ^*，CN3 ^*: clinical blood culture negative samples from BD flasks without LPS addition were used as background controls.

CN1-S ^*，CN2-S ^*，CN3-S ^*: at different dilutions to CN1^*，CN2 ^*，CN3 ^*Standard LPS (1EU/ml) was added for calculation of LPS recovery (%) at different dilutions.

Example 6 comparison of GNB detection time between KT-TALA System and BD BACTEC System

The purpose of this example was to combine blood culture with a KT-TALA assay to detect GNB much earlier than the traditional BD BACTEC system. For verification, 0.5McF of Escherichia coli, Klebsiella pneumoniae (as a test), and Staphylococcus aureus (as a negative control) were mixed at 1:10^-8、1:10 ^-9、1:10 ^-11、1:10 ^-12The same number of bacteria were injected into two BD BACTEC bottles, one for the BD BACTEC detector reporting positive detection time and the other for a 37 ℃ bacteria shaking incubator (functioning similarly to the BD BACTEC detector), sampling was performed every hour starting 3 hours after incubation to determine the first time point at which LPS detection was positive.

The results of the independent experiments (table 4) show that in experiment 1, when the ratio of 1:10^-8The time for LPS positive reporting for the K-LPS-A system was 3 hours earlier (6.5 to 7.5 hours) than for the BD BACTEC system when GNB was inoculated (starting from 0.5 McF). This early detection of LPS and specificity for GNB was further confirmed in experiment 2, indicating that LPS reporting time was 6.5 to 8.5 hours earlier than the bacterial positive reporting time and that LPS was not detected in gram positive staphylococcus aureus medium. The advantage of the early LPS reporting time is more evident when the number of bacteria in the flask is lower, and experiment 3 demonstrates that GNB is present in a range of 1:10^-9,1：10 ^-11,1：10 ^-12(starting at 0.5 McF), LPS-positive reporting times were compared to bacterial-positive reports, respectivelyThe time is 13h, 16h or 17h earlier. This may mean that the BD BACTEC system takes 22-26 hours to detect GNB in the early stages of sepsis with low load bacterial infection, whereas the KT-TALA system (as one of the K-LPS-A systems) only takes 9 hours to detect LPS of GNB, which will help the physician to make appropriate treatment decisions at an earlier time.

TABLE 4 KT-TAL detects GNB in culture flasks faster than BD BACTEC system^*

^*Coli, klebsiella pneumoniae (test) and staphylococcus aureus (negative control) were adjusted to 0.5McF and tested at a 1:10⁸,1：10 ⁹,1：10 ¹¹Or 1:10¹²Further dilution series of ratios (v).

Two equal amounts of bacteria were injected into two BD BACTEC bottles, one placed in the BD BACTEC detector for GNB positive detection reporting time and the other in the laboratory 37 ℃ bacterial shaker for LPS sampling per hour, beginning 3 hours after incubation. The KT-TAL assay was performed to detect LPS of GNB at different time points. The difference in time of positive detection between the two assays was determined by subtracting the two reporting times.

Example 7 the novel method for detecting GNB sepsis much earlier than the conventional BD BACTEC system

To demonstrate that the method of the invention can detect sepsis with GNB infection earlier than the conventional BD back tec system, 10ml of blood from A patient suspected of sepsis were injected into two BD back tec bottles, 5ml each, one for the BD back tec system to report pathogens and the other for the K-LPS-A system assay as samples at different times after incubation. The BD BACTEC system reported positive GNB pathogen at 28.5 hours, whereas the K-LPS-A system detected positive LPS at 12 hours post-incubation, indicating that the novel method of the invention detected GNB 16.5 hours earlier than the conventional BD BACTEC system. The etiologic agent of the infection was identified as Acinetobacter baumannii (acinetobacter baumannii).

TABLE 5 dynamic testing of LPS in culture samples from patients with Acinetobacter baumannii sepsis^*

^*10ml of blood from a patient suspected of sepsis were injected into 2 BD BACTEC bottles, 5ml each, one for the BD BACTEC system reporting pathogens and the other for the LPS assay sampling at the indicated time points. At 28.5 hours, the BD BACTEC system reported pathogen positivity, and LPS was detected 12 hours after culture, with a difference of 16.5 hours. The etiologic agent of the infection was identified as Acinetobacter baumannii (acinetobacter baumannii).

EXAMPLE 8 Effect of the source of horseshoe crab amebocyte lysate on the K-LPS-A System

The limulus reagent is a sterile freeze-dried product prepared from the hemocyte lysate of marine arthropod limulus, contains prothrombin and coagulogen which can be activated by a trace amount of LPS, and is a biological reagent prepared by low-temperature freeze-drying, and the currently used limulus reagent is derived from Tachypleus Tridentatus (TAL) or Limulus polyphemus (LAL).

To determine the effect of the source of horseshoe crab amoebocyte lysate on the K-LPS-A system, in this example, A series of cultured E.coli cultures were collected at 1,2,3, 4, 5 and 9 hours and assayed using Tachypleus tridentatus reagent (TAL) and Limulus polyphemus reagent (LAL), respectively, as follows:

TABLE 6 Effect of the source of Limulus amebocyte lysate on the K-LPS-A System^#

^#Coli cultures assayed at different incubation times with Tachypleus tridentatus reagent (TAL) and Limulus polyphemus reagent (LAL), respectivelyAnd (4) liquid.

The experiment is repeated for three times under the same condition, and the coefficient of variation is less than 5 percent, which shows that the LPS detection of the Tachypleus tridentatus reagent (TAL) and the Limulus polyphemus reagent (LAL) has no difference.

Example 9 Effect of different kinetic assays on the K-LPS-A System

In this example, in order to determine the effect of the detection method on the K-LPS-A system, the international standard (CSE) diluted to A plurality of concentrations (10EU/ml, 5EU/ml, 1EU/ml, 0.5EU/ml) by the dynamic turbidity method, dynamic color method, and end-point color method was selected, and the results were as follows:

TABLE 7 Effect of assay methods on the K-LPS-A System^#

^#And respectively detecting and detecting the escherichia coli culture solution with different culture times by using a dynamic turbidity method, a dynamic color development method and an end-point color development method.

The experiment is repeated for three times under the same condition, and the coefficient of variation is less than 5 percent, which indicates that the LPS detection by the dynamic turbidity method, the dynamic color development method and the end point color development method has no difference.

Discussion of the related Art

Sepsis is a life-threatening systemic infection that requires appropriate and rapid treatment based on rapid pathogen determination. Gram-negative bacteria (GNB) are the major pathogenic pathogens with endotoxin (LPS) as its characteristic surrogate biomarker.

In the present invention, the present inventors first developed a novel rapid and sensitive in vitro detection method.

In the present invention, LPS is first increased by GNB culture, and then LPS of the culture medium is detected by A dynamic endotoxin assay system (K-LPS-A) using A limulus amebocyte lysate (e.g., TAL or LAL). The results show that at high GNB concentrations LPS can be detected 3 hours post culture, whereas BD BACTEC positive reports are at 9.5-10.5 hours, 6.5-7.5 hours earlier; at low GNB concentrations, the BD BACTEC system required 22-26 hours for GNB detection, but the K-LPS-A system required only 9 hours for GNB detection, 13-17 hours earlier.

In summary, the novel method of the present invention for detecting GNB infection is much faster than the conventional BD BACTEC system, and is particularly suitable for detecting the early stages of sepsis with a low bacterial load, which will help to make appropriate treatment decisions at an earlier time.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Claims

A method for detecting endotoxin produced by Gram Negative Bacteria (GNB) in the blood of a septic patient, the method comprising: blood culture, dynamic sampling, dilution, heating, rotation, further dilution, and measurement with a dynamic turbidity reader and limulus amoebocyte lysate, the dynamic turbidity TAL assay (KT-TALA) system comprising:

(A) techniques and devices for blood culture, sampling, dilution, heating, rotation, further dilution and testing in optimal conditions;

(B) special limulus amebocyte lysate reagent;

(C) reader for dynamic turbidity TAL assay.
The method of claim 1, wherein the subject is a human or an animal.
The method of claim 1, wherein the test target is endotoxin Lipopolysaccharide (LPS) produced in blood by gram-negative bacteria (GNB).
The method of claim 1, wherein the test agent is a amoebocyte lysate comprising TAL or LAL.
The method of claim 1, wherein the dynamic sampling is every 1-2 hours.
The method of claim 1, wherein the dynamic turbidity readings are used in the gelation process of LPS-targeted TAL.
The method of claim 1, wherein the entire process for blood culture, sampling and testing is fully automated by the device.
A method for detecting endotoxin produced by Gram Negative Bacteria (GNB) in the blood of a septic patient, the method comprising: blood culture, dynamic sampling, dilution, heating, spinning or centrifugation (spin), further dilution, and measurement with a dynamic endotoxin assay system comprising:

(A) techniques and devices for blood culture, sampling, dilution, heating, centrifugation, further dilution and testing in optimal conditions;

(B) special limulus amebocyte lysate reagent;

(C) a dynamic incubation reader for dynamic turbidity, dynamic color or end-point color.
The method of claim 8, wherein said limulus amebocyte lysate comprises a limulus reagent TAL or a limulus reagent LAL.
The method of claim 8, wherein the method comprises dynamic sampling and dynamic reading.
The method of claim 10, wherein the dynamic readout is a dynamic nephelometry based on the gelation process of LPS-targeted limulus amoebocyte lysate;
the method of claim 10, wherein the dynamic readout is a dynamic color reaction based on the color reaction of a color substrate added during gelation of the LPS-targeted limulus amoebocyte lysate;
the method of claim 10, wherein the dynamic readout is an end-point chromogenic assay based on the chromogenic reaction of LPS-targeted chromogenic substrate added during gelation of limulus amoebocyte lysate.
A detection system, comprising:

(a) techniques and devices for blood culture, sampling, dilution, heating, rotation, further dilution and testing in optimal conditions;

(b) special limulus amebocyte lysate (TAL) reagents;

(c) a reader (KT-TALA) for dynamic turbidity TAL assay; and

(d) limulus amoebocyte lysate (TAL).
A detection system, comprising:

(a) techniques and devices for blood culture, sampling, dilution, heating, rotation (or centrifugation), further dilution and testing in optimal conditions;

(b) special limulus amebocyte lysate reagent; and

(c) dynamic endotoxin assay system.
Use of a test system according to claim 14 or 15 for the preparation of a reagent or kit for the detection of endotoxin produced by gram-negative bacteria (GNB) in the blood of a patient with sepsis.
An in vitro method for detecting endotoxin produced by Gram Negative Bacteria (GNB) in blood, comprising:

(a) providing a blood sample;

(b) carrying out blood culture on the blood sample, and carrying out dynamic sampling and dynamic reading in the culture process; and

(c) determining whether Gram Negative Bacteria (GNB) in the blood produce endotoxin based on the dynamic readings.
The method of claim 17, wherein in step (b), n dynamic samples are taken, n being a positive integer from 4 to 50, preferably n being from 5 to 30, more preferably n being from 6 to 20.
The method of claim 17, wherein in step (b), in the dynamic sampling, comprising: the withdrawn culture was diluted, heated, spun down (spin), and further diluted.
The method of claim 17, wherein in step (b), in the dynamic readout, detection and readout is performed by a dynamic assay for endotoxin; and, said dynamic detection is selected from the group consisting of: dynamic turbidity methods, dynamic color development methods, endpoint color development methods, or combinations thereof.