Improved method for preparing clinical samples for nucleic acid amplification

Abstract

The present disclosure is a method of preparing a clinical sample comprising a host cell, a microorganism, a protein, and a polyanionic polymer nucleic acid amplification inhibitor.

Description

Technical Field

[0001] The present invention relates generally to the field of clinical sample preparation, and in particular to reducing the negative effects on nucleic acid amplification detection caused by certain additives to clinical samples. Background Art

[0002] Hospital-acquired pathogenic microbial infections require rapid identification of the presence of one or more microorganisms to ensure appropriate antimicrobial therapy. On any given day, approximately 1 in 31 hospital patients suffers from at least one hospital-acquired pathogenic microbial infection. In 2015, there were an estimated 687,000 hospital-acquired pathogenic microbial infections, resulting in 72,000 deaths. Administration of broad-spectrum, non-targeted antimicrobial agents to patients with these hospital-acquired microbial infections can lead to antimicrobial resistance.

[0003] Preservation of microorganisms in clinical samples obtained from subjects with pathogenic microbial infections is crucial for subsequent clinical diagnostic testing. For this reason, polyanionic detergents are effective in inhibiting bactericidal processes such as the complement pathway in whole blood and are often used in blood culture bottles to promote the survival and growth of bacteria in samples (Edberg et al., 1976, "Use of sodium polyanethol sulfonate to selectively inhibit aminoglycoside and polymyxin antibiotics in a rapid blood level antibiotic assay," Antimicro. Agents Chemother. 9(3): 414-417; Kocka et al., 1972, "Action of sulfated polyanions used in blood culture on lysozyme, complement, and antibiotics," Ann. Clinc. Lab. Sci. 2(6): 470-473; Traub and Kleber, 1977, "Inactivation of classical and alternative pathway-activated bactericidal activity of human serum by sodium polyanethol sulfonate," J. Clin. Microbiol. 5(3): 278-284).

[0004] Next-generation molecular diagnostics (eg, nucleic acid amplification) for identification of bacteria from clinical blood samples offer a rapid alternative to traditional bacterial culture. However, polyanionic compounds commonly added to clinical samples, such as sodium polyanethol sulfonate (SPS), which inhibits bactericidal processes, and heparin, which inhibits coagulation, have inhibitory effects on nucleic acid amplification techniques (Fredericks and Relman, 1998, "Improved amplification of microbial DNA from blood cultures by removal of the PCR inhibitor sodium polyanethol sulfonate," J. Clin. Microbiol., 36(10): 2810-2816; Qian et al., 2001, "Direct identification of bacteria from positive blood cultures by amplification and sequencing of the 16S rRNA gene: evaluation of BACTEC 9240 instrument true-positive and false-positive results," J. Clin. Microbiol., 39(10: 3578-3585; and Regan et al., 2012, "A sample extraction method for faster, more sensitive PCR-based detection of pathogens in blood culture," J. Mol. Diagn. 14(2): 120-129). In addition to such additives, blood components such as hemoglobin, lactoferrin, heme and immunoglobulins can also interfere with the nucleic acid amplification process. However, the chemical similarity between nucleic acids and polyanionic polymers such as SPS leads to co-purification of the two polymers in a variety of nucleic acid purification techniques, resulting in inhibition of downstream molecular applications (Regan et al., 2012, supra).

[0005] Improved methods are needed to reduce the negative effects of certain common additives and naturally occurring compounds (e.g., SPS, heparin) on nucleic acid amplification-based diagnostics during the processing of clinical samples. Current clinical sample processing methods for downstream nucleic acid amplification currently dilute the final sample from 10× to 1000× to alleviate SPS inhibition. However, these methods also significantly reduce nucleic acid detection sensitivity (Pennington, 2014, "Dealing with amplification inhibitors: reagent choice matters," Promega Corporation). SUMMARY OF THE INVENTION

[0007] The present invention relies in part on such discovery: adding proteases to clinical samples reduces contamination of certain nucleic acid amplification inhibitors in processed clinical samples without otherwise significantly compromising the integrity or quality of the samples, and thereby improves nucleic acid amplification of the samples. Improved nucleic acid amplification also allows for improved detection of rare nucleic acids in mixed samples, such as detection of microbial DNA in clinical samples derived from human patients.

[0008] Therefore, in one aspect, the present disclosure provides a method for preparing a clinical sample from a subject, the clinical sample comprising cells from the subject, microorganisms, and a polyanionic polymer nucleic acid amplification inhibitor bound to at least one protein in the sample. In some embodiments, the method comprises: (a) obtaining a clinical sample comprising cells from the subject, microorganisms, plasma proteins, and a polyanionic polymer nucleic acid amplification inhibitor from the subject, (b) separating the clinical sample into a first fraction comprising cells from the subject and a second fraction comprising microorganisms and a polyanionic polymer nucleic acid amplification inhibitor bound to a protein, (c) adding a protease that degrades the protein to the second fraction, and (d) separating the second fraction into a third fraction comprising the microorganism and a fourth fraction comprising the polyanionic polymer nucleic acid amplification inhibitor.

[0009] In another aspect, the present disclosure provides a method for preparing a clinical sample from a subject, the clinical sample comprising a microorganism and a polyanionic polymer nucleic acid amplification inhibitor that binds to at least one protein in the sample, the method comprising the steps of: (a) obtaining a clinical sample comprising a microorganism and a polyanionic polymer nucleic acid amplification inhibitor from a subject, (b) adding a protease that degrades the protein to the sample, (c) separating the clinical sample into a first fraction comprising cells of the subject and a second fraction comprising the microorganism and the polyanionic polymer nucleic acid amplification inhibitor that binds to the protein, (d) removing the second fraction from the first fraction, and (e) separating the second fraction into a third fraction comprising the microorganism and a fourth fraction comprising the polyanionic polymer nucleic acid amplification inhibitor.

[0010] In another aspect, the present disclosure provides a method for preparing a clinical sample from a subject, the clinical sample comprising microorganisms, cells, and at least one polyanionic polymer nucleic acid amplification inhibitor bound to at least one protein in the sample, the method comprising the following steps: (a) obtaining a clinical sample comprising microorganisms, proteins, and the polyanionic polymer nucleic acid amplification inhibitor from the subject, (b) adding a protease that degrades the protein to the clinical sample, (c) separating the clinical sample into a first fraction comprising the cells of the subject and a second fraction comprising the microorganisms and the polyanionic polymer nucleic acid amplification inhibitor bound to the protein, (d) removing the second fraction from the first fraction, and (e) separating the second fraction into: a third fraction comprising the microorganisms and a fourth fraction comprising the polyanionic polymer nucleic acid amplification inhibitor.

[0011] In another aspect, the present disclosure provides a method for preparing a clinical sample from a subject, the clinical sample comprising a microorganism, cells, and at least one polyanionic polymer nucleic acid amplification inhibitor bound to at least one protein in the sample, the method comprising the following steps: (a) obtaining a clinical sample comprising a microorganism, a protein, and a polyanionic polymer nucleic acid amplification inhibitor from a subject, (b) separating the clinical sample into: a first fraction comprising the cells of the subject and a second fraction comprising the microorganism and the polyanionic polymer nucleic acid amplification inhibitor bound to the protein, (c) removing the second fraction from the first fraction, (d) after step (b) or step (c), adding a protease that degrades the protein in the second fraction to the second fraction, and (e) separating the second fraction into a third fraction comprising the microorganism and a fourth fraction comprising the polyanionic polymer nucleic acid amplification inhibitor.

[0012] In another aspect, the present disclosure provides a method for preparing a clinical sample from a subject, the clinical sample comprising a microorganism and at least one polyanionic polymer nucleic acid amplification inhibitor that binds to at least one protein in the sample, the method comprising the steps of: (a) obtaining a clinical sample comprising a microorganism, a protein, and a polyanionic polymer nucleic acid amplification inhibitor from the subject, (b) selectively lysing the microorganism, (c) adding a protease that degrades the protein to the clinical sample, and (d) separating the clinical sample into a first fraction comprising cells of the clinical sample and a second fraction comprising nucleic acids from the microorganism.

[0013] In some embodiments of each of the aforementioned aspects, the method further comprises the step of amplifying the microbial nucleic acid.

[0014] In some embodiments of each of the aforementioned aspects, the separation is performed by centrifugation. In some embodiments of each of the aforementioned aspects, the separation is performed by microfluidics. In some embodiments of each of the aforementioned aspects, the separation is performed by immunoprecipitation. In some embodiments of each of the aforementioned aspects, the separation is performed by filtration.

[0015] In some embodiments of each of the aforementioned aspects, the obtaining step utilizes a vacuum liquid container. In some embodiments of each of the aforementioned aspects, the obtaining step utilizes a blood culture bottle. In some embodiments of each of the aforementioned aspects, the obtaining step is receiving the sample from a health care provider.

[0016] In some embodiments of each of the aforementioned aspects, the protease is selected from the group consisting of proteinase K, factor Xa, Arg-C protease, Asp-N endopeptidase, Asp-N endopeptidase + N-terminal Glu, 3-bromo-3-methyl-2-(2-nitrophenylthio)-3H-indole (BNPS-skatole), caspase 1, high specificity chymotrypsin, clostripain, cyanogen bromide (CNBr), enterokinase, granzyme B, formic acid, glutamyl endopeptidase, hydroxylamine, iodosylbenzoic acid, LysC, LysN, 2-nitro-5-thiocyanatobenzoic acid (NTCB), pepsin, proline endopeptidase, staphylococcal peptidase I, tobacco etch virus protease, thermolysin, thrombin and trypsin. In some embodiments of each of the aforementioned aspects, the amount of the added protease is 5 units (U) to 5000U. In some embodiments of each of the aforementioned aspects, the protease and the supernatant are incubated at 20°C to 55°C for 10 minutes to 120 minutes.

[0017] In some embodiments of each of the aforementioned aspects, the nucleic acid amplification is isothermal strand displacement amplification, PCR, qPCR, RT-PCR, degenerate oligonucleotide PCR, or primer extension preamplification.

[0018] In some embodiments of each of the aforementioned aspects, nucleic acid amplification is performed by phi29 DNA polymerase, Bst DNA polymerase large fragment, or DNA polymerase I Klenow fragment.

[0019] In some embodiments of each of the aforementioned aspects, the addition of the protease enhances the amplification of the microbial nucleic acid by 5-fold to 100-fold.

[0020] In some embodiments of each of the aforementioned aspects, the method allows detection of microbial nucleic acids present in the original clinical sample at 10 to 100,000 colony forming units / mL (cfu / mL), 10 to 10,000 cfu / mL, or 10 to 100 cfu / mL. In some embodiments of each of the aforementioned aspects, the method allows detection of microbial nucleic acids present in the clinical sample at 2.5 to 100,000 cfu / mL, 2.5 to 10,000 cfu / mL, 2.5 to 1,000 cfu / mL, or 2.5 to 100 cfu / mL. In some embodiments of each of the aforementioned aspects, the method allows detection of microbial nucleic acids present in the clinical sample at 1 to 100,000 cfu / mL, 1 to 1,000 cfu / mL, 1 to 100 cfu / mL, or 1 to 10 cfu / mL. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This illustrates that SPS forms a large complex with albumin, the most abundant protein in blood. The BSA-SPS complex can be visualized in an agarose gel stained with SYBR-Safe (ThermoFisher, Waltham, MA). Lane 1 is a 1 kb DNA ladder (Invitrogen, Carlsbad, CA); lane 2 is 0.25% BSA (Sigma-Aldrich, St. Louis, MO); lane 3 is 0.05% SPS; lane 4 is 0.25% SPS + 0.05% BSA, forming a complex.

[0023] Figure 2 It was demonstrated that SPS-serum protein complex formation was minimized by proteinase K treatment. Lane 1 is a 1 kb DNA ladder; lanes 2 to 3 are samples not treated with proteinase K; lanes 4 to 5 are samples treated with 4 units (U) / mL (8 U total) of proteinase K (New England BioLabs, Ipswich, MA); lanes 6 to 7 are samples treated with 12 U / mL (24 U total) of proteinase K; and lanes 8 to 9 are samples treated with 40 U / mL (80 U total) of proteinase K.

[0024] Figure 3Shown is the degradation of serum proteins in the supernatant after proteinase K treatment. Lane 1 is a protein ladder (SeeBlue Plus2 prestained protein ladder, ThermoFisher, Waltham, MA); Lane 2 is the protein present in the supernatant after centrifugation of a clinical blood sample diluted with DPBS; Lane 3 is the protein present in the supernatant after centrifugation of a clinical blood sample diluted with DPBS and treated with proteinase K at 37°C for 10 minutes.

[0025] Figure 4 It was shown that the viability of Escherichia coli from clinical blood samples collected in SPS vacuum liquid containers treated with proteinase K was not reduced compared to untreated samples.

[0026] Figures 5A to 5B It is shown that the addition of proteinase K treatment to clinical samples containing SPS resulted in an increase in the total DNA yield after DNA amplification after 1.5, 2.5 and 4 hours of amplification time ( Figure 5A ) and bacteria-specific DNA yield ( Figure 5B ) significantly improved.

[0027] Figure 6 It was shown that the addition of proteinase K treatment to samples prepared from blood collected in lithium heparin vacutainers resulted in increased DNA yield following DNA amplification. DETAILED DESCRIPTION OF THE INVENTION

[0029] Clinical samples obtained from patients are crucial in diagnosing a variety of diseases and conditions. The ability to quickly and efficiently prepare clinical samples for diagnostic testing is not only critical to maintaining patient health, but is also more cost-effective and efficient for both the doctor's office and the laboratory that prepares the clinical samples and performs the diagnostic testing.

[0030] Clinical blood samples are routinely obtained from patients suffering from or suspected of having pathogenic microbial infections. The use of additives such as sodium polyanethol sulfonate (SPS) and heparin in blood samples maintains bacterial viability, the ability to recover and quickly identify one or more pathogenic microorganisms, and enables doctors to more reliably predict which antimicrobial agent(s) will be most effective in treating infections (Belding and Kelbanoff, 1972, "Effect of Polyanetholsulfonate on Antimicrobial Systems in Blood," Appl. Microbiol. 24 (5): 691-698). Identification of one or more pathogenic microorganisms requires amplification of microbial nucleic acids relative to the nucleic acid of the object. Since the in vitro amplification of nucleic acids is inhibited by the presence of nucleic acid amplification inhibitors in clinical samples, the current practice is to (1) avoid samples containing SPS all together, which reduces the efficiency of microbial recovery, or (2) culture microorganisms in vivo, which has a significantly longer timeline (see, e.g., US 2018 / 0142231 A1).

[0031] The present disclosure is based in part on the discovery that polyanionic polymer nucleic acid amplification inhibitors can be removed from clinical samples during processing by adding proteases. These nucleic acid amplification inhibitors are non-protein compounds (e.g., polymers and organic compounds such as glycosaminoglycans) that, when present in clinical samples, block subsequent diagnostic tests such as microbial identification.

[0032] definition

[0033] Unless defined otherwise below, all scientific and technical terms used herein are intended to have the same meaning as those of ordinary skill in the art to which they belong. In the event of any conflict, this specification (including definitions) shall prevail. Reference to the technology employed herein is intended to refer to the technology generally understood in the art, including changes in those technologies or replacements of technologies that are equivalent or developed later, which will be apparent to those skilled in the art. In order to more clearly and concisely describe the subject matter of the present invention, the following definitions are provided for certain terms used in the specification and the appended claims.

[0034] As used herein, "preparation" refers to steps performed to modify a clinical sample prior to a diagnostic test. In some embodiments, preparation includes collecting, separating, removing a portion of a sample, and / or adding additional components, such as a protease, to the sample.

[0035] As used herein, "separation" refers to the physical partitioning of a clinical sample into at least two distinct fractions.

[0036] As used herein, "host cell" refers to a cell derived from a host or subject, which is different from a commensal cell (e.g., a microbial cell that is part of a host microorganism), an infected cell (e.g., a pathogenic microorganism), or a contaminating cell (e.g., accidentally introduced during sample collection or preparation). The term "host" may refer to a patient or medical subject, which may be a human or non-human mammal.

[0037] As used herein, "obtaining" refers to extracting a clinical sample from a subject or receiving a clinical sample that has been extracted from a subject. Extracting a clinical sample from a subject can be accomplished by any route known in the art, including, but not limited to, intravenous, intraarterial, intraperitoneal, intracranial, intraspinal, intramuscular, intraurethral, ​​intratracheal, and intranasal. In various embodiments, "extracting" can be accomplished using a syringe, biopsy needle, suction tube, swab, or similar device, or by urination, defecation, expectoration, wound drainage, or lavage.

[0038] "Fraction" used herein refers to different subsamples physically separated from larger and more complex samples. In some embodiments, fractions include host cells, microorganisms, soluble proteins and / or polyanionic polymer nucleic acid amplification inhibitors. It is not necessary to be complete or perfect for sample separation into two or more fractions. For example, centrifuging a whole blood sample into cell and plasma fractions will generally result in a cell fraction or precipitate still comprising some plasma, and a plasma fraction or supernatant still comprising some cells. Degree or purity separation is within the control of those skilled in the art, but perfect separation is neither practical nor necessary.

[0039] As used herein, "centrifugation" refers to the separation of a sample into components based on density, wherein denser components (e.g., blood cells, large particles, etc.) will form a pellet and less dense components (e.g., liquids, small peptides, etc.) will form a supernatant.

[0040] As used herein, "microfluidics" refers to separating a clinical sample into fractions by flowing the clinical sample through microchannels. Microfluidic devices can separate different types of cells based on cell size, isotachoporetic, optical, magnetic, and / or other biophysical properties that distinguish cell types.

[0041] As used herein, "plasma tube" refers to a container for separating plasma. Plasma tubes can be used to separate specific cell types (eg, red blood cells, white blood cells, platelets, microorganisms) into multiple fractions.

[0042] As used herein, "immunoprecipitation" refers to the fixation of specific cell types (e.g., erythrocytes, leukocytes, platelets) or proteins using immunoglobulins or synthetic molecules derived from or based on immunoglobulins (e.g., Fab fragments, F(ab')2 fragments, Fab' fragments, Fd fragments, Fv fragments and scFv fragments, as well as corresponding heavy chain-only antibodies (hcAb) and corresponding fragments from cartilaginous fish and camelid species). Immunoglobulin (or immunoglobulin-like) molecules can be combined with any macromolecule (e.g., protein, glycoprotein) that distinguishes cells or proteins to be separated.

[0043] As used herein, "filtration" refers to the removal of structures from a clinical sample. The structures can be cells, cell fragments, organelles, microorganisms, large proteins or protein complexes (eg, polyanionic polymer nucleic acid amplification inhibitors and proteins).

[0044] As used herein, "removing" a portion of a sample refers to extracting a majority of a portion of a sample, wherein the majority refers to at least 50% of the sample, but may include greater than 60%, greater than 70%, greater than 80%, greater than 90%, greater than 95%, or greater than 99%. Removal can be performed by any method known in the art, including but not limited to pipetting, decantation, and filtration. In some embodiments, removal of a portion is performed by removing a liquid phase (e.g., supernatant).

[0045] As used herein, "clinical sample" refers to a diagnostic sample extracted from a subject, derived from a subject, or otherwise obtained from a subject and processed for diagnostic testing. Clinical samples used herein include blood, sputum, urine, mucus, saliva, tissue abscesses, wound drainage, lymph, lavage fluid, feces, cerebrospinal fluid, or any fluid aspirate or tissue extract of human and / or other mammalian origin (EEHealth, "Microbiology Collection", 2018).

[0046] As used herein, a "polyanionic polymer" is a non-proteinaceous substance having a molecular structure composed of a plurality of similar chemical units bonded together and containing a plurality of negatively charged groups, and having an average molecular weight of at least 500 Daltons (500 Da).

[0047] As used herein, "sodium polyanethole sulfonate" (or "SPS") is a polyanionic polymer commonly used as an anticoagulant in vacuum liquid containers (including blood collection containers). SPS helps preserve bacteria present in clinical blood samples by inhibiting the complement cascade to kill bacteria through innate and humoral immunity. Commercial SPS preparations typically have an average molecular weight of 9 to 11 kDa (e.g., catalog number 444464, Sigma-Aldrich, St. Louis, MO).

[0048] "Heparin" used herein is a glycosaminoglycan comprising an unbranched polysaccharide composed of repeating disaccharide units. Heparin is usually obtained from mammalian mucosal tissues (e.g., bovine lungs or pig intestines), but can also be obtained from other tissues and other mammals. The average molecular weight of commercial heparin preparations is usually 12 to 15 kDa, but natural heparin can be 3 to 30 kDa. The term "heparin" used herein is intended to include any "heparin salt". "Heparin salt" is a compound in which heparin is combined with a positively charged molecule (e.g., sodium heparin, lithium heparin or ammonium heparin).

[0049] "Nucleic acid amplification inhibitor" used herein refers to a compound that is added to a non-natural level to a clinical sample and inhibits DNA polymerase activity when present during a nucleic acid amplification reaction. In some embodiments, the nucleic acid amplification inhibitor is introduced into the clinical sample during sample collection. In some embodiments, the nucleic acid amplification inhibitor is introduced into the clinical sample during sample processing. The nucleic acid amplification inhibitor can be natural (by natural production) or synthetic (not by natural production). The nucleic acid amplification inhibitor can be a non-natural non-protein, such as SPS. The nucleic acid amplification inhibitor can be a natural non-protein, such as heparin.

[0050] As used herein, "soluble" refers to the ability of a protein to remain in solution without precipitating and / or forming insoluble aggregates.

[0051] As used herein, "serum" refers to the blood fraction that contains plasma, all electrolytes, antibodies, antigens, hormones, and any exogenous substances such as drugs and microorganisms. Serum is not intended to contain blood cells or platelets, although residual cells may be present after serum is prepared.

[0052] As used herein, "serum protein" refers to any protein in plasma that is not involved in coagulation (e.g., fibrinogen). Serum proteins include, but are not limited to, albumins such as human serum albumin, and globulins such as alpha globulin, beta globulin, and gamma globulin, as well as transferrin, lactoferrin, globin, and antibodies.

[0053] As used herein, a "microorganism" is a microorganism that requires a microscope to visualize it. Some examples of microorganisms include bacteria, archaea, fungi, protists, viruses, and microscopic animals.

[0054] As used herein, "pathogenic microorganisms" are microorganisms that cause disease. The most common pathogenic microorganisms in humans are viruses and bacteria. Less common pathogenic microorganisms include fungi, protozoa, and helminths.

[0055] As used herein, "protease" refers to an enzyme that breaks down proteins and peptides by hydrolyzing peptide bonds.

[0056] As used herein, a "container" is a vessel used to collect a clinical sample from a subject. In some embodiments, the subject has or is suspected of having a microbial infection. In some embodiments of the present disclosure, the container is a vacuum liquid container (e.g., BD Biosciences, Franklin Lakes, NJ). In some embodiments, the container is a blood culture bottle.

[0057] As used herein, a "vacuum liquid container" is a sterile glass or plastic sample collection tube containing a rubber stopper that maintains a vacuum seal within the tube. This vacuum seal helps collect a predetermined volume of liquid. In some embodiments of the present disclosure, the vacuum liquid container contains sodium polyanethol sulfonate (SPS). In some embodiments, the vacuum liquid container contains heparin.

[0058] As used herein, a "blood culture bottle" is a sterile glass or plastic blood collection container that is used to collect and culture clinical samples from patients suffering from or suspected of having a bacterial infection. Blood culture bottles facilitate the growth of microorganisms in clinical samples for downstream identification of the microbial species present. In some embodiments of the present disclosure, the blood culture bottle comprises sodium polyanethol sulfonate (SPS). In some embodiments, the blood culture bottle comprises heparin.

[0059] " Nucleic acid amplification " used herein refers to the process for amplifying or propagating one or more specific nucleic acid molecule populations from clinical blood samples. In some embodiments, the population is from a pathogen, such as a virus or bacteria. In some embodiments, the population is from a host of a clinical blood sample. The amount of nucleic acid molecules in the population can be amplified in any of several ways including polymerase chain reaction (PCR), strand displacement amplification (SDA) or transcription-mediated amplification (TMA). In some embodiments, nucleic acid amplification is isothermal strand displacement amplification, PCR, qPCR, RT-PCR, degenerate oligonucleotide PCR or primer extension pre-amplification.

[0060] "Polymerase" as used herein refers to an enzyme that utilizes an existing nucleic acid molecule (e.g., DNA or RNA) as a template to catalyze the formation of a new nucleic acid molecule (e.g., DNA or RNA) to produce a complementary (or substantially complementary) polynucleotide sequence in the new molecule. In some embodiments, the polymerase is a strand displacement polymerase that catalyzes the displacement of one strand of a DNA double helix before DNA or RNA synthesis occurs. In some embodiments, the strand displacement polymerase is phi29 DNA polymerase (e.g., catalog number M0269, New England BioLabs, Ipswich, MA), Bst DNA polymerase large fragment (e.g., catalog number M0275, New England BioLabs, Ipswich, MA), or DNA polymerase I Klenow fragment (e.g., catalog number M0210, New England BioLabs, Ipswich, MA).

[0061] Principle of the Invention

[0062] The present invention relies in part on the discovery that polyanionic polymer nucleic acid amplification inhibitors (e.g., SPS, heparin, hyaluronate, dermatan sulfate polyanions, and chondroitin D-glucuronide anions) can be removed from clinical samples by the addition of proteases. This is because polyanionic polymer nucleic acid amplification inhibitors bind to naturally occurring proteins present in clinical samples during nucleic acid amplification. Thus, the present disclosure provides methods for removing polyanionic polymer nucleic acid amplification inhibitors from clinical samples obtained from subjects infected with pathogenic microorganisms.

[0063] Clinical samples

[0064] The methods of the present invention can be implemented on various types of clinical samples obtained from various types of subjects. In some embodiments, the clinical sample is blood, lymph, urine, feces, sputum, mucus, saliva, tissue abscess, wound drainage, lavage fluid, cerebrospinal fluid, or any fluid aspirate or tissue extract of human or other mammalian origin.

[0065] Sample container

[0066] A variety of different sample containers are used in the art, which are applicable to different types of clinical samples. In many cases, the most commonly used and commercially available sample containers are preloaded with preservatives and / or anticoagulants (e.g., SPS, heparin, hyaluronate, dermatan sulfate polyanion, EDTA, and chondroitin D-glucuronic acid anion), which may have the unexpected effect of acting as a nucleic acid amplification inhibitor during the different nucleic acids present in genetic identification or analysis samples. For example, commonly used blood collection containers, blood culture bottles, plasma tubes, and blood culture media may contain heparin (e.g., catalog numbers 364960, 366667, 367871, 367878, 367884, 367886, 367960, 367961, 367962, and 367964 Collection tubes, BD Biosciences, Franklin Lakes, NJ), SPS (e.g., catalog number 364960 Collection tubes, catalog numbers 442022 and 442023, BACTEC TM PLUS media, BD Biosciences, Franklin Lakes, NJ) or potassium EDTA (e.g., Catalog Nos. 367842, 367899, and 368589, PlusPlastic K2EDTA Tubes, BD Biosciences, Franklin Lakes, NJ).

[0067] In the method of the present invention, before implementing the method of the present invention, the samples are usually removed from the sample container in which they are collected and transferred to a new container (e.g., a centrifuge tube or a microfluidic device). However, in some cases, depending on the nature of the sample collection container, one or more steps may be performed in the container in which the sample is collected.

[0068] Proteins that bind to polyanionic polymer nucleic acid amplification inhibitors

[0069] As described above, the present invention relies in part on such discovery: adding protease to clinical samples reduces the contamination of certain nucleic acid amplification inhibitors in treated clinical samples without otherwise significantly compromising the integrity or quality of the sample, and thereby improves nucleic acid amplification of nucleic acids in the sample. Without being bound by any particular theory, it is believed that certain proteins in clinical samples may combine with certain nucleic acid amplification inhibitors to form a complex so that the nucleic acid amplification inhibitor and the protein remain in the same fraction during sample processing. This may have an adverse effect if it prevents the nucleic acid amplification inhibitor from being separated from the nucleic acid population to be amplified.

[0070] In some embodiments, the protein bound to the nucleic acid amplification inhibitor is soluble in physiological fluids and / or solutions for treatment (e.g., supernatants from centrifugation or solutions for microfluidic devices or filtration). For example, in some embodiments, the protein can be a soluble serum protein, such as serum albumin. In some embodiments, the protein can be any other soluble protein that forms a complex with a polyanionic polymer nucleic acid amplification inhibitor. Some non-limiting examples of proteins in clinical samples include human serum albumin, amylase, immunoglobulin, lactoferrin, calprotectin, myeloperoxidase, azurocidin, lactotransferrin, cathelicidin, lactase, myoglobin, hemoglobin, and transferrin.

[0071] Pathogenic microorganisms.

[0072] In some aspects, the disclosure provides methods of amplifying microbial DNA from a clinical sample after treating the clinical sample with a protease.

[0073] In some embodiments, microorganism is bacterium. Although bacterium is usually present in healthy mammals, the balance between different types of bacteria in the host, the balance between bacteria and the host, or the presence of pathogenic bacteria in the host can cause infection. Some non-limiting examples of pathogenic bacteria include: Staphylococcus aureus (S.aureus), Staphylococcus epidermidis (S.epidermidis), Streptococcus agalactiae (S.agalactiae), Enterococcus faecalis (E.faecalis), Enterococcus faecium (E.faecium), Escherichia coli (E.coli), Klebsiella pneumoniae (K.pneumoniae).

[0074] Staphylococcus aureus is a bacterium that is commonly present in the human body and is often found in the nose, respiratory tract and on the skin. Although S. aureus is not always pathogenic, it is a common cause of skin infections, including abscesses, respiratory tract infections and food poisoning. The common method of treating S. aureus infections is to use antibiotics, but the emergence of antibiotic-resistant strains of S. aureus, such as methicillin-resistant S. aureus (MRSA) and vancomycin-resistant S. aureus (VRSA), has become a clinical health challenge worldwide.

[0075] Staphylococcus epidermidis is a bacterium that is normally present in the human body, most often found on the skin. Although S. epidermidis is not usually pathogenic, subjects with compromised immune systems are at risk for developing S. epidermidis infections, and S. epidermidis poses a particular threat to subjects with surgical implants because it can grow on plastic surfaces and spread to the body. S. epidermidis strains are often resistant to antibiotics, including rifamycins, fluoroquinolones, gentamicin, tetracycline, clindamycin, and sulfonamides.

[0076] Streptococcus agalactiae is a bacterium that is not usually pathogenic and is found in the gastrointestinal and urogenital tracts of up to 30% of humans. Pathogenic infections caused by S. agalactiae are a concern for neonates and immunocompromised individuals. S. agalactiae infections in adults can be life-threatening and include bacteremia, soft tissue infections, osteomyelitis, endocarditis, and meningitis. S. agalactiae is increasingly resistant to clindamycin and erythromycin.

[0077] E. faecalis is a bacterium that inhabits the gastrointestinal tract of humans and other mammals. However, E. faecalis can cause endocarditis, sepsis, urinary tract infections, and meningitis. E. faecalis infections can be life-threatening, especially when E. faecalis is resistant to treatment with gentamicin and vancomycin.

[0078] E. coli is a bacterium that inhabits the gastrointestinal tract of humans and other mammals, but it can also be pathogenic, causing conditions such as gastroenteritis, urinary tract infections, neonatal meningitis, hemorrhagic colitis, and bacteremia. E. coli is increasingly resistant to multiple antibiotics, including fluoroquinolones, cephalosporins, and carbapenems.

[0079] Klebsiella pneumoniae is a bacterium that is normally found in the mouth, skin, and intestines of humans and other mammals. However, if inhaled, it can cause destructive changes to the lungs of humans and mammals, particularly to the alveoli. Klebsiella pneumoniae infections are commonly seen in subjects with compromised immune systems, including those with diabetes, alcoholism, cancer, liver disease, chronic obstructive pulmonary disease, glucocorticoid therapy, and renal failure. Klebsiella pneumoniae is resistant to a variety of antibiotics, including fluoroquinolones, cephalosporins, tetracyclines, chloramphenicol, carbapenems, and trimethoprim / sulfamethoxazole. Resistance to azoles is increasing.

[0080] Methods for isolating nucleic acids from clinical samples.

[0081] In one aspect, the present disclosure provides a method for preparing a clinical sample from a subject, the clinical sample comprising cells from the subject, microorganisms, and at least one polyanionic polymer nucleic acid amplification inhibitor bound to at least one protein in the sample, the method comprising the following steps: (a) obtaining a clinical sample comprising cells from the subject, microorganisms, proteins, and the polyanionic polymer nucleic acid amplification inhibitor from the subject; (b) separating the clinical sample into a first fraction comprising the cells of the subject and a second fraction comprising the microorganisms and the polyanionic polymer nucleic acid amplification inhibitor bound to the protein; (c) adding a protease that degrades the protein to the second fraction; and (d) separating the second fraction into a third fraction comprising the microorganisms and (ii) a fourth fraction comprising the polyanionic polymer nucleic acid amplification inhibitor.

[0082] In another aspect, the present disclosure provides a method for preparing a clinical sample from a subject, the clinical sample comprising a microorganism and at least one polyanionic polymer nucleic acid amplification inhibitor that binds to at least one protein in the sample, the method comprising the steps of: (a) obtaining a clinical sample comprising a microorganism and at least one polyanionic polymer nucleic acid amplification inhibitor from a subject, (b) adding a protease that degrades the protein to the sample, (c) separating the sample into a first fraction comprising cells of the subject and a second fraction comprising the microorganism and the polyanionic polymer nucleic acid amplification inhibitor bound to the protein, (d) removing the second fraction from the first fraction, and (e) separating the second fraction into a third fraction comprising the microorganism and a fourth fraction comprising the polyanionic polymer nucleic acid amplification inhibitor.

[0083] Nucleic acid amplification inhibitors.

[0084] In some embodiments, when obtaining clinical samples, there is a nucleic acid amplification inhibitor in the container. In some embodiments, the nucleic acid amplification inhibitor is introduced into the clinical blood sample during sample processing. The nucleic acid amplification inhibitor can be a polyanionic polymer compound, such as sodium polyanethol sulfonate (SPS) or heparin. The nucleic acid amplification inhibitor can also be a protein, such as a bacterial toxin or an immunoglobulin (e.g., IgG).

[0085] In some embodiments, the nucleic acid amplification inhibitor is a polyanionic polymer. In some embodiments, the average molecular weight of the polyanionic polymer nucleic acid amplification inhibitor is greater than 500Da, greater than 1 kilodalton (1kDa), greater than 2kDa, greater than 3kDa, greater than 4kDa, greater than 5kDa, greater than 6kDa, greater than 7kDa, greater than 8kDa, greater than 9kDa or greater than 10kDa. Some non-limiting examples of polyanionic polymers include sodium polyanethole sulfonate, heparin, hyaluronate, dermatan sulfate polyanion and chondroitin D-glucuronic acid anion.

[0086] In some embodiments, the at least one polyanionic polymer is sodium polyanethole sulfonate. As described above, SPS is added to a variety of commercially available sample collection containers, including blood culture bottles and microbial blood collection vacuum liquid containers. SPS interferes with the activity and effectiveness of cationic antimicrobials and counteracts the inhibitory effect of blood on bacteria. However, the presence of SPS in clinical sample containers leads to inhibition of downstream molecular-based assays, including assays using polymerase chain reaction (PCR) amplification of nucleic acids. SPS also preserves the bacteria present in clinical blood samples by inhibiting the complement cascade to kill bacteria through innate immunity and humoral immunity.

[0087] In some embodiments, the polyanionic polymer nucleic acid amplification inhibitor is heparin. Heparin is an anticoagulant that activates antithrombin, thereby blocking the coagulation cascade. As described above, heparin is present in some sample collection containers, including clinical blood sample containers. In some embodiments, heparin is lyophilized. In some embodiments, heparin is spray-dried. Generally speaking, heparin or another anticoagulant is present in a blood sample container intended for collecting whole blood and plasma, because the anticoagulant can prevent the formation of coagulated blood and serum. On the contrary, anticoagulants (such as heparin) are not usually present in blood sample collection containers for coagulated blood and serum. In some embodiments, the heparin in the clinical blood sample container is a heparin salt. In some embodiments, heparin is sodium heparin. In some embodiments, heparin is lithium heparin. In some embodiments, heparin is ammonium heparin.

[0088] Protease

[0089] The present disclosure provides a method for preparing a clinical sample, the method comprising adding a protease to the clinical sample. Some non-limiting examples of proteases that can be used in the present invention include: proteinase K, factor Za, Arg-C protease, Asp-N endopeptidase, Asp-N endopeptidase + N-terminal Glu, BNPS-skatole, caspase 1, high specificity chymotrypsin, clostripain, CNBr, enterokinase, granzyme B, formic acid, glutamyl endopeptidase, hydroxylamine, iodosylbenzoic acid, LysC, LysN, NTCB, pepsin, proline endopeptidase, staphylococcal peptidase I, tobacco etch virus protease, thermolysin, thrombin, trypsin, elastase, plasmin, acrosin, complement C1, keratinase, collagenase, fibrinolytic enzyme, cocoon enzyme, thermolysin and carboxypeptidase A.

[0090] In some embodiments, the protease is proteinase K. Proteinase K is an enzyme produced by the fungus Engyodontium album. Proteinase K preferentially digests proteins after hydrophobic amino acids (e.g., glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, or tryptophan). However, if incubated with proteinase K for a sufficiently long time, the protein will be completely degraded. The activity of proteinase K is stable over a wide pH range (e.g., pH 4 to pH 12), and increasing the reaction temperature from 37°C to 50°C to 60°C may increase the activity of proteinase K several times. Adding proteinase K to a sample with nucleic acids rapidly inactivates proteins that bind and degrade nucleic acids.

[0091] In some embodiments, the protease is selected from the group consisting of proteinase K, factor Za, Arg-C protease, Asp-N endopeptidase, Asp-N endopeptidase + N-terminal Glu, BNPS-skatole, caspase 1, high specificity chymotrypsin, clostripain, CNBr, enterokinase, granzyme B, formic acid, glutamyl endopeptidase, hydroxylamine, iodosylbenzoic acid, LysC, LysN, NTCB, pepsin, proline endopeptidase, staphylococcal peptidase I, tobacco etch virus protease, thermolysin, thrombin, and trypsin.

[0092] In some embodiments, the amount of the added protease is 5 units / mL (U / mL) to 5000U / mL. "Protease Unit" (U) is the international unit of protease catalytic activity, defined as the amount of enzyme that catalyzes the conversion of 1 micromole of substrate per minute under the specified conditions of an acceptable assay method (1U = 1 μmol / min). In some embodiments, the amount of the added protease is at least 5U / mL. In some embodiments, the amount of the added protease is less than or equal to 5000U / mL. In some embodiments, the amount of protease added is 5 U / mL, 100 U / mL, 200 U / mL, 300 U / mL, 400 U / mL, 500 U / mL, 600 U / mL, 700 U / mL, 800 U / mL, 900 U / mL, 1000 U / mL, 1100 U / mL, 1200 U / mL, 1300 U / mL, 1400 U / mL, 1500 U / mL, 1600 U / mL, 1700 U / mL, 1800 U / mL, 1900 U / mL, 2000 U / mL, 2100 U / mL, 2200 U / mL, 2300 U / mL, 2400 U / mL, 2500 U / mL, 2600 U / mL, 2700 U / mL, 2800 U / mL, 2900 U / mL, 3000 U / mL, 3100 U / mL, 3200 U / mL, 3300 U / mL, 3400 U / mL, 3500 U / mL, 3600 U / mL, 3700 U / mL, 3800 U / mL, 3900 U / mL, 4000 U / mL, 4100 U / mL, 4200 U / mL, 4300 U / mL, 4400 U / mL, 4500 U / mL, 4600 U / mL, 4700 U / mL, 4800 U / mL, 4900 U / mL, 500 500U / mL, 2600U / mL, 2700U / mL, 2800U / mL, 2900U / mL, 3000U / mL, 3100U / mL, 3200U / mL, 3300U / mL, 3400U / mL, 3500U / mL, 3600U / mL, 3700U / mL, 3800U / mL, 3900U / mL, 4000U / mL, 4100U / mL, 4200U / mL, 4300U / mL, 4400U / mL, 4500U / mL, 4600U / mL, 4700U / mL, 4800U / mL, 4900U / mL or 5000U / mL.

[0093] In some embodiments, the protease is incubated with the sample (or fraction of the sample) at 20° C. to 60° C. In some embodiments, the protease is incubated at at least 20° C. In some embodiments, the protease is incubated at less than or equal to 60° C. In some embodiments, the protease is incubated at 20° C., 25° C., 30° C., 35° C., 40° C., 45° C., 50° C., 55° C., or 60° C.

[0094] In some embodiments, the protease is incubated with the sample (or fraction of the sample) for 10 minutes to 120 minutes. In some embodiments, the protease is incubated for at least 10 minutes. In some embodiments, the protease is incubated for less than or equal to 120 minutes. In some embodiments, the protease is incubated for 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 70 minutes, 80 minutes, 90 minutes, 100 minutes, 110 minutes or 120 minutes.

[0095] Clinical sample preparation

[0096] In some aspects, the disclosure provides methods for preparing clinical samples. "Preparation" refers to steps performed to modify clinical samples prior to diagnostic testing. In some embodiments, preparation includes obtaining, separating clinical samples, removing a portion of the sample, and / or adding additional components, such as proteases, to the sample.

[0097] The methods of the present invention include separating or partitioning a sample (or subsample) into fractions containing host cells, microorganisms and / or polyanionic polymer nucleic acid amplification inhibitors. Some non-limiting methods of separation include centrifugation, microfluidics, immunoprecipitation and filtration.

[0098] In various embodiments, the host cell can be an erythrocyte, a leukocyte, a platelet, an epithelial cell, a muscle cell, a bone cell, a neuron, a glial cell, a hepatocyte, an immune cell, a kidney cell, a gastric cell, and / or a colon cell.

[0099] In some embodiments, the method comprises a plurality of centrifugation steps. In some embodiments, there are 2 centrifugation steps. In some embodiments, there are 3 centrifugation steps. In some embodiments, there are 4 centrifugation steps. In some embodiments, there are 5 centrifugation steps. In some embodiments, a plurality of centrifugation steps can be performed at the same speed and / or for the same length of time. In some embodiments, a plurality of centrifugation steps can be performed at different speeds and / or for different lengths of time.

[0100] In some embodiments, the sample (or sample fraction) is centrifuged at 500 x g (500 x gravity) to 50,000 x g. In some embodiments, the sample (or sample fraction) is centrifuged at at least 500 x g. In some embodiments, the sample (or sample fraction) is centrifuged at less than or equal to 50,000 x g. In some embodiments, the sample (or sample fraction) is centrifuged at 500×g, 1,000×g, 2,000×g, 3,000×g, 4,000×g, 5,000×g, 6,000×g, 7,000×g, 8,000×g, 9,000×g, 10,000×g, 11,000×g, 12,000×g, 13,000×g, 14,000×g, 15,000×g, 16,000×g, 17,000×g, 18,000×g, 19,000×g, or 20,000×g.

[0101] In some embodiments, the sample (or sample fraction) is centrifuged for 2 minutes to 60 minutes. In some embodiments, the sample (or sample fraction) is centrifuged for at least 2 minutes. In some embodiments, the sample (or sample fraction) is centrifuged for less than or equal to 60 minutes. In some embodiments, the sample (or sample fraction) is centrifuged for 2 minutes, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, or 60 minutes.

[0102] In some embodiments, centrifugation is performed using a vacuum liquid container. In some embodiments, centrifugation is performed using a plasma tube. The plasma tube contains an anticoagulant (e.g., EDTA, sodium citrate, heparin) for separating serum from plasma.

[0103] As described above, sample separation can be performed using microfluidic devices known in the art. Some non-limiting examples of microfluidic devices include inertial microfluidics (Hou et al., 2015, "Direct detection and drug-resistance profiling of bacteremias using inertial microfluidics," Lab Chip, 15 (10): 2297-2307), plug-based microfluidics (Boedicker et al., 2008, "Detecting bacteria and determining their susceptibility to antibiotics by stochastic confinement in nanoliter droplets using plug-based microfluidics," Lab Chip, 8: 1265-1272), on-chip bacterial purification (Mahalanabis et al., 2009, "Cell lysis and DNA extraction of gram-positive and gram-negative bacteria from whole blood in a disposable microfluidic chip," Lab ona Chip 9: 2811-2817; Cady et al., 2005, Sensors and Actuators B: Chemical, 107: 332-341.) and affinity-based capture (Boehm et al., 2007, Sensors and Actuators B: Chemical, 126: 508-514; Xia et al., 2006, "Combined microfluidic-micromagnetic separation of living cells incontinuous flow," Biomedical Microdevices 8: 299-308; Yung et al., 2009, "Micromagnetic-microfluidic blood cleansing" device, "Lab on a Chip, 9: 1171-1177; Lee et al., 2014, "Synthetic ligand-coated magnetic nanoparticles for microfluidicbacterial separation from blood," Nano Letter, 14: 1-5; Cho et al., 2007, "One-steppathogen specific DNA extraction from whole blood on a centrifugalmicrofluidic device," Lab on a Chip, 7: 565-573.). .

[0104] In some embodiments, clinical samples are separated into one or more fractions using microfluidics. In some embodiments, macromolecular complexes of proteins, polyanionic polymer nucleic acid amplification inhibitors and / or host cells are separated into a fraction by microfluidics. In some embodiments, microorganisms and / or host cells are separated into a fraction by microfluidics. Macromolecular complexes, host cells or microorganisms can be labeled to assist separation. In some embodiments, macromolecular complexes, host cells or microorganisms are labeled with fluorophores (e.g., GFP, YFP), affinity tags or antibodies that bind to proteins and / or microorganisms on the surface. In some embodiments, macromolecular complexes, host cells or microorganisms are not labeled before separation. Methods for separating unlabeled macromolecular complexes, host cells and / or microorganisms in clinical samples by microfluidics include, but are not limited to, size exclusion and isotachophoresis. Size exclusion microfluidics separates smaller particles (e.g., microorganisms, proteins, polyanionic polymers) from larger macromolecular particles (e.g., mammalian cells).

[0105] In some embodiments, immunoprecipitation is used to separate clinical samples into one or more fractions. Treating clinical samples with proteases (e.g., proteinase K) can reduce the nonspecific binding of proteins to antibodies during immunoprecipitation by preventing the polyanionic polymer nucleic acid amplification inhibitor from forming complexes with proteins in the clinical sample that can nonspecifically bind to the immunoprecipitation antibody. In some embodiments, clinical samples are treated with proteases prior to immunoprecipitation to reduce the retention of polyanionic polymer nucleic acid amplification inhibitors.

[0106] Immunoprecipitation can utilize antibodies that bind to proteins on microorganisms but not on eukaryotic cells. Immunoprecipitation can utilize antibodies that bind to proteins on eukaryotic cells but not on microorganisms. In some embodiments, immunoprecipitation enriches host cells in clinical samples by binding to microbial cells and removing them. In some embodiments, immunoprecipitation depletes clinical samples of host cells by binding to host cells and removing them.

[0107] Immunoprecipitation can be used in conjunction with other methods of separating clinical samples. In some embodiments, the centrifuged sample is also separated by immunoprecipitation to further reduce the retention of polyanionic polymer nucleic acid inhibitors. In some embodiments, the sample separated by microfluidics is also separated by immunoprecipitation to further reduce the retention of polyanionic polymer nucleic acid inhibitors. In some embodiments, the sample separated by filtration is also separated by immunoprecipitation to further reduce the retention of polyanionic polymer nucleic acid inhibitors.

[0108] In some embodiments, filtration is used to separate clinical samples into one or more fractions. Depending on the pore size of the filter, the filter can remove host cells, microorganisms, or protein complexes (eg, polyanionic polymer nucleic acid amplification inhibitors bound to proteins).

[0109] In the method of the present invention, during sample preparation, protease is added to the clinical sample or the fraction of the sample. In some embodiments, before any separation of the sample separation into fractions, protease is added to the clinical sample. In some embodiments, after one or more steps of separation into fractions, protease is added to the fractions (e.g., supernatant, resuspended precipitate, filtrate).

[0110] Amplification of nucleic acids

[0111] In some aspects, the present invention includes the additional step of amplifying the nucleic acid population present in the original sample. Amplification of the nucleic acid population can be performed before identifying the nucleic acid source (e.g., mammalian DNA, microbial DNA). In some embodiments, the amplified nucleic acid is a microbial nucleic acid. In some embodiments, the amplified nucleic acid is a host nucleic acid. In some embodiments, the nucleic acid is DNA. In some embodiments, the nucleic acid is RNA. Some non-limiting examples of nucleic acid amplification techniques known in the art include: polymerase chain reaction (PCR), quantitative PCR (quantitative PCR, qPCR), reverse transcriptase PCR (reverse-transcriptase PCR, RT-PCR), degenerate oligonucleotide PCR, primer extension pre-amplification, strand displacement amplification (SDA) or transcription-mediated amplification (TMA). In some embodiments, nucleic acid amplification is performed by isothermal strand displacement amplification, polymerase chain reaction (PCR), quantitative PCR (qPCR), real-time PCR (real-time PCR, RT-PCR), degenerate oligonucleotide PCR or primer extension pre-amplification.

[0112] Amplification of a nucleic acid population by any of the foregoing methods requires primers and a polymerase. Some non-limiting examples of polymerases include eukaryotic polymerases, such as polymerase α, polymerase δ, and polymerase ε; bacterial polymerases, such as Thermus aquaticus (Taq), Deep (New England BioLabs, Ipswich, MA), and Therminator TM (New England BioLabs, Ipswich, MA); RNA polymerases, such as RNA polymerase I and RNA polymerase II; and strand-displacing polymerases.

[0113] In some embodiments, nucleic acid amplification is performed by a strand displacement polymerase. A "strand displacement polymerase" is an enzyme that separates the strands of a DNA double helix as it extends a primer template. Some non-limiting examples of strand displacement polymerases include: phi29 DNA polymerase; Bst DNA polymerase, large fragment (New England BioLabs, Ipswich, MA); Bsu DNA polymerase, large fragment (New England BioLabs, Ipswich, MA); Deep DNA polymerase (New England BioLabs, Ipswich, MA); Deep (exo) DNA polymerase (New England BioLabs, Ipswich, MA); DNA polymerase I, large fragment; M-MuLV reverse transcriptase; DNA polymerase (New England BioLabs, Ipswich, MA); DNA polymerase (New England BioLabs, Ipswich, MA); (exo) DNA polymerase (New England BioLabs, Ipswich, MA); SD polymerase; and Klenow fragment. In some embodiments, nucleic acid amplification is performed by phi29 DNA polymerase, Bst DNA polymerase, large fragment, or Klenow fragment. As used herein, "phi29" refers to the replicative polymerase from Bacillus subtilis bacteriophage phi29. The phi29 polymerase has strand displacement and processive synthesis properties, as well as inherent 3'→5' exonuclease proofreading capabilities.

[0114] In the method for the present invention, the interpolation of protease improves the amplification of nucleic acid in clinical samples. In some embodiments, the nucleic acid colony is a microbial nucleic acid. In some embodiments, compared with the sample not treated with protease, the interpolation of protease improves the amplification of nucleic acid by 5 times to 100 times. In some embodiments, compared with the sample not treated with protease, the amplification of nucleic acid improves by 5 times, 10 times, 15 times, 20 times, 25 times, 30 times, 35 times, 40 times, 45 times, 50 times, 55 times, 60 times, 65 times, 70 times, 75 times, 80 times, 85 times, 90 times, 95 times or 100 times. In some embodiments, compared with the sample not treated with protease, the amplification of nucleic acid improves by at least 5 times. In some embodiments, compared with the sample not treated with protease, the amplification of nucleic acid improves by less than 100 times.

[0115] In the method for the present invention, the interpolation of protease improves the separation of nucleic acid from clinical samples. In some embodiments, nucleic acid is a microbial nucleic acid. In some embodiments, compared with a sample not treated with protease, the interpolation of protease improves the separation of nucleic acid by 10 times to 100 times. In some embodiments, compared with a sample not treated with protease, the separation of nucleic acid improves by 10 times, 15 times, 20 times, 25 times, 30 times, 35 times, 40 times, 45 times, 50 times, 55 times, 60 times, 65 times, 70 times, 75 times, 80 times, 85 times, 90 times, 95 times or 100 times. In some embodiments, compared with a sample not treated with protease, the separation of nucleic acid improves by at least 10 times. In some embodiments, compared with a sample not treated with protease, the separation of nucleic acid improves by less than 100 times.

[0116] Application of alternative methods of sample handling.

[0117] In some aspects, the present disclosure provides a method for preparing a clinical sample from a subject, the clinical sample comprising a microorganism, cells, and at least one polyanionic polymer nucleic acid amplification inhibitor bound to at least one protein in the sample, the method comprising the following steps: (a) obtaining a clinical sample comprising a microorganism, a protein, and a polyanionic polymer nucleic acid amplification inhibitor from a subject, (b) adding a protease that degrades the protein to the clinical sample, (c) separating the clinical sample into a first fraction comprising the cells of the subject and a second fraction comprising the microorganism and the polyanionic polymer nucleic acid amplification inhibitor bound to the protein, (d) removing the second fraction from the first fraction, and (e) separating the second fraction into: a third fraction comprising the microorganism and a fourth fraction comprising the polyanionic polymer nucleic acid amplification inhibitor.

[0118] In some aspects, the present disclosure provides a method for preparing a clinical sample from a subject, the clinical sample comprising a microorganism, cells, and at least one polyanionic polymer nucleic acid amplification inhibitor bound to at least one protein in the sample, the method comprising the following steps: (a) obtaining a clinical sample comprising a microorganism, a protein, and a polyanionic polymer nucleic acid amplification inhibitor from a subject, (b) separating the clinical sample into: a first fraction comprising the cells of the subject and a second fraction comprising the microorganism and the polyanionic polymer nucleic acid amplification inhibitor bound to the protein, (c) removing the second fraction from the first fraction, (d) after step (b) or step (c), adding a protease that degrades the protein in the second fraction to the second fraction, and (e) separating the second fraction into a third fraction comprising the microorganism and a fourth fraction comprising the polyanionic polymer nucleic acid amplification inhibitor.

[0119] In some aspects, the present disclosure provides a method for preparing a clinical sample from a subject, the clinical sample comprising a microorganism and at least one polyanionic polymer nucleic acid amplification inhibitor that binds to at least one protein in the sample, the method comprising the following steps: (a) obtaining a clinical sample comprising a microorganism, a protein, and a polyanionic polymer nucleic acid amplification inhibitor from the subject, (b) selectively lysing the microorganism, (c) adding a protease that degrades the protein to the clinical sample, and (d) separating the clinical sample into a first fraction comprising cells of the clinical sample and a second fraction comprising nucleic acids from the microorganism.

[0120] In some embodiments of each of the aforementioned aspects, the separation is performed by centrifugation. In some embodiments of each of the aforementioned aspects, the separation is performed by filtration. In some embodiments of each of the aforementioned aspects, the separation is performed by microfluidic size exclusion. In some embodiments of each of the aforementioned aspects, the separation is performed by plasma tube. In some embodiments of each of the aforementioned aspects, the separation is performed by immunoprecipitation.

[0121] In some embodiments of each of the foregoing aspects, the clinical sample is blood, sputum, urine, mucus, saliva tissue abscess, wound drainage, lymph, lavage fluid, stool, cerebrospinal fluid, or any fluid aspirate or tissue extract of human or other mammalian origin.

[0122] In some embodiments of each of the aforementioned aspects, the at least one polyanionic polymer nucleic acid amplification inhibitor is sodium polyanethole sulfonate. In some embodiments of each of the aforementioned aspects, the at least one polyanionic polymer nucleic acid amplification inhibitor is heparin.

[0123] In some embodiments of each of the aforementioned aspects, the obtaining step utilizes a vacuum liquid container or a blood culture bottle.

[0124] In some embodiments of each of the foregoing aspects, the protease is selected from the group consisting of proteinase K, factor Xa, Arg-C protease, Asp-N endopeptidase, Asp-N endopeptidase + N-terminal Glu, 3-bromo-3-methyl-2-(2-nitrophenylthio)-3H-indole (BNPS-skatole), caspase 1, high specificity chymotrypsin, clostripain, cyanogen bromide (CNBr), enterokinase, granzyme B, formic acid, glutamyl endopeptidase, hydroxylamine, iodosylbenzoic acid, LysC, LysN, 2-nitro-5-thiocyanatobenzoic acid (NTCB), pepsin, proline endopeptidase, staphylococcal peptidase I, tobacco etch virus protease, thermolysin, thrombin and trypsin.

[0125] In some embodiments of each of the aforementioned aspects, the amount of protease added is 5 units / mL (U / mL) to 5000 units / mL (U / mL).

[0126] In some embodiments of each of the aforementioned aspects, the protease and the second fraction are incubated at 20°C to 55°C for at least 10 minutes.

[0127] In some embodiments of each of the aforementioned aspects, the microorganism is a pathogenic microorganism.

[0128] In some embodiments of each of the aforementioned aspects, the method further comprises the step of performing nucleic acid amplification on the nucleic acids in the third fraction. In some embodiments of each of the aforementioned aspects, the nucleic acid amplification is isothermal strand displacement amplification, polymerase chain reaction (PCR), quantitative PCR (qPCR), reverse transcriptase PCR (RT-PCR), degenerate oligonucleotide PCR, or primer extension preamplification.

[0129] In some embodiments of each of the aforementioned aspects, the addition of the protease increases amplification of microbial nucleic acid by at least 5-fold relative to a sample to which the protease is not added. Example

[0130] Example 1: Polyanethol Sulfonate (SPS) Binding to Serum / Plasma Proteins

[0131] Blood collection containers often contain the anticoagulant sodium polyanethole sulfonate (SPS). Although SPS has many advantages - preventing the complement system from lysing bacterial cells, reducing blood cell clumps, and facilitating the separation of blood components into solids (e.g., red blood cells and white blood cells, etc.) and liquids (e.g., plasma, small molecular weight nucleic acid amplification inhibitors, etc.), it is well known that its persistence in the liquid phase inhibits downstream processes, such as nucleic acid amplification.

[0132] One hypothesis as to how SPS persists after separation of blood into cells and plasma is that it forms large complexes with molecules in the liquid phase (e.g., proteins such as albumin, which is the most abundant protein in plasma and serum), causing it to coprecipitate during separation. This hypothesis was tested by premixing 0.05% SPS with 0.25% bovine serum albumin (BSA) (Sigma-Aldrich, St. Louis, MO). The SPS-BSA mixture was then separated by size on an agarose gel stained with SYBR-safe (ThermoFisher, Waltham, MA). Figure 1 ).

[0133] exist Figure 1In the figure, lane 1 is a 1 kb DNA ladder, lane 2 is 0.25% BSA, lane 3 is 0.05% SPS, and lane 4 is a mixture of 0.25% SPS and 0.05% BSA. The solution of 0.05% SPS and 0.25% BSA alone is not visible with SYBR-safe, indicating that SPS and BSA form a large macromolecular complex.

[0134] Example 2: SPS protein complex reduction under proteinase K treatment

[0135] To determine whether protease treatment disrupts the SPS protein complex, whole blood samples from healthy donors were collected in ACD vacuum containers (BD Biosciences, Franklin Lakes, NJ). One milliliter (mL) of whole blood was washed with 1.75 mL of Dulbecco's phosphate-buffered saline (DPBS, Ca-free). +2 and Mg +2 , ThermoFisher, Waltham, MA) and diluted 3-fold with 0.25 mL of 1% SPS in water. A total of 8 blood samples were prepared for analysis.

[0136] Bacteria (Escherichia coli) are added to blood samples with a final concentration of 90,000 colony forming units (CFU) / mL to simulate bacteremia blood samples. Bacteremia blood samples are centrifuged briefly (500 × g, 3 minutes) to separate human blood cells (i.e., red blood cells and white blood cells) from plasma rich in bacteria. Precipitate comprises red blood cells and white blood cells, and supernatant comprises plasma, bacteria and SPS- and SPS- serum protein complexes. 2 milliliters of supernatant are manually removed from bacteremia blood samples by pipetting and transferred to fresh tubes. The initial supernatant (S1) is treated with the protease proteinase K (New England Biolabs, Boston, MA) (0 units (U), 4U, 12U and 40U / mL supernatant) of increasing concentration at 37°C for 5 minutes, then 40 μL of 10% Triton X100 (Sigma-Aldrich, St.Louis, MO) are added to reach a final concentration of 0.2% and further incubated at 37°C for 5 minutes. S1 was then centrifuged at 3000 × g for 5 minutes and excess supernatant was removed. The original precipitated blood sample was resuspended with another two milliliters of 1 × DPBS and then centrifuged again at 500 × g for three minutes. After spinning at 3000 × g, another two milliliters of the second supernatant (S2) were taken out and added to the precipitate from S1. S2 was then treated with 40 μL of 10% Triton X100 at room temperature for 5 minutes and spun down at 3000 × g. Excess supernatant was then aspirated, leaving the final sample.

[0137] All experimental samples (supernatant and proteinase K) and control samples (supernatant without proteinase K) were resuspended in 900 μL TBST (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5% Tween 20 (Sigma P9416)) and further centrifuged at 10,000 × g for 3 minutes to concentrate bacteria and insoluble SPS-serum protein complexes. After centrifugation, the supernatant from this spin was completely aspirated, leaving only the pellet.

[0138] The precipitated samples were resuspended in 10 μL of 1×TE (10 mM Tris-HCl, pH 8.0, 0.1 mM EDTA) and lysed with 10 μL of lysis buffer (50 mM NaOH, 0.4 mM EDTA). The lysed samples were incubated at 75°C for 10 minutes to promote lysis, and then 10 μL of neutralization buffer (40 mM Tris-HCl, pH 8.0) was added. 15 μL of each sample was run on an agarose gel stained with SYBR-safe (ThermoFisher, Waltham, MA) to visualize SPS contamination.

[0139] exist Figure 2 In the experiment, the formation of SPS protein complex was minimized by proteinase K treatment. Lane 1 is a 1 kb DNA ladder, lanes 2 to 3 are samples not treated with proteinase K, lanes 4 to 5 are samples treated with 8 units (U) of proteinase K, lanes 6 to 7 are samples treated with 24 U of proteinase K, and lanes 8 to 9 are samples treated with 80 U of proteinase K. Relative to samples containing proteinase K, bacterial precipitates from samples not containing proteinase K were enriched in SPS and serum proteins ( Figure 2 ). In addition, treatment with proteinase K reduced SPS and serum proteins in the precipitate in a concentration-dependent manner.

[0140] Example 3: Treatment with proteinase K reduces serum proteins in bacteremic blood samples containing SPS

[0141] To determine whether treatment with proteinase K resulted in a significant reduction in soluble protein, 1 mL whole blood samples from healthy donors were collected into vacuum liquid containers containing SPS and washed with 1× DPBS (without Ca +2 and Mg +2 , ThermoFisher, Waltham, MA) was diluted to a final volume of 3 mL. The samples were spiked with 10,000 CFU of Escherichia coli to simulate bacteremic blood samples.

[0142] The bacteremia blood samples were centrifuged at 500 × g to precipitate blood cells. The precipitate contained red blood cells and white blood cells, and the supernatant contained plasma, bacteria, and SPS-serum protein complexes. Two milliliters of supernatant were manually removed from the bacteremia blood samples by pipetting and transferred to a fresh tube, then treated with 40 μL of 10% Triton X100 to a final concentration of 0.2%, and treated with 8 units (U) of proteinase K to a final concentration of 4 U / mL ( Figure 3 All samples were incubated at 37°C for 10 minutes. Samples of the supernatant were taken before and after proteinase K treatment, diluted with BOLT LDS sample buffer (Thermo Fisher, Waltham, MA), and loaded onto BOLT 4% to 12% Bis Tris Plus gels (Thermo Fisher, Waltham, MA) with MES SDS running buffer (Thermo Fisher, Waltham, MA). After the samples were separated on the gel, the protein gel was stained with PageBlue protein staining solution (Thermo Fisher, Waltham, MA) and destained according to the manufacturer's instructions.

[0143] exist Figure 3In the figure, lane 1 is SeeBlue-Plus2 prestained protein standard (ThermoFisher, Waltham, MA), lane 2 is the protein present in the supernatant after centrifugation of a clinical blood sample diluted with DPBS, and lane 3 is the protein present in the supernatant after centrifugation of a clinical blood sample diluted with DPBS and treated with proteinase K at 37°C for 10 minutes.

[0144] like Figure 3 As seen in , proteinase K treatment significantly reduced soluble protein in large clinical sample volumes and, therefore, reduced potential SPS-protein complex formation and contamination in subsequent enrichment pellets.

[0145] Example 4: Proteinase K treatment does not affect bacterial viability

[0146] To examine the effect of proteinase K treatment on bacterial viability, clinical blood samples were collected and treated as in Example 2 with the following modifications. Escherichia coli (E. coli) bacteria were added to diluted blood samples collected in SPS vacuum blood collection tubes at a final concentration of 10,000 CFU / mL. 100 μL of the supernatant fraction treated with proteinase K and 100 μL of the supernatant fraction not treated with proteinase K were cultured on agar plates. Individual colonies were counted after overnight incubation. The viability of E. coli in the supernatant was not significantly inhibited by the treatment with proteinase K ( Figure 4 ). Figure 4 Excellent recovery of live bacteria from clinical blood samples collected in SPS vacuum liquid containers was also demonstrated.

[0147] Example 5: Treatment with proteinase K increases the rate of in vitro DNA amplification of enriched pellets from samples collected with SPS additive.

[0148] In order to examine the effect of proteinase K treatment on DNA amplification efficacy, clinical blood samples were collected in ACD vacuum blood collection tubes and diluted with 1×DPBS and SPS as in Example 2. Bacteria (E. coli) were added to blood samples at a final concentration of 1,000 CFU / mL to simulate bacteremia blood samples. Bacteremia blood samples were briefly centrifuged at 500×g for 3 minutes to separate human blood cells from bacteria-rich plasma. Two milliliters of supernatant from bacteremia blood samples were manually removed by pipetting and transferred to a fresh tube (supernatant 1, S1). The precipitated blood sample was resuspended with another two milliliters of 1×DPBS and then centrifuged again at 500×g for three minutes. Another two milliliters of the second supernatant (S2) were taken out and added to S1. The combined 4 mL supernatant was then treated at 37°C for 10 minutes without proteinase K or 4U / mL proteinase K and a final concentration of 0.2% Triton X100. The sample was then centrifuged at 3000×g for 5 minutes to collect bacteria. Aspirate most of the supernatant, leaving the concentrated sample.

[0149] All experimental samples were resuspended in 900 μL TBST (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5% Tween 20 (Sigma P9416)) and further centrifuged at 10,000×g for 3 minutes to concentrate the bacteria. The supernatant was completely aspirated, leaving only the precipitate.

[0150] The concentrated pellet samples were then subjected to the REPLI-g single cell MDA protocol with 0.5% BSA (Sigma-Aldrich, St. Louis, MO) supplemented with MDA reaction buffer. Time point samples were collected after 1.5, 2.5 and 4 hours of incubation / MDA at 30°C. To quantify bacterial DNA concentration, 16S rRNA gene concentration was measured using a custom 16S rRNA TaqMan probe and TaqManFast Advanced Master Mix. Total amplified DNA was quantified using the Qubit dsDNA HS Assay Kit (ThermoFisher, Waltham, MA).

[0151] Figure 5A It was shown that the addition of proteinase K increased the rate of total DNA amplification by at least 100-fold when compared to samples not treated with proteinase K at the 1.5, 2.5 and 4 hour time points. Figure 5B It was shown that similar to total DNA amplification, bacterial DNA amplification was also enhanced at least 100-fold when compared to samples not treated with proteinase K, as measured by the 16S signal.

[0152] Example 6: Treatment with proteinase K increases the rate of in vitro DNA amplification from samples collected in lithium heparin vacuum containers

[0153] Blood from healthy donors was collected in lithium heparin vacuum blood collection tubes (Becton Dickinson, Franklin Lakes, NJ). 2.5 CFU / mL of E. coli was spiked into 4 mL of blood replicates and diluted with 8 mL of 1×DPBS. Plasma supernatants rich in bacteria were produced by two consecutive centrifugations at 500×g, as described in Example 2. Half of the supernatant was treated with proteinase K (final concentration 4 U / mL) and all supernatants were treated with 10% Triton X-100 (final concentration 0.2%), followed by incubation at 37°C for 10 minutes. All supernatant samples were then precipitated by high-speed centrifugation at 3,000×g for 10 minutes. The precipitate was resuspended in 1× DNase I buffer and treated with DNase I (final concentration of 40 U / mL; New England Biolabs, Boston, MA) for 15 minutes at 37°C. The DNase-treated samples were then pelleted at 10,000 × g for 3 min, washed in 1 mL of TBST, and pelleted again. The supernatant was aspirated and the DNA contained in the pellet was amplified using the REPLI-g single-cell MDA protocol. Figure 6 As shown in , the addition of proteinase K increased the amplification of bacterial DNA by an average of 10-fold relative to control samples not treated with proteinase K ( Figure 6 ).

[0154] Example 7: Synthesis of RNA libraries from microorganisms isolated from patients with bacteremia

[0155] Similar to DNA polymerase, reverse transcriptase function is inhibited by polyanionic polymers (e.g., SPS, heparin) (Kiehl et al., 1973, Anticoagulants as Inhibitors of Reverse Transcriptase Activity, Journal of the National Cancer Institute, 51(5): 1705-1707). Therefore, the absence of polymerase inhibitors is crucial for preparing complementary DNA (cDNA) libraries and subsequent transcriptomes from clinical samples. The use of proteinase K treatment to alleviate the inhibitory effects of polyanionic polymers (e.g., SPS, heparin) can also be used to enable the determination of the transcriptional profile of cells from microorganisms or humans from bacteremic patients. For example, this can be used clinically to predict antibiotic resistance (Khaledi et al., 2016, Transcriptome Profiling of Antimicrobial Resistance in Pseudomonas aeruginosa, Antimicrobial Agents and Chemotherapy, 60(8): 4722-4733).

[0156] cDNA libraries will be prepared from clinical samples using any of a variety of protocols in the art. For example, as described in the claims, clinical samples will be treated with proteinase K and the desired microbial DNA will be isolated by centrifugation. Total RNA in the sample will be extracted using PicoPure RNA Isolation Kit (ThermoFisher, Waltham, MA) with random hexamers or any other suitable primers.

Claims

1. A method for preparing a clinical sample from a subject, the clinical sample comprising a microorganism and at least one polyanionic polymer nucleic acid amplification inhibitor that binds to at least one protein in the sample, the method comprising the steps of: (a) obtaining the clinical sample comprising the microorganism, the at least one protein and the at least one polyanionic polymer nucleic acid amplification inhibitor from the subject; (b) separating the clinical sample into: (i) a first fraction comprising cells of the subject, and (ii) a second fraction comprising the microorganism and the polyanionic polymer nucleic acid amplification inhibitor bound to the at least one protein; (c) adding to the second fraction a protease that degrades the at least one protein; and (d) separating the second fraction into: (i) a third fraction comprising the microorganism, any remaining cells from the subject, and (ii) a fourth fraction comprising the polyanionic polymer nucleic acid amplification inhibitor; wherein the polyanionic polymer nucleic acid amplification inhibitor is sodium polyanethol sulfonate or heparin; and The protease is proteinase K, factor Xa, chymotrypsin, granzyme B, glutamyl endopeptidase, trypsin, LysC, enterokinase, proline endopeptidase, thrombin or staphylococcal peptidase I.

2. The method of claim 1, wherein the separation is performed by centrifugation.

3. The method of claim 1, wherein the separation is performed by filtration.

4. The method of claim 1, wherein the separation is performed by microfluidic size exclusion.

5. The method of claim 1, wherein the separation is performed by a plasma tube, which is a container for separating plasma.

6. The method of claim 1, wherein the separation is performed by immunoprecipitation.

7. The method of claim 1, wherein the clinical sample is blood, sputum, urine, mucus, saliva, tissue abscess, wound drainage, lymph fluid, lavage fluid, stool, or cerebrospinal fluid.

8. The method of claim 1, wherein the obtaining step utilizes a vacuum liquid container or a blood culture bottle.

9. The method of claim 1, wherein the amount of the added protease is 5 units / mL (U / mL) to 5000 units / mL (U / mL).

10. The method of claim 1, wherein the protease and the second fraction are incubated at 20°C to 55°C for at least 10 minutes.

11. The method of claim 1, wherein the microorganism is a pathogenic microorganism.

12. The method of claim 1, further comprising the step of performing nucleic acid amplification on the nucleic acid in the third fraction.

13. The method of claim 12, wherein the nucleic acid amplification is isothermal strand displacement amplification, polymerase chain reaction (PCR), quantitative PCR (qPCR), reverse transcription PCR (RT-PCR), degenerate oligonucleotide primer PCR, or primer extension preamplification.

14. The method of claim 12, wherein the addition of the protease increases the amplification of microbial nucleic acid by at least 5-fold relative to a sample to which no protease was added.

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