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 technology]

[0001] Sepsis due to bloodstream infection (BSI) is a life-threatening condition with increased hospitalization rates. Appropriate and timely detection of sepsis is important, as delayed diagnosis leads to multiple organ failure and increased mortality. Polymerase chain reaction (PCR) provides a rapid method to detect bacterial pathogens in BSI-induced sepsis. One of the most important factors in PCR-based sepsis diagnosis is to reliably amplify and detect low levels of nucleic acid copies arising from pathogens in blood. This requires PCR to achieve high detection sensitivity.

[0002] Molecular diagnostics aim at the rapid detection of trace amounts of pathogens (typically bacteria) in samples such as blood. However, blood is a complex matrix, containing white 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, which contain large amounts 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 allow early detection of trace amounts of bacteria. The large amount of DNA, especially in white blood cells, dramatically increases the background in DNA-based detection methods. Also, the presence of heme from hemoglobin strongly reduces the activity of DNA polymerase. A microliter of human blood contains about 4,000-11,000 white blood cells and about 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 about 10-100,000 bacterial species in a volume of 10 ml of whole blood.

[0004] However, the high amount of human DNA due to the presence of white blood cells and other DNA fragments floating in the blood hinders 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 from lysed mammalian cells form complexes that prevent filtration of the sample. This is especially problematic for small devices. Further dilution of the already large sample volume results in unacceptably long operation steps.

[0005] Molysis™ (Molzym GmbH & Co. KG, Bremen, Germany) selectively lyses mammalian cells using chaotropic and detergent agents. This lysis step is followed by digestion with DNase, which is not affected by the chaotropic / detergent agents. Alternative approaches, such as those marketed by Roche Diagnostics (LightCycler® Septifast™), rely on PCR primer pairs specifically designed to prevent non-specific binding to and amplification of human DNA. However, there is a need in the art for pre-analytical buffers that deplete human DNA without compromising nucleic acid detection from bacterial pathogens. Summary of the Invention

[0006] The method described in this invention allows for the selective lysis of white and red blood cells in a sample, while bacteria and fungi remain intact and unlysed (dead or live).

[0007] Particular and preferred aspects of the invention are set out in the accompanying independent and dependent claims. Features from the dependent claims may be combined with features of the independent claims and with features of other dependent claims as appropriate.

[0008] One aspect of the invention relates to a method for selective lysis of eukaryotic cells, particularly animal cells, in a sample that contains or is suspected of containing a microorganism, the method comprising the steps of providing a sample comprising eukaryotic cells, particularly animal cells, that contain or are suspected of containing a microorganism, adding a non-ionic 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 added detergent and added buffer to the volume of the sample is between 2:1 and 1:10, and incubating the solution for a period of time long enough to lyse the eukaryotic cells, particularly the animal cells.

[0009] In some embodiments, the non-ionic detergent is present at a concentration in the range of 0.1-5% (w / v% or v / v%). In particular embodiments, the non-ionic detergent is present at a concentration in the range of 0.1-2% (w / v% or v / v%), particularly at a concentration in the range of 0.1-1% (w / v% or v / v%). In particular embodiments, the non-ionic detergent is present at a concentration around 1% (w / v% or v / v%).

[0010] In some embodiments, the non-ionic detergent is polidocanol. In some embodiments, the non-ionic detergent is polidocanol at a concentration of 0.1-2% (v / v%), particularly 0.1-1% (v / v%). In some embodiments, the non-ionic detergent is polidocanol at a concentration of around 1% (v / v%).

[0011] In some embodiments, a time period long enough to lyse eukaryotic cells, particularly animal cells, is between 30 seconds and 10 minutes, more particularly between 1 and 8 minutes, more particularly between 2 and 6 minutes.

[0012] In some embodiments, dissolution can be performed at a temperature between 15° C. and 30° C., more specifically between 18° C. and 27° C., more specifically between 20° C. and 25° C. In some embodiments, dissolution can be performed at or near room temperature.

[0013] In some embodiments, the animal cell is a mammalian cell.

[0014] In some aspects, 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 detergent and buffer added to the volume of the sample is 2:1 to 1:5. In other embodiments, the ratio of the volume of detergent and buffer added to the volume of the sample is 1:1 to 1:10. In yet other embodiments, the ratio of the volume of detergent and buffer added to the volume of the sample is 1:1 to 1:5.

[0017] In some embodiments, the final solution has a pH of about 8.5-9.0. In certain embodiments, the final solution has a pH of about 8.7-9.0. In certain embodiments, the final solution has a pH of about 8.8-9.0. In certain embodiments, the final solution has a pH of about 8.9-9.0.

[0018] In some embodiments, the buffer is an alkaline buffer. Here, the alkaline buffer 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 that when mixed with the sample in a ratio according to the invention, the pH of the final solution is around or slightly lower than 9.0. In certain embodiments, the alkaline buffer has a pKa of around 9.0, so that the final solution has a pH of about 9.0 or slightly lower, particularly the final solution has a pH of about 8.5-9.0, more particularly about 8.7-9.0, more particularly about 8.8-9.0, more particularly about 8.9-9.0.

[0019] In certain embodiments, the method may further comprise a "neutralization step." In some embodiments, the step comprises adding an acid or an acidic buffer to obtain a pH of about 7-9 after selective lysis according to the present disclosure. In some embodiments, the step of neutralizing the buffer comprises adding an acid or an acidic buffer to obtain a pH around 7.0.

[0020] In certain embodiments, the above methods are followed by a step of lysing the microorganism.In certain embodiments, the above methods are followed by detecting the microorganism by performing a polymerase chain reaction (PCR) assay. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] In the context of the present invention, "blood cells" relates to mammalian cells present in the blood and includes red blood cells (erythrocytes), white blood cells (leukocytes) and platelets (thrombocytes).

[0022] In the context of the present invention, "whole blood" relates to unprocessed blood containing plasma and cells, potentially treated with an anticoagulant.

[0023] "Sample" refers to an aqueous suspension containing cellular material, including bodily fluids such as lymph, cerebrospinal fluid, blood (whole blood and plasma), saliva, etc., but also includes aqueous fractions of homogenized suspensions of, for example, muscle, brain, liver, or other tissues.

[0024] "Eukaryote" in the present invention relates to any kind of eukaryote except fungi, such as animals, especially blood-containing animals, including invertebrates such as crustaceans, and vertebrates. Vertebrates include both cold-blooded animals (fish, reptiles, amphibians) and warm-blooded animals (birds and mammals). Mammals include especially primates, more particularly humans.

[0025] As used herein, "selective lysis" is obtained when the percentage of microbial cells (e.g., bacterial cells) in a sample (e.g., blood) that remain intact is significantly higher (e.g., 2, 5, 10, 20, 50, 100, 250, 500, or 1000 times higher) compared to the percentage of eukaryotic cells from the organism from which the sample is collected that remain intact.

[0026] As used herein, "microorganisms" relates to bacteria (gram-positive and gram-negative bacteria, as well as bacterial spores) and unicellular fungi such as yeasts and molds, typically as pathogens, present in the organism from which the sample was collected.

[0027] A first aspect of the present invention relates to a method for selective lysis of eukaryotic cells, particularly animal cells, in a sample that contains or is suspected to contain microorganisms such as bacteria. The aim of the method is to increase the sensitivity of the test to detect trace amounts of bacteria in a sample (i.e. less than 10000, 1000, 100 or even fewer microorganisms per ml of sample). As explained in the background of the present invention, DNA from eukaryotic cells, particularly animal cells, in a sample interferes with PCR-based detection methods, and this DNA forms aggregates with proteins and membranes, which increase the viscosity after lysis and dramatically affect 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%) remains viable or still contains bacterial DNA in the cell wall when killed by the treatment. The method according to the present invention addresses the above-mentioned problems.

[0028] The method according to the invention is particularly applicable to any type of sample in which the detection of DNA from microorganisms, in particular bacteria, is compromised by the presence of other cells containing DNA, in particular cells from the host in which the microorganism resides as a pathogen.

[0029] The method according to the invention will now be further described with respect to an embodiment for determining the presence of trace amounts of bacteria in a mammalian blood sample.

[0030] The blood sample can be stored as whole blood or a processed fraction such as plasma or platelet preparation. Typically, the method according to the invention is performed on freshly isolated whole blood. Such samples are generally treated, for example with heparin, EDTA or citrate, to avoid clotting.

[0031] Alternatively, the method is performed on fresh blood by drawing blood directly from a vein into a tube containing detergent and buffer.

[0032] Thus, fresh or preserved blood samples are supplemented with a buffer and a non-ionic detergent. The choice of buffer and its concentration are selected to compensate for the buffer capacity of the provided blood sample and to obtain a pH of around 9.0 or slightly below, in particular a pH of about 8.5-9.0. In particular, the buffer and its concentration are selected to obtain a pH of about 8.7-9.0, more particularly about 8.8-9.0, more particularly 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 required pH change. In particular, the buffer comprises a detergent and is added in a ratio of the volume of detergent and buffer added to the volume of the sample of 2:1 to 1:10. Furthermore, the ratio of the volume of detergent and buffer added to the volume of the sample can be 2:1 to 1:5, 1:1 to 1:10, or 1:1 to 1:5.

[0033] In the context of the present invention, suitable buffers include carbonate buffers, which typically have a pKa around 9.0 and have optimal buffering capacity in the above pH range.

[0034] Suitable detergents are non-ionic detergents, which have a lytic effect on eukaryotic cells, especially animal cells, on the one hand, and a solubilizing effect on DNA and proteins, on the other hand. In a particular embodiment, the non-ionic detergent is polidocanol, also called polydocanol. Polidocanol refers to a chemical compound or composition consisting of a mixture of polyethylene glycol monododecyl ethers with an average of about 9 ethylene oxide groups per molecule. Polidocanol has the molecular formula (C2H4O) as a result of the production process in which lauryl alcohol is reacted with ethylene oxide (ethoxylation). n C 12 H 26 O or HO(CH2CH2O) n (CH2) 11 It can be described by CH3, where n is about or exactly 9, i.e., the median number of ethylene glycol moieties is about 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 the detergent varies from detergent to detergent, but is typically in the range of 0.1-5%, in particular 0.1-2%, more particularly 0.1-1%. Depending on the detergent (solid or liquid), the % refers 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 the blood sample in the presence of the buffer and detergent is carried out for 10 minutes or less, 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 around room temperature.

[0037] The method according to the invention has the advantage that selective dissolution is obtained in less than 10 minutes at temperatures below 30° C. The method can therefore generally be carried out at ambient temperature without the need to heat the sample.

[0038] Optionally, after lysis, the pH of the lysed sample is brought to a neutral value (i.e., a pH around 7.0) by adding an acid or an acidic buffer in a neutralization step. It has been found that samples lysed at neutral pH can be stored for extended periods of time (up to 1, 2, 6, 12, or even 24 hours) without further lysis of the bacterial cells and without drastic changes in the fluid properties of the lysed sample.

[0039] In general, the methods according to the invention comprise a step of separating intact bacterial cells from the sample, typically carried out by centrifugation or filtration.

[0040] Although preferred embodiments, specific structures and configurations, and materials for the devices according to the present invention have been discussed 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] The present invention describes a novel buffer that depletes human cells and DNA without compromising nucleic acid detection from bacterial pathogens. The formulation of the buffer is 1000 mM Na2CO3 / 1% polidocanol, pH 9.0. In the existing literature, carbonate / non-ionic detergent buffers at pH 9.5 and above have shown selective lysis of human cells to deplete human DNA. Surprisingly, the present invention depletes human DNA at pH 9.0. Compared to carbonate / non-ionic detergents that work at pH 9.5 and above, the present invention has advantages for protocols that use RNA-based PCR detection specifically for sepsis. RNA is especially prone to degradation under basic pH conditions. Therefore, the lower pH 9.0 in the present buffer system helps to minimize the risk of RNA degradation compared to carbonate / non-ionic detergents at pH 9.5 and above. This trend is observed when comparing the performance of 1000 mM Na2CO3 / 1% polidocanol, pH 9.0 buffer with 1000 mM Na2CO3 / 1% polidocanol, pH 9.6 buffer (Tables 2 and 3). Additionally, a higher stability of carbonate in pH 9.0 buffer can be observed.

[0042] Example 1: Pre-analysis to remove human DNA Pseudomonas aeruginosa strains were grown to log phase in LB medium and an appropriate volume corresponding to 1000 CFU / mL was added to freshly collected human whole blood. Blood containing the added bacteria was combined with the selected buffer (Table 2) in a 1:1 ratio, mixed and kept at room temperature for 10 min to allow the human chromatin to be fragmented. After the incubation step, an equal volume of neutralization buffer (1 M Tris-HCL, pH 4.5) was added and the samples were centrifuged at 3220 g for 15 min. The supernatant was discarded and the cell pellet was resuspended in 1 mL of cobas® PCR medium (4.2 M guanidine HCL, 50 mM Tris-HCL, pH 7.5) containing 1% β-mercaptoethanol. Samples were transferred to a MagNa Lyser instrument (Roche Diagnostics) for cell lysis, which was performed at maximum speed for 70 s. The lysed samples were loaded onto a cobas® 6800 system and total nucleic acids were isolated in 50 μL of elution buffer using the standard cobas® assay workflow.

[0043] Example 2: Reverse transcription PCR assay The assay contains two primers and one probe against a Pseudomonas aeruginosa specific tuf target and two primers and one probe against a human β-globin target. The primer and probe sequences are shown in Table 1. The reactions were performed on a Lightcycler® 480 instrument (Roche Diagnostics) using a temperature profile consisting of a uracil-DNA N-glycosylase incubation step (50° C. for 120 s, 94° C. for 5 s), a pre-PCR step (1 cycle: 55° C. for 120 s, 60° C. for 360 s, 65° C. for 240 s), 5 cycles of 95° C. for 5 s and 55° C. for 30 s (1st measurement at the end of each cycle), followed by 45 cycles of 91° C. for 5 s and 58° C. for 25 s (2nd measurement at the end of each cycle) and ended with a cooling step (40° C. for 120 s). The real-time PCR assay mixture consisted of 15 μL of cobas® PCR Generic Master Mix containing 100–300 nM primers and 50–200 nM probe, 10 μL of 3 mM Mn 2+ , and 25 μL of total nucleic acid template. Custom Optimization Algorithm Testing Framework (ATF) software was used to calculate cycle threshold (Ct) values ​​for the PCR assay. [Table 1]

[0044] Example 3: Experimental Results [Table 2] [Table 3]

Claims

1. 1. A method for selective lysis of white and red blood cells in a mammalian blood sample containing or suspected of containing microorganisms, said method comprising: a) providing a blood sample containing blood cells which contain or are suspected of containing a microorganism; b) adding a non-ionic detergent and a carbonate buffer to the blood sample to obtain a solution having a pH of 8.5 to 9.0, the ratio of the volume of the detergent and buffer added to the volume of the blood sample being between 2:1 and 1:10; c) incubating the solution for a time period long enough to lyse the blood cells. wherein the non-ionic detergent is polidocanol and is present at a concentration in the range of 0.1-5% (w / v % or v / v %).

2. The method of claim 1 , wherein the non-ionic detergent is present at a concentration of about 1% v / v %.

3. The method according to claim 1 or 2, wherein the microorganism is a bacterium or a fungus.

4. 4. The method of claim 1, further comprising: d) neutralizing the buffer with an acid to obtain a pH around 7.

0.

5. The method of claim 4, further comprising the step of e) isolating the microorganism from the sample.

6. A method according to any one of claims 1 to 5, further comprising f) lysing the microorganism.

7. The method of any one of claims 1 to 6, further comprising g) detecting said microorganism by performing a polymerase chain reaction (PCR) assay.