Method of pooling blood samples

Abstract

To provide methods of pooled analysis of samples containing red blood cells, such as whole blood cell samples.SOLUTION: The present invention relates to a method for detecting a target in a sample aliquot, comprising the steps of: providing a sample aliquot from each of a plurality of blood cell samples, thereby providing a plurality of sample aliquots; separately performing a lysing reaction on each of the sample aliquots, the lysing step comprising contacting each of the sample aliquots with a lysis reagent comprising (i) a buffer, (ii) lithium lauryl sulfate (LLS), and at least one of a chloride containing salt and an anticoagulant selected from the group consisting of EDTA, EDTA-Na2, EGTA, and combinations thereof, and the reagent having a pH that is greater than 5.5, and whereby at least a part of the blood cells in each of the sample aliquots lyse; combining the lysed sample aliquots into a single pooled sample to form a pooled lysate; separating a target from the pooled lysate; and performing a reaction to detect the separated target from the pooled lysate, the detection of the presence of the target in the pooled lysate indicating the presence of the target in at least one of the initially provided sample aliquots.SELECTED DRAWING: None

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of priority of U.S. Provisional Application No. 62 / 160,591, filed May 12, 2015, which is incorporated herein by reference.

Background Art

[0002] (Background) Blood transfusion plays an essential role in patient management. The blood used for transfusion is often collected from many volunteers. Such samples are screened for infectious diseases (e.g., HIV - 1, HIV - 2, hepatitis B virus, hepatitis C virus, dengue virus, West Nile virus, Treponema pallidum, Anaplasma phagocytophilum, and especially Trypanosoma cruzi and Plasmodium species depending on the regional prevalence).

[0003] Screening for additional pathogens is desirable. For example, human babesiosis is an emerging infectious disease that is a tick - borne intra - erythrocytic infection. Babesia microti is the most common cause of human babesiosis in the United States and is widely prevalent in the northeastern and north - central midwestern states. This species is associated with most cases of transfusion - transmitted babesiosis (TTB) in the United States and is currently the most frequently reported transfusion - transmitted disease to the FDA. 159 well - documented cases of B. microti TTB were reported in the United States between 1979 and 2009 (Herwaldt et al., Ann Intern Med 2011). 87% of the infected infusions occurred in 7 specific states in the United States. 12 transfusion - related deaths associated with TTB have occurred since 2005.

[0004] Since the proportion of infected samples is low, screening can be simplified by pooling the samples. If the pooled sample is infected, the pool can be deconvoluted to identify the source of infection in the individual samples, but if the pool is not infected, all the samples contributing to the pool can be assumed to be free of infection without individual testing.

[0005] Nucleic acid testing in minipools (of 6 - 24) is routinely used to screen donated blood for viruses (e.g., HBV, HIV) after separating whole blood into plasma or serum. However, plasma and serum cannot be used to detect red blood cell pathogens. Furthermore, whole blood cannot be pooled due to its tendency to clot when samples from different patients are mixed. Blood clots can also interfere with subsequent steps in the assay by changing the consistency of the sample and clogging automated equipment.

Summary of the Invention

Means for Solving the Problems

[0006] (Abstract) A method for detecting a target in a sample aliquot, the method comprising: providing the sample aliquot from each of a plurality of blood cell samples, thereby providing a plurality of sample aliquots; separately performing a lysis reaction on each of the sample aliquots, wherein the lysis step comprises each of the sample aliquots and the following: (i) a buffer, (ii) lithium lauryl sulfate (LLS), and a chloride-containing salt and EDTA, EDTA-Na2, EGTA, and combinations thereof selected from the group consisting of including the step of contacting with a lysis reagent comprising at least one of the anticoagulants to be selected, wherein the reagent has a pH higher than 5.5, whereby at least a portion of the blood cells in each of the sample aliquots is lysed; the step of combining the lysed sample aliquots into a single pooled sample to form a pooled lysate; the step of separating a target from the pooled lysate; and the step of performing a reaction to detect the separated target from the pooled lysate, wherein detecting the presence of the target in the pooled lysate indicates the presence of the target in at least one of the initially provided sample aliquots, a method is disclosed herein. In some embodiments, the method further includes, when the target is detected in the pooled lysate, individually testing separate blood samples to identify which of the blood cell samples contain the target. In some aspects, it is desirable that blood cell samples that do not contain the target and thus samples determined to contain the target are discarded. In a non-limiting example of this aspect, blood cell samples for use in a blood bank are discarded when it is determined that the target is present in the sample. In some aspects, it is desirable that the blood cell samples have the target and thus samples determined to contain the target are retained. In a non-limiting example of this aspect, blood cell samples used to obtain a therapeutic target are retained when it is determined that the target is present in the sample.

[0007] A method for separating a target from a plurality of sample aliquots, the method comprising: (a) providing sample aliquots from each of a plurality of whole blood samples, thereby providing a plurality of sample aliquots; (b) A step of separately performing a lysis reaction on each of the sample aliquots, wherein the lysis step comprises contacting each of the sample aliquots with the following: (i) a buffer, (ii) lithium lauryl sulfate (LLS), and (iii) at least one anticoagulant selected from the group consisting of chloride-containing salts and EDTA, EDTA-Na2, EGTA, and combinations thereof; in a lysis reagent, wherein the reagent has a pH higher than 5.5, whereby at least a portion of the blood cells in each of the sample aliquots is lysed; (c) combining the lysed sample aliquots into a single pooled sample to form a pooled lysate; and (d) separating a target from the pooled lysate. A method is disclosed. In some embodiments, the method further comprises contacting the pooled lysate with a solid support configured to immobilize the target; and separating the immobilized target from the pooled lysate. In some embodiments, the target is a nucleic acid target, and the solid support comprises an attached immobilization probe. In some embodiments where the target is a nucleic acid target, the method further comprises contacting the pooled lysate with a capture probe comprising a first segment complementary to the nucleic acid target and a second segment complementary to the immobilization probe. In some aspects, the method further comprises providing hybridization conditions favorable for the formation of a hybridization complex between the first segment of the capture probe and the nucleic acid target. In some aspects, the method further comprises providing hybridization conditions favorable for the formation of a hybridization complex between the second segment of the capture probe and the immobilization probe attached to the solid support. In some embodiments, the solid support is a magnetic bead solid support. In some embodiments, the solid support is a silica solid support. In some aspects, the silica solid support is glass wool. In some aspects, the silica solid support is beads. In some embodiments, the solid support is housed within a column.

[0008] In one embodiment of the above method, the target is a nucleic acid target, and the pooled lysate is tested for the presence or absence of the nucleic acid target using nucleic acid amplification and detection reactions. In one aspect, the amplification reaction is an isothermal amplification reaction. In one aspect, the amplification reaction includes performing a transcription-mediated amplification reaction to generate an amplification product, where the detection reaction includes detecting the amplification product with a detection probe. In one aspect, the amplification reaction and the detection reaction are performed simultaneously. In one embodiment, one or more of the steps of the above method are performed by an automated device. In one aspect, the amplification reaction and the detection reaction are performed by an automated device. In one aspect, the step of separating the target from the pooled lysate is performed by an automated device. In one aspect, the lysing reaction, pooling reaction, target separation reaction, and nucleic acid analysis reaction are all performed by an automated device. In one aspect, the automated device is an integrated device that performs all of the automated steps. In one aspect, the automated device is a collection of spatially separated devices, each performing one or more of the automated steps. In one embodiment of the above method, the lysing reaction is performed on at least 4 sample aliquots. In one embodiment of the above method, the lysing reaction is performed on at least 16 sample aliquots. In one embodiment of the above method, the lysing reaction is performed on at least 20 sample aliquots. In one embodiment of the above method, the lysing reaction is performed on a maximum of 200 sample aliquots. In one embodiment of the above method, the lysing reaction is performed on from about 4 sample aliquots to about 200 sample aliquots.

[0009]

[0010] ​In one embodiment of the above method, the buffer is sodium bicarbonate. In one aspect, the buffer is sodium bicarbonate and the reagent comprises a chloride-containing salt which is ammonium chloride. In one embodiment, the buffer is sodium bicarbonate, the reagent comprises a chloride-containing salt which is ammonium chloride, and the pH of the reagent is from about 7.0 to about 8.0. In one aspect, the ammonium chloride is present in the reagent at a concentration of from about 100 mM to about 500 mM, or at a concentration of about 250 mM. In one aspect, the sodium bicarbonate is present in the reagent at a concentration of from about 5 mM to about 30 mM, or at a concentration of about 14 mM. In one aspect, the reagent comprises EDTA-Na2 present in the reagent at a concentration of about 1 mM, EGTA present in the reagent at a concentration of about 1 mM, or a combination of EDTA-Na2 and EGTA, wherein the EDTA-Na2 is present in the reagent at a concentration of about 1 mM and the EGTA is present in the reagent at a concentration of about 1 mM, and is an anticoagulant. In one aspect, the lysis reagent comprises (i) about 14 mM sodium bicarbonate, (ii) about 5% (v / v) LLS, (iii) a chloride-containing salt and an anticoagulant, wherein the chloride-containing salt is ammonium chloride, the ammonium chloride is present in the reagent at a concentration of about 250 mM, and wherein the anticoagulant is EDTA, the EDTA is present in the reagent at a concentration of from about 0.1 mM to about 10 mM, and wherein the pH of the reagent is from about 7.2 to about 7.5. Ranges include all integers and decimals within the range.

[0011] In one embodiment of the above method, the buffer is a TRIS buffer. In one aspect, the reagent contains a chloride salt that is magnesium chloride, and the magnesium chloride is present in the reagent at a concentration of about 20 mM to about 35 mM, or at a concentration of about 30 mM. In one aspect, the above TRIS buffer is present in the reagent at a concentration of about 75 mM to about 150 mM, or at a concentration of about 100 mM. In one aspect, the above reagent contains a TRIS buffer and further contains a chloride salt that is magnesium chloride, and the magnesium chloride is present in the reagent at a concentration of about 20 mM to about 35 mM, or at a concentration of about 30 mM. In one aspect, the lysis reagent contains about 100 mM TRIS buffer and about 6% (v / v) LLS, where the pH of the reagent is about 7.5. The range includes all integers and decimals within the range.

[0012] In one embodiment of the above reagent, the LLS is present in the reagent at a concentration of about 4% (v / v) to about 15% (v / v), or at a concentration of about 10% (v / v), or at a concentration of about 8% (v / v), or at a concentration of about 6% (v / v), or at a concentration of about 5% (v / v). In one aspect, the buffer is a sodium bicarbonate buffer. In one aspect, the buffer is a TRIS buffer. The range includes all integers and decimals within the range.

[0013] In one embodiment, the reagent contains a sodium phosphate buffer, about 10% (v / v) LLS, and an anticoagulant, where the anticoagulant is EDTA-Na2 present in the reagent at a concentration of about 1 mM, EGTA present in the reagent at a concentration of about 1 mM, or a combination of EDTA-Na2 and EGTA, where the EDTA-NA2 is present in the reagent at a concentration of about 1 mM and the EGTA is present in the reagent at a concentration of about 1 mM.

[0014] In one embodiment, the blood cell sample is a whole blood sample. In one aspect, the blood cell sample is a human whole blood sample. In one aspect, one or more of the blood cell samples are human whole blood samples, and one or more of the blood cell samples are non-human whole blood samples. In one aspect, the blood cell sample contains red blood cells. In one aspect, the blood cell sample contains white blood cells.

[0015] In one embodiment, each sample aliquot is contacted with the lysis reagent at a volume ratio of sample aliquot to lysis reagent of about 1:2 to about 1:10 (v / v). In one aspect, the volume ratio of sample aliquot to lysis reagent is selected from the group consisting of 1:2 (v / v), 1:3 (v / v), 1:4 (v / v), 1:5 (v / v), 1:6 (v / v), 1:7 (v / v), 1:8 (v / v), 1:9 (v / v) and 1:10 (v / v). In one aspect, the volume ratio of sample aliquot to lysis reagent is about 1:3 (v / v). In one aspect, the volume ratio of sample aliquot to lysis reagent is about 1:4 (v / v). The range includes all integers and decimals within the range.

[0016] In one embodiment, the target is a host-derived target. In one embodiment, the target is a pathogen-derived target. In one aspect, the target is released from red blood cells. In one aspect, the target is a protein target. In one aspect, the target is a nucleic acid target. In one aspect, the target is an RNA target. In one aspect, the target is a ribosomal RNA target. In one aspect, the target is a pathogen-derived target derived from a pathogen selected from the group consisting of hepatitis virus, human immunodeficiency virus, dengue virus, West Nile virus, flavivirus, Zika virus, and parasites. In one aspect, the target is a pathogen-derived target derived from a parasite selected from the group consisting of parasites derived from the genus Babesia, parasites derived from the genus Plasmodium, parasites derived from the genus Trypanosoma, parasites derived from the genus Leishmania, parasites derived from the genus Anaplasma, parasites derived from the genus Toxoplasma, Babesia microti, Babesia divergens, Babesia duncani, Plasmodium falciparum, Plasmodium malariae, Plasmodium ovale, Plasmodium vivax, and Plasmodium knowlesi.

[0017] In one embodiment of the method where the target is a nucleic acid target, the separating step comprises contacting the pooled lysate with a capture probe and an immobilization probe, the capture probe having a first segment complementary to the nucleic acid target and a second segment complementary to the immobilization probe, wherein the nucleic acid target binds to the capture probe, and wherein the binding The captured probe binds to the immobilized probe. In one aspect, the immobilized probe is attached to a solid support. In one aspect, the pooled lysate is contacted with a solid support configured to immobilize the target; and the immobilized target is separated from the pooled lysate. In one aspect, hybridization conditions favorable for the formation of a hybridization complex between the first segment of the capture probe and the nucleic acid target are provided. In one aspect, hybridization conditions favorable for the formation of a hybridization complex between the second segment of the capture probe and the immobilized probe attached to the solid support are provided. In one aspect, the solid support is a magnetic bead solid support. In one aspect, the solid support is a silica solid support. In one aspect, the silica solid support is glass wool. In one aspect, the silica solid support is beads. In one aspect, the solid support is housed within a column. In one aspect, the method is performed without a centrifugation step.

[0018] In one embodiment, the entire volume of the aliquot of the lysed sample is combined. In one aspect, 25% or less of the volume of the aliquot of the lysed sample is combined. In one aspect, at least 50% of the blood cells are lysed within 5 minutes or less after contacting the sample aliquot with the lysis reagent.

Mode for Carrying Out the Invention

[0019] (Definition) "Pathogen" includes viruses, bacteria, protozoa, fungi, and other microorganisms that cause diseases in humans and other animals. Exemplary pathogens include, but are not limited to, hepatitis virus, human immunodeficiency virus, dengue virus, West Nile virus, flavivirus, Zika virus, and parasites. Exemplary parasites include, but are not limited to, parasites derived from the genus Babesia, parasites derived from the genus Plasmodium, parasites derived from the genus Trypanosoma, parasites derived from the genus Leishmania, parasites derived from the genus Anaplasma, parasites derived from the genus Toxoplasma, Babesia microti, Babesia divergens, Babesia duncani, Plasmodium falciparum, Plasmodium malariae, Plasmodium ovale, Plasmodium vivax, and Plasmodium knowlesi.

[0020] As used herein, the term "target" can be a single type of molecule (e.g., a specific protein or nucleic acid derived from a pathogen or a host cell). Alternatively, the target can be a class of molecules (e.g., any protein target or nucleic acid target derived from a pathogen or a host cell). For example, a target that is a single type of nucleic acid target derived from a pathogen is a ribosomal RNA target derived from a parasite (e.g., 18S derived from a Babesia organism) be a rRNA target), or the target, which is a class of nucleic acids derived from a pathogen, can be total RNA derived from a whole blood sample suspected of being infected with the pathogen. The target derived from the host cell can be a therapeutic target (e.g., an antibody or a protein (such as a coagulation factor)). Multiple distinct targets (e.g., an RNA target and a protein target, or two distinct RNA targets (e.g., two different mRNA targets, or an mRNA target and an rRNA target)) can also be analyzed. The target includes an endogenous component of the host's blood cells (host-derived target) and a component that results from a pathogenic infection of infected red blood cells and is typically encoded by the infecting pathogen (i.e., a "pathogenic" or "pathogen-derived" target).

[0021] As used herein, a "lysing reagent" is a reagent (often provided in solution form) that is effective for inducing lysis of blood cells in a sample including a whole blood sample, a sample containing red blood cells, a sample containing white blood cells, and a sample containing red blood cell products (e.g., pelleted red blood cells). In some examples, the lysing reagent preferentially lyses red blood cells. Preferentially lysing red blood cells over other cellular components of the blood means that the percentage of lysed red blood cells is higher than the percentage of other cellular components present in the sample being analyzed (other cell types are evaluated in aggregates). Many lysing reagents are useful for the methods of pooling lysates described herein. Preferred lysing reagents consist of: a buffer, lithium lauryl sulfate (LLS), and at least one anticoagulant selected from the group consisting of chloride-containing salts and EDTA, EDTA-Na2, EGTA, and combinations thereof; wherein the above reagents have a pH higher than 5.5.

[0022] As used herein, "sample aliquot" refers to a smaller volume of a sample. Typically, the sample aliquot is taken from a larger sample volume for testing. Each of the sample aliquots is preferably dissolved separately using a lysis reagent as described herein. The dissolved sample aliquots are then combined to form a pooled lysate. The pooled lysate includes other components such as non-target substances released from within the blood cells, and any targets present in one or more of the now-dissolved sample aliquots, along with undissolved blood cells. As used herein, multiple sample aliquots refers to two or more sample aliquots, each of which is drawn from two or more samples. The sample aliquots are preferably not combined until after the sample aliquots have been mixed with the lysis reagent under conditions that promote lysis of at least some of the blood cells in the sample aliquots.

[0023] An anionic detergent is a compound that has a negatively charged, anionic head group and a long hydrocarbon tail, and is often provided as a salt with an alkali metal or ammonium ion.

[0024] An anticoagulant inhibits the clotting of whole blood. Examples of anticoagulants include heparin and calcium chelating agents. Heparin activates antithrombin III, which inhibits the activity of thrombin and other proteases involved in blood clotting. Calcium chelating agents (e.g., EDTA (ethylenedinitrilo) tetraacetic acid), EDTA-Na2 (disodium ethylenediaminetetraacetate dihydrate), EGTA (ethylene-bis(oxyethylenenitrilo) tetraacetic acid), and citrate) bind calcium ions required for blood clotting.

[0025] A buffer refers to a weak acid or weak base used to maintain the pH of a solution. Preferred buffers for use herein include, but are not limited to, sodium bicarbonate, TRIS (2-amino-2-(hydroxymethyl)-1,3-propanediol), sodium hydrogen phosphate, and sodium dihydrogen phosphate.

[0026] A nucleic acid refers to a multimeric compound (including conventional RNA, DNA, hybrid RNA-DNA, and their analogs) containing nucleotides or analogs having nitrogenous heterocyclic bases or base analogs that are joined together to form a polymer.

[0027] This nitrogenous heterocyclic base may be referred to as a nucleobase. Nucleobases can be conventional DNA bases or RNA bases (A, G, C, T, U), base analogs, etc. (For example, The Biochemistry of the Nucleic Acids 5-36; Adams et al., ed., 11.sup.th ed., 1992; van Aerschott et al., 1995, See Nucl. Acids Res. 23(21): 4363-70; Nair et al., 2001, Nucleosides Nucleotides Nucl. Acids, 20(4-7):735-8; Hill et al., 1998, Proc. Natl. Acad. Sci. USA 95(8):4258-63; Lin and Brown, 1992, Nucl. Acids Res. 20(19):5149-52; Okamoto et al., 2002, Bioorg. Med. Chem. Lett. 12(1):97-9; Nguyen et al., 1998, Nucl. Acids Res. 26(18):4249-58; Kiopffer & Engels, 2005, Nucleosides Nucleotides Nucl. Acids, 24(5-7) 651-4; Babu & Wengel, 2001, Chem. Commun. (Camb.) 20: 2114-5; Hrdlicka et al., 2005, J. Am. Chem. Soc. 127(38): 13293-9; U.S. Patent No. 5,378,825; WO93 / 13121; Gamper et al., 2004, Biochem. 43(31): 10224-36; and Berger et al., 2000, Nucl. Acids Res. 28(15): 2911-4). Many derivatized and modified nucleobases or analogs are commercially available (e.g., Glen Research, Sterling, Va.).

[0028] Nucleobase units attached to sugars may be referred to as nucleobase units or monomers. The sugar moiety of the nucleic acid can be ribose, deoxyribose, or a similar compound (e.g., having 2'-methoxy or 2'-halide substitution). Nucleotides and nucleosides are examples of nucleobase units. This nucleobase unit can be linked by various linkages or conformations (phosphodiester, phosphorothioate or methylphosphonate linkages, peptide-nucleic acid linkages (PNA; Nielsen et al., 1994, Bioconj. Chem. 5(1): 3-7; PCT number WO 95 / 32305), and locked nucleic acid (LNA) conformations where the nucleotide monomer having a bicyclic furanose unit is locked in an RNA-mimicking sugar conformation (Vester et al., 2004, Biochemistry 43(42):13233-41; Hakansson & Wengel, 2001, Bioorg. Med. Chem. Lett. 11 (7):935-8), or combinations of such linkages in a nucleic acid strand). Nucleic acids can contain one or more "abasic" residues. That is, its backbone contains no nitrogenous bases at one or more positions (U.S. Patent No. 5,585,481).

[0029] Nucleic acids may contain only conventional RNA or DNA sugars, bases, and linkages, or may contain both conventional components and substitutions (e.g., conventional RNA bases having 2'-O-methyl linkages, or mixtures of conventional bases and analogs). Inclusion of PNA, 2'-methoxy- or 2'-fluoro-substituted RNA, or structures that affect the overall charge, charge density, or steric association of the hybridization complex (e.g., oligomers containing charged linkages (e.g., phosphorothioates) or neutral groups (e.g., methylphosphonates)) can affect the stability of the duplex formed by the nucleic acid.

[0030] The oligomers are substantially the same in overall length and other properties, but at least a part of the oligomers comprise a "random polymer" sequence which is a population of oligomers in which at least a portion of the oligomers are synthesized by random incorporation of different bases for a particular length (e.g., a random assortment of all four standard bases (A, T, G, and C) in a DNA oligomer, or a random assortment of some bases (U or T and G) in a defined portion of a larger oligomer). The resulting oligomers are actually a population of oligomers in which a finite number of members are determined by the length and number of bases that make up the random portion (e.g., in a population of oligomers containing a 6nt random sequence synthesized by using two different bases there are 2 6 species of oligomers).

[0031] Nucleic acid complementarity means that the nucleotide sequence of one strand of a nucleic acid hydrogen bonds to another sequence on the opposing nucleic acid strand due to the orientation of the bases of the nucleic acid bases. Complementary bases are typically A and T and C and G for DNA, and C and G, and U and A for RNA. Complementarity can be complete (i.e., exact) or substantial / sufficient. Complete complementarity between two nucleic acids means that the two nucleic acids can form a double strand in which every base in the double strand is bound to a complementary base by Watson-Crick pairing. "Substantial" or "sufficient" complementarity means that the sequence of one strand is not perfectly and / or completely complementary to the sequence of the opposing strand, but sufficient binding occurs between the bases on these two strands to form a stable hybrid complex under a set of hybridization conditions (e.g., salt concentration and temperature). Such conditions can be estimated by using the sequence and standard mathematical calculations to estimate the Tm of the hybridized strands or by the empirical determination of Tm using conventional methods. Tm refers to the temperature at which 50% of the population of hybridization complexes formed between two nucleic acid strands is denatured. At temperatures below the Tm, formation of the hybridization complex is favored, whereas at temperatures higher than the Tm, melting or separation of the strands in the hybridization complex is favored. Tm can be estimated, for example, for nucleic acids with a known G+C content in aqueous 1M NaCl solution by using Tm = 81.5 + 0.41(% G+C), although other known Tm calculation methods take into account the structural characteristics of the nucleic acid.

[0032] "Isolate" or "separate" or "purify" means removing one or more components from a complex mixture (e.g., a sample). Preferably, the step of isolating, separating or purifying removes at least 70%, preferably at least 90%, and more preferably at least 95% w / w of the target nucleic acid from other sample components. The step of isolating, separating or purifying may optionally include additional washing steps to remove non-target sample components.

[0033] The "release" of a capture hybrid refers to separating one or more components of the capture hybrid from each other, for example, separating a target nucleic acid from a capture probe and / or a capture probe from an immobilization probe. The release of the target nucleic acid strand separates the target from the other components of the capture hybrid and makes the target available for binding to a detection probe. These other components of the capture hybrid can, without affecting the target detection, leave, for example, the capture probe strand bound to the immobilization probe on the capture support.

[0034] A "label" refers to a molecular moiety that directly or indirectly detects or generates a detectable response or signal, for example, by catalyzing a reaction that generates a detectable signal. Labels include luminescent moieties (e.g., fluorescent, bioluminescent, or chemiluminescent compounds), radioisotopes, members of specific binding pairs (e.g., biotin and avidin), enzymes or enzyme substrates, reactive groups, or chromophores (e.g., dyes or particles that produce a detectable color).

[0035] A "capture probe" includes a first segment that contains a target complementary region of an array, and a second segment for attaching this capture probe (or a hybridization complex containing this capture probe) to an immobilization probe. This first segment is configured to be substantially complementary to a specific target nucleic acid sequence such that the first segment and the target nucleic acid can hybridize to form a stable (i.e., having a detectable melting point) double-strand under hybridization conditions (e.g., those described in the examples). Alternatively, this first segment can be configured to non-specifically bind to nucleic acid sequences in a sample under hybridization conditions (see WO 2008 / 016988). This second segment includes a region of a sequence that is complementary to the sequence of the immobilization probe. Preferably, the chimeric capture probe is a nucleic acid homopolymer (e.g., poly-A or poly-T) that is covalently bound to the target complementary region of the capture probe and hybridizes to the complementary homopolymer of the immobilization probe (e.g., poly-T or poly-A, respectively) under appropriate conditions as previously described (U.S. Patent No. 6,110,678 to Weisburg et al.). The capture probe may further include a third segment that acts as a closing sequence to inactivate unbound target capture probes in the capture reaction. This third segment may be adjacent to the first segment that is opposite the second segment (e.g., capture sequence: target hybridizing sequence: closing sequence), or this third segment may be adjacent to the second segment that is opposite the first segment (e.g., closing sequence: capture sequence: target hybridizing sequence). See WO2006 / 007567 and US2009-0286249.

[0036] The "immobilized probe" contains a nucleic acid that is directly or indirectly bound to a support. This nucleic acid is complementary to the nucleic acid in the capture probe, but may or may not have the same length (number of nucleotide units) as that in the capture probe. The nucleic acid in this immobilized probe preferably contains at least 6 consecutive nucleotide units, and may contain, for example, 10 to 45 or 10 to 40 or 10 to 30 or 10 to 25 or 15 to 25 (including both ends) L-nucleotide units. The nucleic acid is preferably a homopolymer, more preferably a homopolymer of adenine or thymine. A preferred form of the immobilized probe is a homopolymer of 14 thymine residues for use in combination with a capture probe containing a second segment having a homopolymer of adenine residues, or contains a homopolymer of 14 thymine residues. The nucleic acid portion of the immobilized probe is typically provided in single-stranded form or, if not, is denatured into single-stranded form before or during use.

[0037] As used herein, "solid support" is any of a variety of materials useful as a support for immobilized probes (e.g., matrices or particles made from nitrocellulose, nylon, silica, polyacrylate, mixed polymers, polystyrene, silane polypropylene, and substances that can be magnetically attracted). Solid supports in the form of sheets, beads / spheres, wool / fibers and other common shapes are useful in this method. Monodisperse magnetic beads are preferred supports because they are relatively homogeneous in size and can be easily recovered from solution, preferably by applying a magnetic force to the reaction vessel in an automated system. The solid support may be widely dispersed in a slurry or may be packed in a column. The immobilized probe may be directly coupled to its capture support, for example, by using any of a variety of covalent bonds, chelation, or ionic interactions, or may be indirectly coupled via one or more linkers attached to the support. This linker is not intended to hybridize to the capture probe, but is intended to act as a spacer between the nucleic acid of the immobilized probe and its support and may contain one or more nucleotides.

[0038] A "detection probe" is a nucleic acid or other molecule that specifically binds to a target sequence and this binding directly or indirectly gives rise to a detectable signal indicating the presence of this target sequence. A detection probe need not be labeled to generate a detectable signal (for example, an electrical impulse resulting from the binding of the probe to its target sequence can be a detectable signal). A "labeled probe" is a probe that contains a label or is directly or indirectly bound to a label (for example, Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd ed., Chapt. 10; U.S. Patent No. 6,361,945, Becker et al.; U.S. Patent No. 5,658,737, Nelson et al.; U.S. Patent No. 5,656,207, Woodhead et al.; U.S. Patent No. 5,547,842, Hogan et al.; U.S. Patent No. 5,283,174, Arnold et al.; U.S. Patent No. 4,581,333, Kourilsky et al.; U.S. Patent No. 5,731,148, Becker et al.). For example, a detection probe can include a non-nucleotide linker and a chemiluminescent label attached to this linker (U.S. Patent Nos. 5,185,439, 5,585,481, and 5,639,604, Arnold et al.). Examples of detection probes include oligonucleotides about 5 to 50 nucleotides in length having an attached label that is detected in a homogeneous reaction (for example, one that uses differential hydrolysis of the label on bound or unbound probes).

[0039] The detection probe can have a nucleotide sequence that is in the same sense or the opposite sense as the target sequence, depending on the format of the assay. The detection probe can hybridize to the capture probe and to the same or a different segment of the target sequence. Some detection probes have an attached chemiluminescent marker, for example, an acridinium ester (AE) compound (U.S. Patent Nos. 5,185,439, 5,639,604, 5,585,481, and 5,656,744). In some detection probes, the acridinium ester label is attached to the central region of the probe near the region of the A and T / U base pairs by using a non-nucleotide linker that constrains the amine of the nucleotide base on both sides of the AE and provides a site for intercalation (U.S. Patent Nos. 5,585,481 and 5,656,744, Arnold et al.). Alternatively, the AE label can be attached to the 3' or 5' end of the detection probe, and this detection probe is used with a second oligomer that hybridizes adjacent to the detection probe on the target nucleic acid to limit the effect of nearby amines provided by the target nucleic acid. In some detection probes, an AE label located at or near a site of mismatch with a related non-target polynucleotide sequence allows discrimination between the target sequence and a related sequence that can differ by only one nucleotide. This is because the double-stranded region around the mismatch site is destabilized enough to render the AE on the probe hybridized to the related non-target sequence sensitive to hydrolysis degradation. HIV-1 and HCV can be detected using a modified form of the PROCLEIX (登録商標) ULTRIO assay. This modification requires replacing the D-polyA and D-polyT sequences in the capture probe and the immobilization probe with L-polyA and L-polyT, respectively.

[0040] "Hybridization conditions" refers to the cumulative environment in which one nucleic acid strand binds to a second nucleic acid strand through complementary strand interactions and hydrogen bonding to form a hybridization complex. Such conditions include the chemical components and their concentrations (e.g., salts, chelating agents, formamide) of an aqueous or organic solution containing nucleic acids, as well as the temperature of this mixture. Other factors (e.g., the length of the incubation time or the dimensions of the reaction chamber) can contribute to that environment (e.g., Sambrook et al., Molecular C loning, A Laboratory Manual, 2.sup.nd ed., pp. 1.90-1.91, 9.47-9.51, 11.47-11.57 (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989)).

[0041] Specific binding of a target capture oligomer to one or more target nucleic acids means the binding between one defined sequence in the first segment of the target capture oligomer that forms a stable duplex and a segment that is exactly or substantially complementary on the target nucleic acid. Such binding is detectably stronger (higher signal or melting temperature) than binding to other nucleic acids in the sample that lack a segment exactly or substantially complementary to that single defined target capture oligomer sequence. Nonspecific binding of a target capture oligomer to a target nucleic acid means that the target capture oligomer can bind to a population of target sequences that do not share a segment having exact or substantial complementarity to a single defined target capture oligomer sequence. For example, this can be achieved by using a randomized sequence in the first segment of the capture probe.

[0042] The "release" of a capture hybrid refers to separating one or more components of the capture hybrid from each other (e.g., separating a target nucleic acid from a capture probe and / or a target capture oligomer from an immobilization probe). The release of the target nucleic acid strand separates the target from the other components of the capture hybrid and makes the target available for binding to a detection probe. These other components of the capture hybrid can, without affecting target detection, leave, for example, the target capture oligomer strand bound to the immobilization probe on the capture support.

[0043] "Sensitivity" is the proportion of truly positive cases that are correctly identified as such (e.g., the percentage of infected blood samples that have an infection). Sensitivity can also be characterized by the lower limit of detection. For example, at a pathogen concentration of 1 particle / mL blood before pooling, the assay detects at least 95% of the infected samples. The lower the detection limit, the higher the assay sensitivity. Specificity measures the proportion of truly negative cases that are correctly identified (e.g., the percentage of non-infected blood samples that are correctly identified as not having an infection).

[0044] References to ranges of values also include integers within the range and sub-ranges defined by the integers within that range. References to any numerical value or range of values are to be understood as encompassing any such variations as may be essential when evaluating other representative conditions of use during measurement.

[0045] Detailed Description I. General The present disclosure provides a method for pooled analysis of a sample containing blood cells (e.g., a whole blood cell sample). The method is particularly useful for screening whole blood samples collected from a number of individuals intended for use in, for example, blood transfusions. Prior to pooling, an aliquot of such a sample is individually contacted with a lysis reagent (described further below) to lyse the red blood cells in the sample. After pooling, the combined lysate or a specimen thereof is tested for the presence of a target characteristic of a pathogen. By lysing prior to pooling, multiple target molecules (e.g., RNA) can be released prior to pooling, increasing the probability that an infected sample is detected relative to the background of non-infected samples pooled together. The method is particularly useful for identifying pathogens of red blood cells, but can also be used to perform a complete panel of tests that at least includes all of the representative targets described in the background.

[0046] II. Lysis Reagent The lysis reagent (which was first described in co-pending application WO 2016 / 064887, filed Oct. 20, 2015, incorporated by reference) includes at least a buffer, lithium lauryl sulfate (LLS), and at least one anticoagulant selected from the group consisting of a chloride-containing salt and EDTA, EDTA-Na2, EGTA, and combinations thereof.

[0047] In some embodiments, the buffer is a sodium bicarbonate buffer. In some embodiments, the buffer is a TRIS (2-amino-2-(hydroxymethyl)-1,3-propanediol) buffer. In some embodiments, the buffer is a sodium bicarbonate buffer. In some embodiments, the buffer is a sodium phosphate buffer. In some embodiments, the buffer is a sodium bicarbonate buffer in a reagent at a concentration of about 5 mM to about 30 mM, about 10 mM to about 20 mM, about 10 mM to about 15 mM, or about 15 mM to about 20 mM. In some embodiments, the buffer is a TRIS buffer in a reagent at a concentration of about 75 mM to about 150 mM, about 75 mM to about 125 mM, about 100 mM to about 125 mM, or about 90 mM to about 110 mM. In some embodiments, the buffer is a sodium phosphate (Na3PO4) buffer in a reagent at a concentration of about 5 mM to about 30 mM, about 10 mM to about 20 mM, about 10 mM to about 15 mM, or about 15 mM to about 20 mM. In some embodiments, the concentration of sodium phosphate in the reagent is about 8 mM to about 40 mM, about 10 mM to about 33 mM, about 15 mM to about 30 mM, about 30 mM, or about 15 mM. In some embodiments, the concentration of sodium monophosphate in the reagent is about 8 mM to about 40 mM, about 10 mM to about 33 mM, about 15 mM to about 30 mM, about 30 mM, or about 15 mM. In some embodiments, the concentration of disodium phosphate in the reagent is about 8 mM to about 40 mM, about 10 mM to about 33 mM, about 15 mM to about 30 mM, about 30 mM, or about 15 mM. The ranges include all integers and decimal (partial number) values within the range.

[0048] In some embodiments, the anticoagulant is one or more of EDTA ((ethylenedinitrilo) tetraacetic acid), EDTA-Na2 (disodium ethylenediaminetetraacetate dihydrate), EGTA (ethylene-bis(oxyethylenenitrilo) tetraacetic acid), heparin, or citrate. In some embodiments, the anticoagulant comprises EDTA in a reagent at a concentration of about 0.05 mM to about 15 mM, about 0.1 mM to about 10 mM, or about 0.5 mM to about 5 mM. In some embodiments, the anticoagulant is EDTA in a reagent at a concentration of about 0.05 mM to about 15 mM, about 0.1 mM to about 10 mM, or about 0.5 mM to about 5 mM. In some embodiments, the anticoagulant is EDTA-Na2 in a reagent at a concentration of about 0.05 mM to about 15 mM, about 0.1 mM to about 10 mM, or about 0.5 mM to about 5 mM. In some embodiments, the anticoagulant is EGTA in a reagent at a concentration of about 0.05 mM to about 15 mM, about 0.1 mM to about 10 mM, or about 0.5 mM to about 5 mM. The ranges include all integers and decimals within the ranges.

[0049] In some embodiments, the salt comprises one or more of the following ions: sodium ion, potassium ion, ammonium ion, magnesium ion, lithium ion, and chloride ion. In some embodiments, the salt is magnesium chloride, ammonium chloride, potassium chloride, or sodium chloride. In some embodiments, the salt comprises chloride ion and one of magnesium ion, sodium ion or potassium ion, and the concentration of the salt in the reagent is about 10 mM to about 50 mM, about 15 mM to about 40 mM, or about 20 mM to about 35 mM. In some embodiments, the salt is ammonium chloride in a reagent at a concentration of about 100 mM to about 500 mM, about 200 mM to about 350 mM, or about 250 mM to about 300 mM. The ranges include all integers and decimals within the ranges.

[0050] In some embodiments, the surfactant is one of lithium lauryl sulfate (LLS), nonylphenoxypolyethoxylethanol (NP 40), sodium dodecyl sulfate (SDS), and Triton-X 100. In some embodiments, the surfactant is an anionic surfactant. In some embodiments, the surfactant is LLS or SDS. In some embodiments, the surfactant is present in the reagent at a concentration higher than about 1.5% (v / v). In some embodiments, the surfactant is present in the reagent at a concentration less than about 15.5% (v / v). In some embodiments, the surfactant is present in the reagent at a concentration of about 2% to about 15% (v / v). In some embodiments, the surfactant is present in the reagent at a concentration of about 2% to about 15% (v / v). In some embodiments, the surfactant is LLS, and the concentration of LLS in the reagent is about 2% to about 15% (v / v), about 4% to about 10% (v / v), or about 5% to about 8% (v / v). In some embodiments, the surfactant is LLS and is present in the reagent at about 14 mM to about 50 mM. The ranges include all integers and decimals within the ranges.

[0051] In some embodiments, the pH of the reagent is higher than pH 5.5. In some embodiments, the pH of the reagent is lower than pH 10.5. In some embodiments, the pH of the reagent is about 6.0 to about 10.0. In some embodiments, the pH of the reagent is about 6.5 to about 8.0, or about 7.0 to about 8.0, or about 7.2 to about 7.6, or about 6.7 to about 7.5, or about 6.7, or about 7.3 or about 7.5. The ranges include all integers and decimals within the ranges.

[0052] In some embodiments of the reagent, the concentration of sodium bicarbonate is 14 mM, the concentration of ammonium chloride is 250 mM, the concentration of LLS is 8% (v / v), the concentration of EDTA is from about 0.1 mM to about 10 mM, and the pH is from 7.2 to 7.6. In some embodiments of the reagent, the buffer is selected from the group consisting of sodium bicarbonate, sodium phosphate, and TRIS, the surfactant is from about 5% to about 10% (v / v), the pH is from about 6.5 to about 8.0, and the salt is selected from the group consisting of magnesium chloride, ammonium chloride, and potassium chloride. In some embodiments of the reagent, the buffer is selected from the group consisting of sodium phosphate and TRIS, and the salt is selected from the group consisting of magnesium chloride and ammonium chloride. In some aspects of this embodiment, the concentration of the anticoagulant is from about 0 mM to about 1 mM. In some further aspects of this embodiment, the surfactant is LLS at a concentration of from about 6% to about 10% (v / v). In some further aspects of this embodiment, the buffer is TRIS at a concentration of from about 90 mM to about 110 mM, and the pH of the reagent is from about 7.2 to about 7.5. In some further aspects of this embodiment, the anticoagulant is at a concentration of from about 0.1 mM to about 5 mM and is EGTA, EDTA, EDTA-Na2, or a combination thereof.

[0053] The lysis reagent functions to lyse blood cells and protect the released targets from degradation by nucleases or proteases in the lysate and is compatible with subsequent steps (e.g., target capture, amplification, detection, and / or sequencing) for the use of those targets. The lysis reagent is particularly suitable for the analysis of RNA from pathogens that infect red blood cells (such as parasites like Babesia and Plasmodium species). The lysis reagent is compatible with methods of nucleic acid detection without removing the lysis reagent from the sample.

[0054] (III. Use of the Lysis Reagent) Blood cells can be obtained from any available source (e.g., whole blood or any fraction thereof that contains red blood cells (e.g., pelleted red blood cells)). Whole blood can be human whole blood, non-human whole blood, or a combination thereof.

[0055] The lysis reagent can be mixed with the blood cells for a time sufficient to induce cell lysis and cause release of the desired target from the cells. Exemplary times for maintaining the blood cells in mixture with the lysis reagent include 1 to 30 minutes, 2 to 15 minutes, 3 to 10 minutes, 4 to 6 minutes, or 5 minutes. Preferably, the time is at least 5 minutes. Preferably, the mixture is free of visible particles after lysis.

[0056] The temperature at which the lysis reagent is incubated with the blood cells can vary. The temperature is preferably selected to maximize the degree and rate of lysis and minimize degradation of the target or prevent inhibition of subsequent processing. Exemplary temperature ranges include 0 to 50 °C, 5 to 45 °C, 10 to 40 °C, 15 to 37 °C, 20 to 30 °C, 22 to 27 °C, or 25 °C. Ambient temperature is appropriate. Lysis of the blood cells should release an amount of target molecule sufficient to be detectable by the methods described herein. Preferably, lysis results in lysis of at least 50%, 60%, 70%, 80%, 90%, or 100% of the blood cells in the sample being lysed. The range includes all integers and decimals within the range.

[0057] The ratio at which the blood cell sample is combined with the lysis reagent can affect the degree and rate of cell lysis, as well as the protection of the target molecule from degradation after release from the lysed cells. Exemplary ratios at which the blood cell sample is mixed with the above lysis reagent include ratios within the range of about 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, or between 1:1 and 1:5 or 1:10 (v / v; sample: reagent). A preferred ratio is that the sample aliquot is mixed with the lysis reagent at a ratio of about 1:3 (v / v sample: reagent). Another preferred ratio is that the sample aliquot is mixed with the lysis reagent at a ratio of about 1:4 (v / v sample: reagent). When the above sample contains red blood cells isolated from whole blood (e.g., pelleted red blood cells), these red blood cells can be mixed with the above lysis reagent at exemplary ratios within the range of 1:1, 1:2, 1:3, 1:4, 1:5, 1:10, or between 1:1 and 1:10 (v / v; red blood cell: reagent). The range includes all integers and decimals within the range.

[0058] IV. Target Targets released from blood cells by the lysis reagent of the present invention can include host-derived targets and pathogen-derived targets. Targets include protein targets (e.g., peptide targets and antibody targets), nucleic acid targets (e.g., DNA targets or RNA targets), whole particles, lipids, and carbohydrates. The RNA target can be a ribosomal RNA (rRNA) target, a messenger RNA (mRNA) target, or a heterogeneous nuclear RNA (hnRNA) target. Preferred targets among pathogen-derived targets are ribosomal RNA targets, particularly 18S rRNA targets, 5S rRNA targets, 5.8S rRNA targets, or 28S rRNA targets. The target can be released from white blood cells, red blood cells, or can be present in plasma. The target includes therapeutic targets. The therapeutic target is preferably a host-derived target (e.g., a peptide target (e.g., thrombin) and an antibody target).

[0059] Exemplary pathogenic organisms include hepatitis virus, human immunodeficiency virus, dengue virus, West Nile virus, flavivirus, Zika virus, and parasites. Exemplary parasites include parasites derived from the genus Babesia, parasites derived from the genus Plasmodium, parasites derived from the genus Trypanosoma, parasites derived from the genus Leishmania, parasites derived from the genus Anaplasma, parasites derived from the genus Toxoplasma, Babesia microti, Babesia divergens, Babesia duncani, Plasmodium falciparum, Plasmodium malariae, Plasmodium ovale, Plasmodium vivax, and Plasmodium knowlesi.

[0060] (V. Assay) Target molecules released from the lysis of a sample aliquot containing blood cells are subjected to analysis. The target molecules may or may not be separated from the lysis reagent (by centrifugation or other means) prior to analysis. Omission of the separation step can facilitate an efficient workflow in performing the assay. The type of assay depends on the target. The assay can be a real-time or endpoint assay.

[0061] (A. Nucleic Acid) Analysis of nucleic acid targets often involves the steps of capture, amplification, and detection. Alternatively, the amplification and detection methods can be performed without prior target capture. Preferably, amplification, as well as detection and target capture (if performed), occur without separating the target molecules from the lysis reagent. Thus, the entire process can be performed in one container.

[0062] (1. Target Capture Assay) Exemplary target capture assays are performed using one or more capture probes, immobilization probes, samples, and appropriate media as follows to enable hybridization of the target capture oligomers to the target nucleic acid and to the immobilization probes. The target sample (preferably herein, the target sample is a pooled lysate) can be heated (e.g., from 65°C to 95°C) prior to performing the assay to denature any nucleic acid in double-stranded form. Its components can be mixed in any order. For example, the target capture oligomer can be added to the target sample and hybridized to the nucleic acid target in the target sample before adding the immobilization probe. However, for automated assays, it is preferred to minimize the number of addition steps by supplying the target capture oligomer and the immobilization probe simultaneously or substantially simultaneously. In this case, the hybridization order can be controlled by performing a first hybridization under conditions that can form a duplex between the target capture oligomer of the first segment and the nucleic acid target, but above the melting temperature of the duplexes formed between the first segment and the second segment of the capture probe and between the target capture oligomer and the immobilization probe. Next, a second hybridization is performed under reduced stringency, preferably under conditions below the melting temperature of the duplex formed between the target capture oligomer of the first segment and the nucleic acid target. Under the second hybridization conditions, a duplex can be formed between the second segment of the capture probe and the immobilization probe. Stringency can be reduced by lowering the temperature of the assay mix. For example, a higher stringency hybridization can be performed at or near 60°C, and a lower stringency hybridization can be performed by cooling to room temperature or 25°C. Stringency can also be reduced by lowering the salt concentration or adding or increasing the concentration of a chaotropic solvent. In some methods, all steps (except possibly the initial denaturation step of the double-stranded target at a higher temperature) can be performed isothermally.

[0063] Nucleic acid target: After formation of the capture probe: immobilized probe hybrid (capture complex), the capture complex is separated from other sample components. One way to separate the capture complex from other sample components is to physically separate the solid support containing the capture complex from other sample components by using any of various known methods (e.g., centrifugation, filtration, or magnetic attraction of a magnetic capture support). The above separation is preferably carried out at a temperature below the melting temperature of the double strand formed between the nucleic acid target, the target capture oligomer, and the immobilized probe. In some methods, the above separation is carried out at a temperature lower than the melting temperature of this stem-loop structure but within 10 °C of this melting temperature (e.g., at 60 °C) in order to maintain the ability to distinguish between hybridization conditions and the need to distinguish between a target nucleic acid that matches and a target nucleic acid that does not match. Another method includes a step that allows the capture complex to remain attached to the solid support placed in the column while the sample components elute from the column and into one or more containers. The column containing the capture complex is then subjected to elution conditions to allow the captured nucleic acid target to elute into a separate container (see, for example, U.S. Patent No. 6,110,678). To further facilitate isolation of the target nucleic acid from other sample components that non-specifically adhere to any part of the capture hybrid, the capture hybrid can be washed one or more times to dilute and remove other sample components. Washing is carried out with a suitable aqueous solution (e.g., a solution containing Tris and EDTA. See, for example, U.S. Patent No. 6,110,678) and under suitable conditions (e.g., the T of the above components

[0064] of the above components). mAt a higher temperature), the capture hybrid can be dissociated into its individual components and then the conditions can be re-adjusted to allow for re-formation of the capture hybrid. However, to make handling easier and minimize the process, washing is preferably carried out by rinsing the intact capture hybrid attached to the capture support in solution by using the conditions that maintain this capture hybrid. Preferably, when carried out, the capture of the target nucleic acid in washing isolates at least 70%, preferably at least 90%, and more preferably about 95% of the above target nucleic acids from other sample components. The isolated nucleic acid can be used for many downstream processes such as nucleic acid amplification.

[0065] The target capture assay can also be performed as part of a real-time biphasic target capture and amplification method. In such a method, 500 μL of sample and 400 μL of target capture reagent (TCR) are added to a reaction tube. The TCR includes magnetic particles, components for lysing organisms present in the sample, capture oligos, a T7 start promoter, and an internal calibration factor. The fluid in the reaction tube is mixed at a specific rate over a specific time to ensure that the mixture is homogeneous. The reaction tube is then transferred to a 43.7 °C transition incubator to preheat the fluid in the reaction tube. The reaction tube is then transferred to an annealing incubator set at 64 °C. During incubation at 64 °C, any organisms present in the sample that were not previously lysed by the lysis reagent are lysed, causing release of the target. The reaction tube is then transferred to a transfer incubator to initiate a cool-down process and is further cooled within a cooling gradient (17 °C to 19 °C), resulting in binding of the T7 start promoter and capture of both the target and the internal calibration factor to the magnetic particles via the capture oligos. The reaction tube is transferred to a magnetic parking station where the tube is subjected to a magnet that attracts the magnetic particles to the side of the tube before entering a wash station. In the wash station, potential interfering substances are removed from the reaction by washing the magnetic particles.

[0066] 2. Amplification Nucleic acid analytes can be amplified using methods such as isothermal amplification reactions (e.g., transcription-mediated amplification (TMA), nucleic acid sequence-based amplification (NASBA), loop-mediated isothermal amplification, polymerase spiral reaction (PSR) (Liu, W. et al. Polymerase Spiral Reaction (PSR): A novel isothermal nucleic acid amplification method. Sci. Rep. 5, 12723; (2015)), ligase chain reaction, and other isothermal amplification methods), or temperature cycling amplification reactions (e.g., polymerase chain reaction (PCR), quantitative PCR (qPCT), real-time PCR (rt-PCT), or other temperature cycling amplification methods), or other amplification methods. Detection of the amplified RNA analyte product can be performed during (in real-time) or after (endpoint) amplification.

[0067] (i. Transcription-mediated amplification) TMA has been described previously (e.g., U.S. Pat. Nos. 5,399,491, 5,554,516, 5,824,518, and 7,833,716; and also, e.g., F. Gonzales and S. McDonough. Applications of Transcription-Mediated Amplification to Quantification of Gene Sequences. Gene Amplification. 1998 Ed. Francois Ferre, Birkhauser, Boston. PP. 189-204). In TMA, a target nucleic acid containing the sequence to be amplified is provided as a single-stranded nucleic acid (e.g., ssRNA or ssDNA). Any conventional method for converting double-stranded nucleic acid (e.g., dsDNA) to single-stranded nucleic acid can be used. A promoter primer specifically binds to the target nucleic acid at its target sequence, and reverse transcriptase (RT) uses the target strand as a template to extend the 3′ end of the promoter primer to generate a cDNA copy, yielding an RNA:cDNA duplex. RNase activity (e.g., RNase H of the RT enzyme) digests the RNA of the RNA:cDNA duplex, and a second primer specifically binds to its target sequence in this cDNA that is downstream from the promoter-primer end. Then, RT synthesizes a new DNA strand by using this cDNA as a template to extend the 3′ end of the second primer to create a dsDNA containing a functional promoter sequence. RNA polymerase specific for the functional promoter initiates transcription to generate approximately 100 to 1000 RNA transcripts (amplified copies or amplicons) complementary to the first target strand. This second primer specifically binds to its target sequence in each amplicon, and RT creates cDNA from this amplicon RNA template to generate an RNA:cDNA duplex.RNase digests the amplicon RNA from the RNA:cDNA duplex, and the target-specific sequence of this promoter primer binds to its complementary sequence in the newly synthesized DNA. RT extends the 3'-end of the promoter primer and the 3'-end of the cDNA to create a dsDNA containing a functional promoter to which RNA polymerase binds and transcribes additional amplicons complementary to the target strand. An autocatalytic cycle that repeatedly uses these steps during the reaction results in amplification of approximately one billion-fold of the original target sequence. Optionally, the amplicon can be detected during amplification (real-time detection) or at the end-point of the reaction (end-point detection) by using a probe that specifically binds to a sequence contained in the amplicon. Detection of the signal arising from the bound probe indicates the presence of the target nucleic acid in the sample.

[0068] TMA can also be performed as part of a real-time biphasic target capture and amplification method. In such a method, TMA can be performed by adding the amplification reagents (50 μL / test) to a reaction tube containing the captured target molecules and mixing in an amplification loading station. The amplification reagents contain the oligos and components necessary to make nucleic acids. The reaction tube is transferred to a transfer incubator at 43.7 °C to raise the temperature of the liquid in the reaction tube, and then it is returned to the amplification loading station where the enzyme (25 μL / test) is added. The reaction tube is transferred to an amplification incubator set at 42.7 °C and left in the incubator for 5 minutes during which the first round of amplification is initiated. The reaction tube is returned to the amplification loading station where the promoter reagent (25 μL / test) is added. The reaction tube is returned to the amplification incubator for additional rounds of target amplification. This promoter reagent includes an oligo and a torch. This torch is complementary to the above target or internal calibration factor, and when bound, emits fluorescence and generates a signal in real time. The signals regarding the above target and internal calibration factor preferably have different wavelengths and can be distinguished.

[0069] (ii. Polymerase Chain Reaction) Alternatively, PCR amplification (e.g., reverse transcriptase or real-time PCR) can be used for amplification. PCR can be performed with or without prior release of the target nucleic acid from the capture complex. This PCR reaction can be performed in the same container (e.g., a microcentrifuge tube) as the capture step. This PCR reaction involves thermocycling between a high temperature of about 95 °C (e.g., 90 - 99 °C) for dissociation and a low temperature of about 60 °C (e.g., 40 - 75 °C, or 50 - 70 °C or 55 - 64 °C) for annealing. Typically, the number of complete thermocycles is at least 10, 20, 30 or 40. PCR amplification is performed using one or more primer pairs. The primer pair used for PCR amplification includes two primers complementary to the opposing strands of the target nucleic acid adjacent to the region whose sequence is desired to be determined. To sequence most of the viral genome (e.g., more than 50, 75 or 99%), the primers are preferably located near the ends of the viral genome. For amplification of related molecules (e.g., mutant forms of the same virus present in a patient sample), the primers are preferably complementary to conserved regions of the target nucleic acid that are likely to be present in most members of the population. PCR amplification is described below: PCR Technology: Principles and Applications for DNA Amplification (edited by H.A. Erlich, Freeman Press, NY, NY, 1992); PCR Protocols: A Guide to Methods and Applications (edited by Innis et al., Academic Press, San Diego, CA, 1990); Mattila et al., Nucleic Acids Res. 19, 4967 (1991); Eckert et al., PCR Methods and Applications 1, 17 (1991); PCR (edited by McPherson et al., IRL Press, Oxford); and U.S. Patent No. 4,683,202.

[0070] (3. Detection) Detection of the nucleic acid target can be performed either during amplification (real-time) or after amplification (endpoint), after capture and using any known method. The amplification products of RNA are often in the form of DNA resulting from RT-PCR or RNA copies resulting from TMA. The amplified nucleic acids can be detected in solution or they can be concentrated in or on a matrix and detected by detecting a label associated with the nucleic acid (e.g., an intercalating agent such as ethidium bromide). Some detection methods use a probe complementary to a sequence in the amplified product to detect the presence of a probe:product complex or use a complex of probes to amplify a signal detected from the amplified product (e.g., U.S. Patent Nos. 5,424,413, 5,451,503, and 5,849,481). Other detection methods use probes where signal generation is linked to the presence of the target sequence. Because a change in signal occurs only when the labeled probe binds to the amplified product, as in molecular beacons, molecular torches, or hybridization switch probes (e.g., U.S. Patent Nos. 5,118,801, 5,210,015, 5,312,728, 5,538,848, 5,541,308, 5,656,207, 5,658,737, 5,925,517, 6,150,097, 6,361,945, 6,534,274, 6,835,542, and 6,849,412; and U.S. Publication No. 2006 / 0194240 A1) is. Such a probe typically has a label (e.g., a fluorophore) attached to one end of the probe, and an interaction compound (e.g., a quencher) attached to another position of the probe such that when the probe is in a certain conformation ( "closed") that inhibits signal generation from the label when the probe is not hybridized to the amplified product, a detectable signal is generated when the probe hybridizes to the amplified product that changes its conformation (to "open"). Detection of a signal from a probe labeled directly or indirectly that specifically associates with the amplified product indicates the presence of the amplified target nucleic acid.

[0071] (4. Sequencing) Following amplification, the target nucleic acid can be detected qualitatively or quantitatively, or alternatively, can be sequenced instead of being detected qualitatively or quantitatively. Purification can be performed, if desired, on a silica column (e.g., Qiagen gravity flow column). The target nucleic acid binds to the column, where it can be washed and then eluted. Alternatively, purification can be performed using a nucleic acid probe-based purification system (e.g., U.S. Patent Nos. 6,110,678 or 8,034,554, US 2013 / 0209992 or US 2009 / 0286249, or WO 2012 / 037531 or WO 2013 / 116774). The amplified target DNA can also be adapted to several sequencing formats by attachment of adapters. The amplified DNA can be tailed by Klenow-mediated addition of nucleotides (usually homopolymers), followed by annealing and ligation to an oligonucleotide complementary to the added tail. Depending on the sequencing platform used, special adapters are ligated to the template prior to sequencing. For example, SMRT Bell adapters are ligated to sample templates for sequencing on Pacific Biosciences’ PacBio RS sequencer (see, e.g., Travers et al. Nucl. Acids Res. (2010) 38 (15): e159).

[0072] The amplified target nucleic acid is suitable for sequence analysis by various techniques. Capture of the target nucleic acid can lead to several different formats of so-called next-generation and third-generation sequencing methods. Such methods can sequence millions of target templates in parallel. Such methods are particularly useful when the target nucleic acid is a heterogeneous mixture of variants. Among its many advantages, parallel sequencing of variants provides a profile of drug resistance mutations in the sample, even if drug mutations are present in a relatively small proportion within this sample.

[0073] Some next-generation sequencing methods amplify by emulsion PCR. Target nucleic acids immobilized on beads via target capture oligomers provide a suitable starting material for emulsion PCR. The beads are mixed with PCR reagents and emulsion oil to create individual microreactors containing a single bead (Margulies et al., Nature 437, 376-80 (2005)). The emulsion is then disrupted, and the individual beads with amplified DNA are sequenced. This sequencing can be, for example, pyrosequencing performed using a Roche 454 GS FLX sequencer (454 Life Sciences, Branford, CT 06405). Alternatively, the sequencing can be, for example, ligation / detection performed using an ABI SOLiD Sequencing System (Life Technologies, Carlsbad, CA 92008). In another variation, the target nucleic acid is eluted from the beads with target capture oligomers and immobilized at different positions on an array (e.g., HiScanSQ (Illumina, San Diego, CA 92121)). The target nucleic acid is amplified by bridge amplification and sequenced in array format by template-directed incorporation of labeled nucleotides (Illumina). In another approach, the target nucleic acid is eluted from the target capture oligomers and a single molecule is analyzed by detecting nucleotide incorporation by polymerase in real time (single molecule real time sequencing or SMRT sequencing). The nucleotide can be a labeled nucleotide that emits a signal when incorporated (e.g., Pacific Biosciences, Eid et al., Sciences 323 pp. 133-138 (2009)) or an unlabeled nucleotide (in which case the system measures chemical changes upon incorporation (e.g., Ion Torrent Personal Genome Machine (Life Technologies)). It is amplified by bridge amplification and sequenced in array format by template-directed incorporation of labeled nucleotides (Illumina). In another approach, the target nucleic acid is eluted from the target capture oligomers and a single molecule is analyzed by detecting nucleotide incorporation by polymerase in real time (single molecule real time sequencing or SMRT sequencing). The nucleotide can be a labeled nucleotide that emits a signal when incorporated (e.g., Pacific Biosciences, Eid et al., Sciences 323 pp. 133-138 (2009)) or an unlabeled nucleotide (in which case the system measures chemical changes upon incorporation (e.g., Ion Torrent Personal Genome Machine (Life Technologies)).

[0074] The captured target nucleic acid can be sequenced by any technique, but third-generation, next-generation or massively parallel methods offer significant advantages over Sanger and Maxam Gilbert sequencing. Several groups have described ultra-high-throughput DNA sequencing procedures (see, for example, Cheeseman, U.S. Patent No. 5,302,509, Metzker et al., Nucleic Acids Res. 22: 4259 (1994)). Pyrosequencing approaches that use the four natural nucleotides (including the bases adenine (A), cytosine (C), guanine (G), or thymine (T)) and several other enzymes for sequencing DNA are now widely used for mutation detection (Ronaghi, Science 281, 363 (1998); Binladin et al., PLoS ONE, issue 2, e197 (February 2007); Rehman et al., American Journal of Human Genetics, 86, 378 (March 2010); Lind et al., Next Generation Sequencing: The solution for high-resolution, unambiguous human leukocyte antigen typing, Hum. Immunol. (2010), doi 10.1016 / jhumimm.2010.06.016 (in press); Shafer et al., J Infect Dis. 1;199(5):610 (2009)). In this approach, detection is based on the pyrophosphate (PPi) released during the DNA polymerase reaction, the quantitative conversion of pyrophosphate to adenosine triphosphate (ATP) by sulfurylase, and subsequent generation of visible light by firefly luciferase. More recent studies have focused mostly on synthetic methods that ligate a photocleavable chemical moiety linked to a fluorescent dye to cap the 3'-OH group of deoxynucleoside triphosphates (dNTPs) for DNA sequencing (Welch et al, Nucleosides and Nucleotides 18, 197 (1999) & European Journal, 5:951-960 (1999); Xu et al., U.S. Patent No. 7,777,013; Williams et al., U.S. Patent No. 7,645,596; Kao et al, U.S. Patent No. 6,399,335; Nelson et al., U.S. Patent Nos. 7,052,839 and 7,033,762; Kumar et al., U.S. Patent No. 7,041,812; Sood et al, U.S. Patent Application No. 2004-0152119; Eid et al, Science 323, 133 (2009)). In the sequencing-by-synthesis methodology, the DNA sequence is derived by measuring the pyrophosphate release when testing the DNA / polymerase complex using each deoxyribonucleotide triphosphate (dNTP) separately and sequentially. See Ronaghi et al, Science 281: 363 365 (1998); Hyman, Anal. Biochem. 174, 423 (1988); Harris, U.S. Patent No. 7,767,400.

[0075] (B. Other targets) Antibodies, proteins, particles, and other targets can be detected by formats such as immunoprecipitation, Western blotting, ELISA, radioimmunoassay, competitive assay, and immunometric assay. See Harlow & Lane, Antibodies: A Laboratory Manual (CSHP NY, 1988); U.S. Patent Nos. 3,791,932; 3,839,153; 3,850,752; 3,879,262; 4,034,074, 3,791,932; 3,817,837; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; and 4,098,876. Sandwich assays are a preferred format (see U.S. Patent Nos. 4,376,110; 4,486,530; 5,914,241; and 5,965,375).

[0076] Competitive assays can also be used. In some methods, the target antigen in a sample competes with an exogenously supplied, labeled target antigen for binding to an antibody detection reagent. The amount of labeled target antigen that binds to the antibody is inversely proportional to the amount of target antigen in the sample. The antibody can be immobilized to facilitate separation of the bound complex from the sample prior to detection.

[0077] Tangential flow devices can also be used to detect targets. A fluid is applied to a test strip that has been treated with a sample in which a target may be present. Labeled binding molecules can be captured as they pass through the strip and enter a specific zone containing the sample with the target.

[0078] (VI. Sensitivity) The method of the present invention can provide high detection sensitivity for a target from a sample aliquot containing blood cells (e.g., whole blood, white blood cells, red blood cells, or other preparations of red blood cells). For pathogen-derived RNA targets, the sensitivity can be expressed as the minimum number of pathogen RNA copies present in a volume of whole blood. The volume of whole blood can be one that can be brought into direct contact with the lysis reagent or one that can be used to prepare a blood fraction (e.g., pelleted red blood cells), which in turn is brought into contact with this lysis reagent. Preferably, the method detects the presence of pathogenic RNA in whole blood with a sensitivity of about 2×10 3 copies of ribosomal RNA / mL (equivalent to 1 parasite / 1 mL) whole blood or better, 2×10 3 copies / 5 mL whole blood or better, 2×10 3 copies / 10 mL whole blood or better, 2×10 3 copies / 50 mL whole blood or better, or 2×10 3 copies / 100 mL whole blood or better. Preferably, the method detects the presence of pathogenic RNA in whole blood with a sensitivity of about 8×10 3 copies of ribosomal RNA / mL (equivalent to 4 parasites / 1 mL) whole blood or better, 8×10 3 copies / 5 mL whole blood or better, 8×10 3 copies / 10 mL whole blood or better, 8×10 3 copies / 50 mL whole blood or better, or 8×10 3 copies / 100 mL whole blood or better. Preferably, the method detects the presence of pathogenic RNA in whole blood with a sensitivity of about 24×10 3 copies of ribosomal RNA / mL (equivalent to 12 parasites / 1 mL) whole blood or better, 24×10 3 copies / 5 mL whole blood or better, 24×10 3 copies / 10 mL whole blood or better, 24×10 3 copies / 50 mL whole blood or better, or 24×10 3Detect with a sensitivity of copy / 100 mL of whole blood or better.

[0079] VII. Workflow Aliquots individually lysed from multiple blood samples are pooled. Different blood cell samples Typically, are from different subjects and often from different humans. Exemplary volumes of individual blood samples are about 200 - 500 ml. Sample aliquots for pooling typically represent a small percentage of the blood cell sample (e.g., 1 μL - 2 ml or 100 μL - 1 ml, or less than 1% or less than 0.1% of the volume of the blood cell sample). Aliquots can be pooled after individual lysis from 2, 4, 8, 16, 24, or 200 samples (among others). In some methods, aliquots lysed from at least 4 samples are pooled. In some methods, aliquots lysed from 2 - 200, 4 - 24, 14 - 16, or 4 - 20 samples are pooled. After lysis, the individual lysed sample aliquots can be pooled in whole or in part. Pooling in whole means that the entire volume of each lysed sample aliquot is pooled. Pooling means that the specimens (i.e., fractions) of each lysed sample aliquot are pooled to form the pooled lysate. Then, analysis can be performed to detect targets that may be present in the blood cell sample. The analysis can search for targets of pathogens that infect red blood cells (e.g., Babesia spp. and Plasmodium spp.) only, or also for targets of any pathogens that may be present in plasma or serum (e.g., HIV-1 and -2, hepatitis B, hepatitis C, West Nile, dengue, syphilis Treponema, and Trypanosoma cruzi, Plasmodium spp. and Anaplasma), or for therapeutic targets. Such targets can be analyzed separately or together (e.g., by multiplex amplification and detection on a microarray).

[0080] The sensitivity of the method results, in part, from lysing blood cells before taking one or more samples, where only a portion of the original sample is then advanced for subsequent analysis. Such sampling can occur by using only a portion of each lysed sample aliquot for pooling, or by using only a fraction of the pooled lysate for analysis or other dilution. The fractionation performed by any or all sampling occurring between lysis and detection can be less than or equal to 0.5 (i.e., 50%), 0.25 (25%), 0.2 (20%), 0.125 (12.5%), 0.1 (10%), 0.0625 (6.25%), 0.05 (5%), 0.0416 (4.16%), or 0.01 (1%) of the initial volume of the aliquot ultimately analyzed. Since the number of blood cells containing the target can be of the same order or less than the reciprocal of the extraction ratio, for sampling of the sample without lysis, the probability that a cell containing no target at all is advanced for subsequent analysis from the sample aliquot is high. For example, if the sample has 2 infected cells per ml and 0.1 ml of the sample is used for analysis, on average, only 1 / 5 of the sample used for analysis contains the particles enabling a positive result. However, the situation is different if lysis is performed before sampling to release the target molecule from the blood cells. If each blood cell yields 1000 different target molecules upon lysis, the number of target molecules greatly exceeds the reciprocal of the fraction of the sample taken, and the probability of detecting the target is high.

[0081] If the target is from an undesirable pathogen in the blood cell sample and the pooled lysate is negative for the target being tested, the blood cell samples that gave rise to the pool can be used (e.g., for transfusion or held for such use). Conversely, if the pooled lysate is found to be positive for the undesirable target, individual aliquots from the pre-combined samples can be tested to deconvolute which of the pooled samples gave the pathogen. The blood cell sample that gave the pathogen is removed from the collection of blood cell samples and these other blood cell samples can be used (e.g., for transfusion). If the target is desirable, the test is similar except that, in deconvoluting, the blood cell sample containing the target can be maintained and used (e.g., harvesting the protein target from the blood cell sample and / or its donor).

Example

[0082] Example 1 This example compares a method of lysing infected hamster blood and then diluting the lysed sample with additional lysis buffer, with a method where the same hamster blood is first diluted with normal blood and then lysed. Condition A (lyse-dilute), 10 μL of whole blood from a Babesia-infected hamster (59% of whose cells were infected) was lysed in 20 μL of PTM medium (250 mM ammonium chloride, 14 mM sodium bicarbonate, 10 mM EDTA, and 8% lithium lauryl sulfate (LLS), pH in the range of 7.2 - 7.6), and then serially diluted in PTM. Condition B (dilute-lyse), 10 μL of the same infected hamster blood was first serially diluted with human whole blood and then lysed with PTM (1:3 (v / v)). The parasitemia of the infected hamster blood was determined by microscopy and by PCR. Here, the PCR results from the infected hamster blood were compared to the PCR results of an in vitro transcription dilution curve. The parasitemia of the infected hamster blood was determined to be 43,130 parasites / mL (p / mL), and the dilution rates for both Conditions A and B resulted in the following: 4,313 p / mL, 431 p / mL, 43 p / mL, 4.8 p / mL, 1.6 p / mL, 0.5 p / mL, 0.18 p / mL, 0.06 p / mL, 0.02 p / mL, and 0.01 p / mL. Lysis was performed for 5 minutes at ambient temperature. Five 500 μL aliquots from each sample in the above dilutions were tested for Babesia using a target capture and real-time transcription-mediated amplification (TMA) assay. % Reactivity was determined such that an amplification / detection reaction with an RLU signal higher than 100,000 was positive for the detection of the Babesia target.

[0083] Target capture was performed as generally described in U.S. Patent No. 6,110,678. Babesia 18S rRNA was amplified and detected in each sample as generally described in U.S. Patent Nos. 5,399,491, 5,554,516, 5,824,518, and 7,833,716. The primers used to amplify Babesia 18S rRNA in the samples were as follows:

Table 1

[0084] The results are shown in Table 2. This compares the % sample reactivity with the number of particles / ml at the dilutions tested. For samples diluted in whole blood, the reactivity decreases to zero between final concentrations of 4.8 particles / ml and 1.6 particles / ml. In contrast, when samples are diluted in PTM, 100% reactivity is obtained up to at least a final concentration of 0.18 particles / ml. This corresponds to approximately 200 18S ribosomal RNA copies / ml. Furthermore, at 0.06 p / mL and 0.02 p / mL, samples diluted in PTM provided 60% reactivity and 40% reactivity, respectively.

Table 2

[0085] Twenty-four Babesia-positive clinical RBC samples were obtained from the American Red Cross (ARC). These clinical samples were each placed in Adsol solution (a preservative solution containing saline, adenine, dextrose, and mannitol (Fenwal Laboratories, Deerfield, Illinois)) and stored at -20 °C upon receipt. The above samples were thawed on the test day and thoroughly mixed by inverting before manipulation. Each sample could be prepared to be tested individually (testing individual donors or IDT) and to be tested in pools using the pre-lysed pooling method.

[0086] Individual lysis of RBC samples To lyse the clinical samples, 500 μL aliquots of the samples were mixed with 3.5 mL of parasite transport medium (PTM), and then mixed by gently inverting at least 3 times at ambient temperature and allowed to stand for 5 minutes. This lysate is the IDT lysate sample.

[0087] The PTM used in this example contains 250 mM ammonium chloride, 14 mM sodium bicarbonate, 10 mM EDTA, and 8% lithium lauryl sulfate (LLS), and the pH is in the range of 7.2 - 7.6.

[0088] Fifteen normal negative samples from whole blood donors were obtained from Bioreclamation Inc. (Long Island, NY) and lysed by using a ratio of 1 mL aliquot of whole blood to 3 mL of PTM. Aliquots of the lysed samples from each negative donor were set aside for use as negative controls.

[0089] Pool preparation 1:4, 1:8, and 1:16 donor pools were prepared using the IDT lysate for each clinical sample. The total volume of each pooled sample was 4.8 mL. · The 1:4 pool was prepared by combining 1200 μL of the lysed clinical sample and 1200 μL of each of 3 lysed negative donors. · The 1:8 pool was prepared by combining 600 μL of the lysed clinical sample and 600 μL of each of 7 lysed negative donors. · The 1:16 pool was prepared by combining 300 μL of the lysed clinical sample and 300 μL of each of 15 lysed negative donors.

[0090] Once the samples were prepared, they were mixed by inverting several times before testing.

[0091] Three replicates of 500 μL each of the individual IDT lysates, pooled samples, and negative control samples were tested on the automated Panther system using the Babesia TMA assay described above. A B. microti IVT panel at 100 c / mL was included as a positive control.

[0092] The results are shown in Table 3 below. The data indicate that Babesia infection could be detected in all clinical samples where the pooling method had a pooling factor of 1:4 and in the majority of samples with a pooling factor up to 1:16. The sensitivity associated with the pooling method, followed by target capture and TMA, was comparable to that of PCR for individual samples.

Table 3

[0093] Example 3 In this example, B. microti-infected hamster blood was diluted to 1 parasite / mL, pooled, and tested using nucleic acid amplification and detection for the limit of detection in the pooled samples. The approximate parasitemia of the B. microti-infected hamster blood was calculated using microscopy. Based on the initial parasitemia estimate, serial dilutions of B. microti-infected hamster blood were prepared in human whole blood. Nine separate 1 mL aliquots were made at hamster blood dilution levels estimated to have approximately 1 p / mL of B. microti. Each of these separate aliquots was lysed by combining the 1 mL aliquot with 3 mL of hemolysis solution (an aqueous solution containing approximately 100 mM TRIS, 25 - 30 mM MgCl2, 6% (v / v) lithium lauryl sulfate (pH between approximately 7.3 - 7.6)). These nine lysates were tested directly at 1:20 and 1:200 pools.

[0094] Each 1:20 pool of the nine lysates was prepared by combining 250 μL of the lysate into an additional 4.75 mL of lysis reagent. The 1:200 pool was prepared by combining 500 μL of this 1:20 pool with 4.5 mL of lysis reagent. The nine aliquots of the lysate were tested in five replicates using isothermal amplification and detection assays on an automated Panther system (Hologic, Inc., San Diego, CA). The 1:20 and 1:200 pools made from each of the nine lysates were tested in seven replicates using isothermal amplification and detection assays on an automated Panther system. The results are shown in Table 4.

Table 4

[0095] These data indicate that the 1:20 pool is 100% reactive in samples containing as little as about 1 p / mL. Furthermore, the 1:200 pool showed good sensitivity but was not 100% reactive. Thus, for assays with a detection limit sensitivity requirement of 100%, the lysate pool results shown in this example indicate that at least the 1:20 pool meets that requirement. For assays with a sensitivity requirement of less than 100%, this example shows that a dilution pool of about 1:200 provides significantly better results.

[0096] Although the invention has been described in detail for purposes of clarity of understanding, certain modifications can be made within the scope of the appended claims. All publications, including accession numbers, websites, etc., cited in this application, and patent documents are hereby incorporated by reference in their entirety for all purposes to the same extent as if each had been individually incorporated by reference. Versions of sequences, websites, or other references that may exist at various times are meant to be the versions associated with the above references as of the effective filing date to the extent that such versions can exist at various times. The effective filing date means the earliest priority date on which the accession number at issue is disclosed. Unless otherwise apparent from the context, any element, embodiment, step, feature, or aspect of the invention can be practiced in combination with any other.

Claims

1. A method for detecting the presence of a pathogen in an aliquot of a whole blood sample, said method comprising: (a) providing an aliquot from each of a plurality of whole blood samples, thereby providing a plurality of whole blood sample aliquots; (b) separately contacting each of the whole blood sample aliquots of said plurality of whole blood sample aliquots with a lysis reagent, thereby lysing at least a portion of the blood cells in each of said plurality of whole blood sample aliquots; (c) pooling said lysed whole blood sample aliquots of step (b) to form a pooled lysate; (d) testing said pooled lysate for the presence of a pathogen, thereby identifying the presence of said pathogen in said pooled lysate as indicative of the presence of said pathogen in at least one of said whole blood sample aliquots, wherein: said whole blood sample is directly mixed with said lysis reagent, and analysis can be performed without removing said lysis reagent from said pooled lysate.

2. The method of claim 1, wherein said whole blood sample is liquid.

3. The method of claim 1, excluding methods using whole blood dried on filter paper.

4. The method of claim 1, comprising only one lysis step, which is step (b), and no other lysis steps.

5. The method of claim 1, wherein step (d) is performed immediately after step (c) without a washing step therebetween.

6. The method of claim 1, wherein step (d) can be performed entirely in one container.

7. The method of claim 1, wherein no centrifugation step is performed between step (a) and step (b).

8. The method of claim 1, wherein in step (b), each of the whole blood sample aliquots of said plurality of whole blood sample aliquots is contacted with said lysis reagent at a volume ratio of whole blood sample aliquot to lysis reagent of 1:2 to 1:10 (v / v).

9. The method of claim 1, wherein in step (b), each of the whole blood sample aliquots of said plurality of whole blood sample aliquots is contacted with said lysis reagent at a volume ratio of whole blood sample aliquot to lysis reagent of 1:3 (v / v).

10. The lysis reagent of step (b) comprises: (i) a buffer, (ii) lithium lauryl sulfate, and (iii) a chloride-containing salt and one or both of anticoagulants selected from the group consisting of EDTA, EDTA-Na ２ , EGTA, and combinations thereof The method according to claim 1, comprising and having a pH higher than 5.

5.

11. The lysis reagent in step (b) (i) a buffer selected from the group consisting of sodium bicarbonate at a concentration of 5 mM to about 30 mM and a TRIS buffer at a concentration of 75 mM to 150 mM, (ii) lithium lauryl sulfate, and (iii) a chloride-containing salt and one or both of anticoagulants selected from the group consisting of EDTA, EDTA-Na ２ , EGTA, and combinations thereof The method according to claim 1, comprising and having a pH higher than 5.

5.

12. The lysis reagent in step (b) (i) a buffer, (ii) lithium lauryl sulfate at a concentration of 4% (v / v) to 15% (v / v), and (iii) a chloride-containing salt and one or both of anticoagulants selected from the group consisting of EDTA, EDTA-Na ２ , EGTA, and combinations thereof The method according to claim 1, comprising and having a pH higher than 5.

5.

13. The lysis reagent in step (b) (i) a buffer, (ii) lithium lauryl sulfate, and (iii) a chloride-containing salt, which is magnesium chloride present in the lysis reagent at a concentration of -20 mM to 35 mM, and EDTA-Na present in the dissolution reagent at a concentration of -1 mM ２ , EGTA present in the dissolution reagent at a concentration of 1 mM, or EDTA-Na ２ is present in the dissolution reagent at a concentration of 1 mM, and EDTA-Na in which EGTA is present in the dissolution reagent at a concentration of 1 mM ２ and a combination of EGTA, an anticoagulant one or both of The method according to claim 1, comprising and having a pH higher than 5.

5.

14. The lysis reagent in step (b) (i) a buffer, which is a TRIS buffer at a concentration of 75 mM to 150 mM, (ii) lithium lauryl sulfate at a concentration of 4% (v / v) to 15% (v / v), and (iii) a chloride-containing salt, which is magnesium chloride present in the lysis reagent at a concentration of 20 mM to 35 mM The method according to claim 1, comprising and having a pH higher than 5.

5.

15. The pooled lysate in step (c) contains from 4 to 200 lysed whole blood sample aliquots, the method according to claim 1.

16. The pooled lysate in step (c) contains at least 4, 8, 16 or 20 lysed whole blood sample aliquots, the method according to claim 1.

17. In step (c), the entire volume of each of the lysed whole blood sample aliquots is pooled, or up to 25% of the volume of each of the lysed whole blood sample aliquots is pooled, the method according to claim 1.

18. The test step (d) tests for the presence of a pathogen-nucleic acid target released from blood cells by the lysis reagent, the method according to claim 1.

19. Test step (d) is the method according to claim 1, which tests for the presence of a pathogen-RNA target released from blood cells by the lysis reagent.

20. Test step (d) is the method according to claim 1, which includes performing a nucleic acid amplification and detection reaction to detect the presence of a pathogen-nucleic acid target released from blood cells by the lysis reagent.

21. Test step (d) is the method according to claim 1, which includes performing transcription-mediated amplification of a pathogen-nucleic acid target released from blood cells by the lysis reagent and detecting the obtained amplification product with a detection probe.

22. Test step (d) is a step of testing for the presence of a pathogen-nucleic acid target released from blood cells by the lysis reagent and contacting the pooled lysate with a capture probe and an immobilization probe, where the capture probe has a first segment complementary to the nucleic acid target and a second segment complementary to the immobilization probe, and where the nucleic acid target binds to the capture probe, and where the bound capture probe binds to the immobilization probe, according to the method of claim 1.

23. - A step of contacting the pooled lysate with a solid support configured to immobilize a target derived from a pathogen released from blood cells by the lysis reagent, where the solid support is a magnetic bead solid support or a silica solid support, and - A step of separating the immobilized target from the pooled lysate further included in the method according to claim 1.

24. The method is performed without a centrifugation step, according to the method of claim 1.

25. Test step (d) tests for the presence of a target derived from a pathogen released from the blood cells by the lysis reagent, and the target derived from the pathogen is a) a virus selected from the group consisting of hepatitis virus, human immunodeficiency virus, dengue virus, West Nile virus, flavivirus, and Zika virus b) A parasite selected from the group consisting of the genera Babesia, Plasmodium, Trypanosoma, Leishmania, Anaplasma, Toxoplasma, Babesia microti, Babesia divergens, Babesia duncani, Plasmodium falciparum, Plasmodium malariae, Plasmodium ovale, Plasmodium vivax, and Plasmodium knowlesi The method according to claim 1, derived from a pathogen selected from the group consisting of **Claim 26** In step (d), if it is determined that the pathogen is present in at least one of the whole blood sample aliquots, the method further comprises individually testing the blood sample aliquots to identify the presence of the pathogen in each of the blood sample aliquots. The method according to claim 1

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