Selective lysis of human blood cells

Abstract

The present invention relates to a method for the specific lysis of eukaryotic cells in a sample containing microorganisms such as bacteria and fungi by incubating the sample in a buffer around pH 9 containing a non-ionic detergent.

Description

【Background Art】 【0001】 Sepsis caused by bloodstream infection (BSI) is a life-threatening condition accompanied by an increase in hospitalization rate. Since a delay in diagnosis leads to an increase in multiple organ failure and mortality, appropriate and timely detection of sepsis is important. Polymerase chain reaction (PCR) provides a rapid method for detecting bacterial pathogens in sepsis induced by BSI. One of the most important factors in PCR-based sepsis diagnosis is to reliably amplify and detect low levels of nucleic acid copies generated from pathogens in the blood. This requires PCR to achieve high detection sensitivity. 【0002】 Molecular diagnosis aims at rapid detection of trace amounts of pathogens (typically bacteria) in samples such as blood. However, blood is a complex matrix containing blood cells (leukocytes) for the adaptive immune system, red blood cells (erythrocytes) for oxygen transport, and platelets (thrombocytes) for wound healing. This complicates the direct detection of pathogens in samples such as whole blood containing a large amount of cellular material. 【0003】 In PCR-based methods, the amount of bacteria in a fresh blood sample is theoretically high enough to detect the bacteria present in such a sample without further culturing. However, a large amount of blood is required to enable early detection of trace amounts of bacteria. In particular, the large amount of DNA in leukocytes dramatically increases the background in DNA-based detection methods. Also, the presence of heme from hemoglobin strongly reduces the activity of DNA polymerase. Microliters of human blood contain approximately 4,000 - 11,000 leukocytes and approximately 150,000 - 400,000 platelets. The concentration of DNA in blood is 30 - 60 μg / ml. It is extremely difficult to detect the presence of approximately 10 - 100,000 bacterial species in a 10 ml volume of whole blood. 【0004】 However, the presence of large amounts of human DNA, due to the presence of leukocytes and other DNA fragments suspended in the blood, interferes with the sensitivity of PCR-based detection for sepsis diagnosis. Apart from interfering with the PCR reaction itself, the amount of mammalian DNA increases the viscosity of the sample. Furthermore, proteins and membranes derived from lysed mammalian cells form complexes that hinder sample filtration. This is particularly problematic for small devices. Further dilution of already large sample volumes results in unacceptably long operating steps. 【0005】 Molysis (trademark) (Molzym GmbH & Co. KG, Bremen, Germany) selectively lyses mammalian cells using chaotropic and washing agents. This lysis step is followed by digestion with DNase, which is unaffected by the chaotropic / washing agents. Alternative approaches, such as those commercially available from Roche Diagnostics (LightCycler® Septifast®), rely on PCR primer pairs specifically designed to prevent nonspecific binding to human DNA and amplification of human DNA. However, in this art, there is a need for pre-analytical buffers that deplete human DNA without impairing nucleic acid detection from bacterial pathogens. [Overview of the project] 【0006】 The method described in the present invention allows for the selective lysis of leukocytes and erythrocytes in a sample while bacteria and fungi remain intact and undissolved (dead or alive). 【0007】 Specific preferred embodiments of the present invention are described in the appended independent and dependent claims. Features from the dependent claims can be combined with features from the independent claims and, as may be, with features from other dependent claims. 【0008】 One aspect of the present invention relates to a method for the selective lysis of eukaryotic cells, particularly animal cells, in a sample containing or suspected to contain microorganisms. The method comprises the steps of: providing a sample containing eukaryotic cells, particularly animal cells, containing or suspected to contain microorganisms; adding a nonionic detergent and a buffer to the sample to obtain a solution having a pH of about 9.0 or slightly lower, wherein the ratio of the volume of the added surfactant and the added buffer to the volume of the sample is 2:1 to 1:10; and incubating the solution for a period of time sufficient to lyse the eukaryotic cells, particularly animal cells. 【0009】 In some embodiments, the nonionic detergent is present at a concentration in the range of 0.1 to 5% (w / v% or v / v%). In certain embodiments, the nonionic detergent is present at a concentration in the range of 0.1 to 2% (w / v% or v / v%), and particularly at a concentration in the range of 0.1 to 1% (w / v% or v / v%). In certain embodiments, the nonionic detergent is present at a concentration of approximately 1% (w / v% or v / v%). 【0010】 In some embodiments, the nonionic detergent is polidocanol. In some embodiments, the nonionic detergent is polidocanol at a concentration of 0.1-2% (v / v%), particularly 0.1-1% (v / v%). In some embodiments, the nonionic detergent is polidocanol at a concentration of around 1% (v / v%). 【0011】 In some embodiments, a sufficiently long time to lyse eukaryotic cells, particularly animal cells, is 30 seconds to 10 minutes, more specifically 1 to 8 minutes, and more specifically 2 to 6 minutes. 【0012】 In some embodiments, dissolution can be carried out at a temperature of 15°C to 30°C, more specifically 18°C ​​to 27°C, and more specifically 20°C to 25°C. In some embodiments, dissolution can be carried out at or near room temperature. 【0013】 In some aspects, animal cells are mammalian cells. 【0014】 In some embodiments, the sample is a mammalian blood sample. In certain embodiments, the mammalian blood sample is a whole blood sample. In certain embodiments, the mammalian blood sample is a plasma sample or a platelet preparation. 【0015】 In some embodiments, the microorganism is a bacterium or a fungus. 【0016】 In certain embodiments, the ratio of the volume of the added detergent and buffer to the volume of the sample is 2:1 to 1:5. In other embodiments, the ratio is 1:1 to 1:10. In yet another embodiment, the ratio is 1:1 to 1:5. 【0017】 In some embodiments, the final solution has a pH of approximately 8.5–9.0. In certain embodiments, the final solution has a pH of approximately 8.7–9.0. In certain embodiments, the final solution has a pH of approximately 8.8–9.0. In certain embodiments, the final solution has a pH of approximately 8.9–9.0. 【0018】 In some embodiments, the buffer is an alkaline buffer, which may have a pKa value of about 9.0. In certain embodiments, the buffer is sodium carbonate. In certain embodiments, the buffer has sufficient buffering capacity so that when mixed with the sample in the ratio according to the present invention, the pH of the final solution is around 9.0 or slightly lower. In certain embodiments, since the alkaline buffer has a pKa of about 9.0, the final solution has a pH of about 9.0 or slightly lower, and in particular the final solution has a pH of about 8.5 to 9.0, more specifically about 8.7 to 9.0, more specifically about 8.8 to 9.0, and more specifically 8.9 to 9.0. 【0019】 In certain embodiments, the method may further include a “neutralization step.” In some embodiments, the step includes adding an acid or acidic buffer after selective dissolution according to the Disclosure to obtain a pH of about 7–9. In some embodiments, the buffer neutralization step includes adding an acid or acidic buffer to obtain a pH of around 7.0. 【0020】 In certain embodiments, the above method is followed by a step of lysing the microorganisms. In certain embodiments, the above method is followed by detection of the microorganisms by performing a polymerase chain reaction (PCR) assay. [Modes for carrying out the invention] 【0021】 In relation to the present invention, "blood cells" refers to mammalian cells present in the blood, and includes red blood cells (erythrocytes), blood cells (leukocytes), and platelets (thrombocytes). 【0022】 In connection with the present invention, "whole blood" refers to untreated blood containing plasma and cells, potentially treated with an anticoagulant. 【0023】 "Sample" refers to an aqueous suspension containing cell material, and includes bodily fluids such as lymph, cerebrospinal fluid, blood (whole blood and plasma), and saliva, but also includes aqueous fractions of homogenized suspensions of, for example, muscle, brain, liver, or other tissues. 【0024】 In this invention, "eukaryote" refers to any type of eukaryote, excluding fungi, such as animals, especially animals containing blood, and includes invertebrates such as crustaceans and vertebrates. Vertebrates include both cold-blooded animals (fish, reptiles, amphibians) and warm-blooded animals (birds and mammals). Mammals include primates in particular, and more specifically humans. 【0025】 As used in the present invention, "selective lysis" is obtained when, in a sample (e.g., blood), the percentage of microbial cells (e.g., bacterial cells) in the sample that remain intact is significantly higher (e.g., 2, 5, 10, 20, 50, 100, 250, 500, or 1000 times or more) compared to the percentage of eukaryotic cells from the organism from which the sample is collected that remain intact. 【0026】 As used in the present invention, "microorganism" typically relates to bacteria (Gram-positive and Gram-negative bacteria, and bacterial spores) as well as single-celled fungi such as yeast and mold present in the organism from which the sample is collected, typically as pathogens. 【0027】 A first aspect of the present invention relates to a method for the selective lysis of eukaryotic cells, particularly animal cells, in a sample that contains or is suspected of containing microorganisms such as bacteria. The aim of this method is to increase the sensitivity of the test in order to detect trace amounts of bacteria in the sample (i.e., 10,000, 1000, less than 100, or even fewer microorganisms per ml of sample). As explained in the background art of the present invention, DNA from eukaryotic cells, particularly animal cells, in a sample interferes with PCR-based detection methods. This DNA forms aggregates with proteins and membranes, which increases the viscosity after lysis and has a dramatic impact on the filtration of the lysed sample. To solve this problem, eukaryotic cells, particularly animal cells, are selectively lysed, so that a significant portion of the microorganisms (i.e., more than 20%, more than 40%, more than 60%, more than 80%, more than 90%, or more than 95%) either remain alive or, if killed by the treatment, still contain bacterial DNA within the cell wall. The method described in the present invention addresses the above problems. 【0028】 The method described in the present invention is particularly applicable to any type of sample in which the detection of DNA from microorganisms, particularly bacteria, is impaired by the presence of other cells containing DNA, particularly cells from the host in which the microorganisms are present as pathogens. 【0029】 Here, an embodiment for examining the presence of trace amounts of bacteria in a mammalian blood sample will be further described for the method described in the present invention. 【0030】 Blood samples can be stored as whole blood or as processed fractions such as plasma or platelet preparations. Typically, the method of the present invention is performed on freshly isolated whole blood. Such samples are generally treated with, for example, heparin, EDTA, or citrate to avoid coagulation. 【0031】 Alternatively, this method is performed on fresh blood by directly collecting blood from a vein in a tube containing a cleaning agent and buffer solution. 【0032】 Therefore, fresh or stored blood samples are supplemented with buffer and nonionic washing agents. The selection of the buffer and its concentration is chosen to compensate for the buffering capacity of the blood sample being provided and to obtain a pH around 9.0 or slightly lower, particularly a pH of about 8.5–9.0. Specifically, the buffer and its concentration are chosen to obtain a pH of about 8.7–9.0, more specifically about 8.8–9.0, and more specifically 8.9–9.0. Similarly, the buffer is sufficiently concentrated so that a buffer volume of up to 200%, 150%, 100%, 50%, 20%, or 10% of the sample volume is added to the sample to obtain the desired pH change. Specifically, the buffer contains a washing agent, and the ratio of the volume of added washing agent and buffer to the volume of sample is 2:1–1:10. Furthermore, the ratio of the volume of added washing agent and buffer to the volume of sample may be 2:1–1:5, 1:1–1:10, or 1:1–1:5. 【0033】 In relation to the present invention, a suitable buffer typically includes a carbonate buffer having a pKa of around 9.0 and optimal buffering capacity within the above pH range. 【0034】 A suitable detergent is a nonionic detergent that has a lytic effect on eukaryotic cells, particularly animal cells, and a solubilizing effect on DNA and proteins. In certain embodiments, the nonionic detergent is polidocanol, also known as polydocanol. Polydocanol relates to a chemical compound or composition consisting of a mixture of polyethylene glycol monododecyl ethers having an average of about nine ethylene oxide groups per molecule. Polydocanol is produced as a result of a method of reacting lauryl alcohol with ethylene oxide (ethoxylation), resulting in a molecular formula (C2H4O) n C 12 H 26 O or HO (CH2CH2O) n (CH2) 11 It can be represented by CH3, where n is approximately or exactly 9, i.e., the median number of ethylene glycol moieties is approximately or exactly 9. The use of polidocanol for the purification of nucleic acids is disclosed in U.S. Patent No. 8,192,958, which is incorporated herein by reference in its entirety. 【0035】 The most effective concentration of a detergent varies depending on the detergent, but is typically in the range of 0.1–5%, particularly 0.1–2%, and more specifically 0.1–1%. Depending on the detergent (solid or liquid), the percentages refer to w / v% or v / v%, respectively. In one particular embodiment, the buffer for selective lysis of human blood cells is 1000 mM sodium carbonate (Na2CO3) / 1% polidocanol, pH 9.0. 【0036】 Incubation of blood samples in the presence of buffer and washing agent is carried out for no more than 10 minutes, preferably 30 seconds to 10 minutes, more preferably about 1 to 3, 1 to 5, 1 to 8, 2 to 6, or 1 to 10 minutes, at a temperature of 10 to 30°C, more preferably near room temperature. 【0037】 The method according to the present invention has the advantage of achieving selective dissolution in less than 10 minutes at a temperature of less than 30°C. Therefore, this method can generally be carried out at ambient temperature without the need to heat the sample. 【0038】 Optionally, after dissolution, an acid or acidic buffer can be added during the neutralization step to bring the pH of the dissolved sample to a neutral value (i.e., around pH 7.0). It has been found that samples dissolved at a neutral pH can be stored for a long period (up to 1, 2, 6, 12, or even 24 hours) without further lysing bacterial cells or dramatically altering the fluid properties of the dissolved sample. 【0039】 Generally, the method according to the present invention includes a step of separating intact bacterial cells from a sample, which is typically carried out by centrifugation or filtration. 【0040】 While preferred embodiments, specific structures and configurations, and materials of the apparatus according to the present invention have been described herein, it should be understood that various changes or modifications in form and detail can be made without departing from the scope and spirit of the invention. [Examples] 【0041】 This invention describes a novel buffer that depletes human cells and DNA without impairing nucleic acid detection from bacterial pathogens. The buffer formulation is 1000 mM Na2CO3 / 1% polidocanol, pH 9.0. Existing literature has shown that carbonate / nonionic detergent buffers with pH 9.5 or higher selectively lyse human cells to deplete human DNA. Surprisingly, this invention depletes human DNA at pH 9.0. Compared to carbonate / nonionic detergents that act at pH 9.5 or higher, this invention has advantages for protocols that specifically use RNA-based PCR detection for sepsis. RNA tends to degrade, especially under basic pH conditions. Therefore, the lower pH 9.0 in this buffer system helps minimize the risk of RNA degradation compared to carbonate / nonionic detergents with pH 9.5 or higher. This trend is observed when comparing the performance of 1000 mM Na2CO3 / 1% polidocanol, pH 9.0 buffer with that of 1000 mM Na2CO3 / 1% polidocanol, pH 9.6 buffer (Tables 2 and 3). Furthermore, higher stability of the carbonate can be observed in the pH 9.0 buffer. 【0042】 Example 1: Pre-analysis for removing human DNA A strain of Pseudomonas aeruginosa was grown in LB medium until the logarithmic phase, and an appropriate volume equivalent to 1000 CFU / mL was added to freshly collected human whole blood. The blood containing the added bacteria was mixed with a selected buffer (Table 2) in a 1:1 ratio and incubated at room temperature for 10 minutes to fragment the human chromatin. After incubation, an equal volume of neutralizing buffer (1M Tris-HCl, pH 4.5) was added, and the sample was centrifuged at 3220g for 15 minutes. The supernatant was discarded, and the cell pellet was resuspended in 1 mL of cobas® PCR medium (4.2M guanidine HCl, 50 mM Tris-HCl, pH 7.5) containing 1% β-mercaptoethanol. The sample was transferred to a MagNa Lyser instrument (Roche Diagnostics) for cell lysis and performed at maximum speed for 70 seconds. The lysed sample was loaded into a cobas® 6800 system, and the total nucleic acid was isolated in 50 μL of elution buffer using a standard cobas® assay workflow. 【0043】 Example 2: Reverse Transcription PCR Assay The assay includes two primers and one probe for a Pseudomonas aeruginosa-specific tuf target, and two primers and one probe for a human β-globin target. The sequences of the primers and probes are shown in Table 1. The reaction was performed using a Lightcycler® 480 instrument (Roche Diagnostics) with a temperature profile consisting of a uracil-DNA N-glycosylase incubation step (120 seconds at 50°C, 5 seconds at 94°C), a pre-PCR step (1 cycle: 120 seconds at 55°C, 360 seconds at 60°C, 240 seconds at 65°C), 5 cycles of 5 seconds at 95°C and 30 seconds at 55°C (first measurement at the end of each cycle), followed by 45 cycles of 5 seconds at 91°C and 25 seconds at 58°C (second measurement at the end of each cycle), and was terminated by a cooling step (120 seconds at 40°C). The real-time PCR assay mixture consists of 15 μL of cobas® PCR generic master mix containing 100-300 nM primers and 50-200 nM probes, and 10 μL of 3 mM Mn 2+ The sample consisted of a total nucleic acid template and 25 μL of a sample. The cycle threshold (Ct) value of the PCR assay was calculated using Custom Optimized Algorithm Test Framework (ATF) software. [Table 1] 【0044】 Example 3: Experimental Results [Table 2] [Table 3]

Claims

[Claim 1] A method for selective lysis of leukocytes and erythrocytes in a mammalian blood sample containing or suspected to contain microorganisms, wherein the method is a) A step of providing a blood sample containing blood cells that contain or are suspected to contain microorganisms. b) A step of adding a nonionic detergent and a carbonate buffer to the blood sample to obtain a solution having a pH of 8.5 to 9.0, wherein the ratio of the volume of the added detergent and buffer to the volume of the blood sample is 2:1 to 1:

10. c) Incubating the solution for a sufficiently long time to dissolve the blood cells. The method comprising, wherein the nonionic detergent is polidocanol and is present in a concentration within the range of 0.1 to 5% (w / v% or v / v%). [Claim 2] The method according to claim 1, wherein the nonionic detergent is present at a concentration of approximately 1% v / v%. [Claim 3] The method according to claim 1 or 2, wherein the microorganism is a bacterium or a fungus. [Claim 4] d) The method according to any one of claims 1 to 3, further comprising neutralizing the buffer with an acid to obtain a pH of approximately 7.

0. [Claim 5] e) The method according to claim 4, further comprising the step of separating the microorganism from the sample. [Claim 6] f) The method according to any one of claims 1 to 5, further comprising lysing the microorganism. [Claim 7] g) The method according to any one of claims 1 to 6, further comprising detecting the microorganism by performing a polymerase chain reaction (PCR) assay.

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