Sample preparation methods and systems

Abstract

A method for preparing a biological sample suitable for use in a subsequent in vitro nucleic acid amplification reaction involves combining the biological sample with an alkaline composition that lyses cells and denatures DNA to form a first liquid composition. The first liquid composition is then mixed with a buffer, a surfactant, and a solid support that captures the DNA to form a second liquid composition. The buffer, surfactant, and solid support can be delivered as components of a single reagent. The captured DNA strands can be used as templates for subsequent nucleic acid amplification and detection reactions with improved sensitivity.

Description

[Technical field]

[0001] Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 62 / 691,487, filed June 28, 2018, the entire disclosure of which is incorporated herein by reference.

[0002] The present disclosure relates to the field of nucleic acid isolation. More specifically, the present disclosure relates to a method for preparing DNA from a biological sample. Even more specifically, the present disclosure relates to an automated method and system for isolating DNA from a biological sample and then amplifying the isolated DNA. [Background technology]

[0003] Currently, in vitro nucleic acid amplification techniques are commonly used to synthesize and detect extremely small nucleic acid targets that are likely to disappear. These techniques traditionally use one or more oligonucleotide primers and a nucleic acid polymerizing enzyme to synthesize copies of one or both strands of a nucleic acid template. Many different methods have been used to prepare biological samples prior to the amplification procedure.

[0004] Differences in the efficiency of sample preparation techniques present challenges for diagnostic assays that detect amplified nucleic acids. The fact that a single technique lyses different organisms, causing the release of nucleic acids with different efficiencies, means that specialized techniques may be required to process different organisms. U.S. Patent No. 8,420,317 details an "alkaline shock" sample preparation technique that can be used to isolate both RNA and DNA from a wide range of organisms, including many organisms that are difficult to lyse. Methods that use detergents, or a combination of detergents and alkali, represent alternative approaches to isolate amplifiable nucleic acids from, for example, bacterial organisms prior to in vitro amplification.

[0005] Automated systems are frequently used to prepare nucleic acids from biological samples and can streamline laboratory workflows by performing additional nucleic acid amplification and detection steps. Here, the number and type of on-board reagents can be a significant limitation when working with assays that are not packaged or configured as individual tests. In other words, the number and type of reagents available to perform sample preparation steps can constrain the range of sample preparation methods that can be performed. Thus, without an increased set of on-board reagents, the usefulness of automated sample processing instruments is limited.

[0006] Therefore, there is a need for a technology that can increase the efficiency of nucleic acid isolation, and therefore can increase the detectability of specific targets in nucleic acid amplification reactions.There is a further need to increase the detectability of one or more targets in multiplex amplification reactions without substantially sacrificing the detectability of other targets in the same reaction.The disclosed technology addresses these needs.

[0007] Indeed, the technology disclosed herein provides a convenient method for preparing biological samples to be tested for the presence of nucleic acid targets using in vitro nucleic acid amplification, which advantageously provides reliable DNA isolation results even from difficult-to-lyse organisms while dramatically improving the detectability of specific nucleic acid targets. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] U.S. Pat. No. 8,420,317 Summary of the Invention [Means for solving the problem]

[0009] In one aspect, the present disclosure relates to a method for processing a biological sample. Generally speaking, the method includes the steps of: (a) mixing the biological sample with an alkaline composition that lyses cells and denatures DNA to form a first liquid composition, the first liquid composition having a pH in the range of about pH 12.0 to about pH 13.5; (b) mixing the first liquid composition with a pH buffered surfactant reagent to form a second liquid composition having a pH lower than about pH 9.5, the pH buffered surfactant reagent comprising a pH buffer, a surfactant, and solid support particles that capture DNA; and (c) isolating the solid support particles and any DNA captured thereon from the second liquid composition.

[0010] In some embodiments, the first liquid composition may be substantially free of surfactants.

[0011] In some embodiments, the biological sample comprises viable bacterial cells, and the method may further comprise culturing the viable bacterial cells to increase the number of bacterial cells.

[0012] In some embodiments, the second liquid composition can have a pH in the range of about pH 8.0 to about pH 9.2.

[0013] In some embodiments, each of steps (a)-(c) is performed on an automated instrument that includes a robotic fluid transfer device.

[0014] In some embodiments, the method further comprises (d) performing an in vitro nucleic acid amplification reaction using the DNA isolated in step (c) as a template and detecting a product of the in vitro nucleic acid amplification reaction. For example, detecting a product of the in vitro nucleic acid amplification reaction can be used to indicate the presence of a species of Gram-positive bacteria in a biological sample.

[0015] In some embodiments, step (d) is also performed on an automated instrument.

[0016] In some embodiments, particularly those using an automated instrument that includes a robotic fluid transfer device, step (a) includes combining the biological sample and an alkaline composition in a reaction vessel to create a first liquid composition, and step (b) includes adding a pH buffered surfactant reagent to the reaction vessel containing the first liquid composition to create a second liquid composition, and each of steps (a) and (b) is performed using the robotic fluid transfer device of the automated instrument.

[0017] In some embodiments, again particularly those using automated equipment including robotic fluid transfer devices, the automated equipment further includes a transport mechanism that moves the reaction vessel from one location within the automated equipment to another location within the automated equipment.

[0018] In some embodiments, after step (b) and before step (c), there is a step of incubating the first liquid composition for a period of from 1 minute to 10 minutes.

[0019] In some embodiments, the incubating step comprises heating the first liquid composition.

[0020] In some embodiments, the incubating step includes transporting the reaction vessel from a first location of the automated instrument to a second location of the automated instrument, the second location of the automated instrument being at a higher temperature than the temperature of the first location of the automated instrument.

[0021] In some embodiments, the solid support particles include magnetically attractable particles.

[0022] In some embodiments, the solid support particles in step (b) are solid support particles that capture DNA regardless of base sequence, and step (c) involves washing the solid support particles to remove any material not immobilized thereon, and then retaining the washed solid support particles, thereby isolating the captured DNA.

[0023] In some embodiments, the alkaline composition comprises a strong base in aqueous solution at a concentration of about 0.1N to about 2.2N.

[0024] In some embodiments, the pH of the first liquid composition ranges from about pH 12.5 to about pH 13.2.

[0025] In some embodiments, the pH of the second liquid composition ranges from about pH 7.6 to about pH 8.8.

[0026] In some embodiments, the alkaline composition comprises a strong base at a concentration of about 1.0N to about 1.7N.

[0027] In some embodiments, the strong base is any of NaOH, KOH, and LiOH.

[0028] In some embodiments, all steps of the method are performed under automated process control.

[0029] definition The following terms have the following meanings for purposes of this disclosure, unless expressly stated to the contrary herein:

[0030] As used herein, a "biological sample" is any tissue or polynucleotide-containing material obtained from a human, animal, or environmental sample. Biological samples according to the disclosed technology include peripheral blood, plasma, serum, or other bodily fluids, bone marrow or other organs, biopsy tissue, clinical or screening swabs (e.g., nasal swabs), or other materials of biological origin. Biological samples can be contained in liquid transport media to facilitate transport and processing. Examples of biological samples include or contain bacteria, such as gram-positive bacteria.

[0031] As used herein, an "alkaline composition" is an aqueous solution containing a strong base. A strong base can be completely ionized or dissociated in solution to provide hydroxide ions. Generally, strong bases are formed from hydroxides of alkali metals or alkaline earth metals. Examples of strong bases include KOH, NaOH, and LiOH.

[0032] As used herein, a "first liquid composition" resulting from mixing a biological sample with an alkaline composition is said to be "substantially surfactant-free" if the surfactant concentration is less than 0.001% (w / v). This allows for the presence of trace amounts of surfactant in the first liquid composition, such as may be carried over from previous sample transport or processing steps.

[0033] As used herein, "alkaline shock" refers to the temporary high pH produced by first combining a biological sample with a pH buffer and a detergent to provide a first composition, and then mixing the first composition with a volume of an alkaline composition.

[0034] As used herein, "polynucleotide" means either RNA or DNA, and any synthetic nucleotide analog or other molecule that may be present in a sequence and that does not prevent hybridization of the polynucleotide with a second molecule having a complementary sequence.

[0035] As used herein, a "detectable label" is a chemical species that can be detected or that can produce a detectable response. Detectable labels according to the disclosed technology can be directly or indirectly attached to a polynucleotide probe and include radioisotopes, enzymes, haptens, chromophores such as dyes or particles that impart detectable color (e.g., latex beads or metal particles), luminescent compounds (e.g., bioluminescent, phosphorescent or chemiluminescent moieties) and fluorescent compounds.

[0036] "Homogeneous assay" refers to a detection procedure that does not require physical separation of hybridized from unhybridized probes prior to determining the extent of specific probe hybridization.

[0037] As used herein, "nucleic acid amplification," or simply "amplification," refers to an in vitro procedure for obtaining multiple copies of a target nucleic acid sequence, its complement, or fragments thereof.

[0038] "Target nucleic acid" or "target" refers to a nucleic acid that comprises a target nucleic acid sequence. Optionally, the target nucleic acid sequence that is amplified is located between two oppositely disposed amplification oligonucleotides and includes a portion of the target nucleic acid that is sufficiently complementary to each of the amplification oligonucleotides.

[0039] "Target nucleic acid sequence" or "target sequence" or "target region" refers to a specific deoxyribonucleotide or ribonucleotide sequence that comprises all or a portion of the nucleotide sequence of a single-stranded nucleic acid molecule, and complementary deoxyribonucleotide or ribonucleotide sequences thereto. As used herein, an "oligonucleotide" or "oligomer" is a polymeric chain of at least two, and typically about 5 to about 100, chemical subunits, each of which includes a nucleotide base moiety, a sugar moiety, and a linking moiety that joins the subunits in a linear spatial arrangement. Common nucleotide base moieties are guanine (G), adenine (A), cytosine (C), thymine (T), and uracil (U), although other rare or modified nucleotide bases that can hydrogen bond are known to those of skill in the art. Oligonucleotides may optionally include analogs of any of the sugar moieties, base moieties, and backbone components. Preferred oligonucleotides of the present technology fall in the size range of about 10 to about 100 residues. Oligonucleotides may be purified from naturally occurring sources, but are preferably synthesized using any of a variety of well-known enzymatic or chemical methods.

[0040] As used herein, a "probe" refers to an oligonucleotide that interacts with a target nucleic acid to form a detectable complex. A probe may optionally include a detectable moiety that may be attached to the end(s) of the probe or may be internal. A probe's "target" generally refers to a sequence contained within an amplified nucleic acid sequence that specifically hybridizes to at least a portion of the probe oligonucleotide using standard hydrogen bonding (i.e., base pairing). A probe may include a target-specific sequence and, optionally, other sequences that are non-complementary to the target sequence to be detected. Particular examples of probes include invasive probes and primary probes, as disclosed in published patent application identified as US2018 / 0163259 A1, the entire disclosure of which is incorporated by reference.

[0041] "Amplification oligonucleotide" refers to an oligonucleotide that can participate in a nucleic acid amplification reaction to result in the synthesis of multiple copies of a template nucleic acid sequence or its complement. In an amplification reaction, it is common to use at least two amplification oligonucleotides, at least one of which functions as an amplification primer.

[0042] As used herein, an "amplification primer", or more simply, a "primer", is an oligonucleotide that hybridizes to a target nucleic acid or its complement and can be extended in a template-dependent primer extension reaction. For example, an amplification primer can be an optionally modified oligonucleotide that can hybridize to a template nucleic acid and has a 3' end that can be extended by DNA polymerase activity. In general, a primer has a downstream target-complementary sequence and, optionally, an upstream sequence that is not complementary to the target nucleic acid. The optional upstream sequence can, for example, function as an RNA polymerase promoter or include a restriction endonuclease cleavage site.

[0043] "Target capture" or simply "capture" refers to a general process for capturing polynucleotides from a solution phase onto a solid support. Optionally, target capture can be mediated by the formation of a hybrid duplex between a target nucleic acid from a solution and an oligonucleotide bound directly or indirectly to a solid support such as a bead (e.g., a microbead) or a particle (e.g., a microparticle). For simplicity, "particle" is used herein to refer generally to beads, microbeads, and microparticles.

[0044] "Capture oligonucleotide" refers to at least one nucleic acid oligonucleotide that provides a means for specifically binding a target sequence and an immobilized oligonucleotide by base pair hybridization. A capture oligonucleotide preferably comprises two binding regions, a target sequence binding region and an immobilized probe binding region, which are usually contiguous on the same oligonucleotide, but a capture oligonucleotide may also comprise a target sequence binding region and an immobilized probe binding region that are present on two different oligonucleotides that are linked together by one or more linkers. For example, the immobilized probe binding region may be present on a first oligonucleotide, and the target sequence binding region may be present on a second oligonucleotide, and the two different oligonucleotides are linked by hydrogen bonds with a linker that is a third oligonucleotide that comprises a sequence that specifically hybridizes with the sequences of the first and second oligonucleotides.

[0045] "Immobilized oligonucleotide" or "immobilized nucleic acid" and variants thereof refer to a nucleic acid that directly or indirectly binds a capture oligonucleotide to an immobilized support. An immobilized probe is an oligonucleotide attached to a solid support that facilitates separation of the bound target sequence from unbound material in a sample. An "immobilizable" oligonucleotide is an oligonucleotide that can become immobilized to a solid support by complementary base interactions with an oligonucleotide directly immobilized to the solid support.

[0046] "Separate" or "purify" or "isolate" means that one or more components of a biological sample are removed from one or more other components of the sample. Sample components generally include nucleic acids in an aqueous phase, which may also include substances such as proteins, carbohydrates, lipids, and labeled probes. Preferably, the separation or purification step removes at least about 70%, more preferably at least about 90%, and even more preferably at least about 95% of the other components present in the sample. The disclosed method allows for the isolation of DNA from a biological sample after certain processing steps have been performed.

[0047] As used herein, a "multiplex" assay is a type of assay that measures multiple analytes (two or more) in a single run of the assay.

[0048] "Consisting essentially of" means that additional component(s), composition(s), or method step(s) may be included in the compositions or kits or methods described herein that do not substantially alter the basic and novel characteristics of the techniques of the present invention. Such characteristics include the ability to selectively detect target nucleic acids in biological samples such as nasal swabs or other swabs that contain bacteria therein. Any component, composition, or method step that has a substantial effect on the basic and novel characteristics of the present disclosure would deviate from this term. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0049] Disclosed herein is a method for isolating DNA from a biological sample. This method can be used to isolate DNA from viral, bacterial, or eukaryotic sources, and can increase the sensitivity of amplification-based assays performed using isolated nucleic acid as a template. Advantageously, this method can be used to isolate DNA from organisms that are difficult to lyse by other methods. Furthermore, this method can be used to isolate DNA that is substantially free of RNA.

[0050] Generally speaking, the advantages of the disclosed approach can be realized by combining a biological sample with an aqueous solution of a strong base (wherein the solution of the strong base is substantially free of pH buffer and surfactant) and then neutralizing the alkaline combination with a pH buffer in the presence of a surfactant. The advantages of the process can also be realized by integrating a target capture step, for example, by including a target capture reagent (e.g., magnetically attractable beads, optionally containing immobilized polynucleotides) in the reaction mixture when the neutralization step is occurring. This is significantly different from the "alkaline shock" sample preparation technique described in co-owned U.S. Patent No. 7,510,837, which avoids prolonged direct exposure of the biological sample to strongly alkaline conditions. Similarly, the disclosed technique allows for combining or mixing a base-treated sample undergoing processing with a surfactant during the pH neutralization step when target capture is also occurring. As shown by the evidence presented below, if the order of reagent addition is reversed, the specific advantages of the disclosed technique are not achieved.

[0051] The observation that prompted the development of the disclosed technology was regarding the reduced sensitivity of amplification assays for detecting certain types of bacteria that are known to be difficult to lyse. For example, Methicillin-resistant Staphylococcus aureus (MRSA) bacteria lyse inefficiently under certain conditions, and a prototype PCR assay for detecting MRSA could give erroneous results when nucleic acid from the lysis was used as a template. This was the case when using a sample preparation technique that relies on contacting a biological sample with a pH-buffered detergent solution containing a target capture reagent to create a first liquid composition, and then mixing the first liquid composition with an alkaline composition (e.g., an aqueous solution containing a strong base) to obtain a second liquid composition suitable for capture or immobilization of the released nucleic acid to a solid support. This "alkaline shock" technique is described in U.S. Pat. No. 7,510,837, the disclosure of which is incorporated herein by reference. A modified lysis protocol involving initial treatment of the biological sample with an alkaline detergent solution followed by pH neutralization using a pH buffered reagent resulted in improved results in the detection of Staphylococcus aureus and MRSA, but still did not provide adequate lysis of another target (C. difficile). Increasing the concentration of strong base in the alkaline detergent reagent resulted in changes in physical properties that made the reagent undesirable for laboratory use. Because of the desirability of sensitive detection of bacterial target nucleic acids using a universal protocol, there has been interest in developing new techniques that can be used to liberate DNA from a wide range of target organisms.

[0052] Biological samples As noted above, the term "biological sample" encompasses a wide variety of sample types. Of particular interest, however, are bacteria that are typically difficult to lyse. Exemplary bacteria that exhibit this characteristic include gram-positive bacteria.

[0053] Those skilled in the art will recognize that Gram-positive bacteria characteristically have a thick peptidoglycan layer on the bacterial cell wall. Peptidoglycan is a polymer of sugars and amino acids that forms a mesh-like layer on the outside of the plasma membrane of most bacteria. The peptidoglycan layer of the cell wall of Gram-positive bacteria is generally more than twice as thick as that of Gram-negative bacteria. This makes Gram-positive bacteria substantially more resistant to dissolution and release of nucleic acids.

[0054] Although the disclosed sample preparation or processing technique can be used to isolate DNA from a wide variety of organisms, the technique has particular advantages when used to isolate DNA from Gram-positive bacteria. In a preferred embodiment, the isolation procedure is carried out on an automated sample preparation instrument. Optionally, the automated sample preparation instrument performs nucleic acid amplification and monitors the formation of amplification products. The Gram-positive bacteria used to illustrate the utility of the improved sample preparation technique were Staphylococcus aureus, Clostridium difficile, and Streptococcus agalactiae (group B streptococcus).

[0055] Preferred pH Buffers and pH Ranges Buffers useful for carrying out the sample preparation method preferably have pKa values ​​in the range of about 6.0 to about 9.0. An exemplary pH buffer used to demonstrate the utility of the disclosed technology is HEPES (N-2-hydroxyethylpiperazine-N=-2-ethanesulfonic acid), which has a pKa of 7.55 at 20°C and has the strongest buffering capacity in the range of pH 6.8 to pH 8.2. Of course, the success of this technology is not limited by the use of any particular pH buffer.

[0056] According to a preferred method, sample preparation is performed in a multi-step procedure. A biological sample, optionally contained in a liquid transport medium, is first combined with an aliquot of concentrated alkaline reagent to effect cell lysis and denature DNA. This alkaline treatment can be performed for a period of 1 second to 1 hour, and can optionally be performed at an elevated temperature. After the treatment period, the alkaline mixture is combined with a pH buffer, a surfactant, and a reagent to facilitate capture of the nucleic acid to the solid support. Optionally, these different components can be combined with the alkaline mixture in a specified order (e.g., pH buffer / surfactant / target capture reagent; or pH buffer / target capture reagent / surfactant). Optionally, the pH buffer and surfactant are combined with each other and delivered as a single reagent, followed by the target capture reagent. Optionally, all three different components are first combined with each other and then delivered as a single reagent to the alkaline mixture (i.e., the combination of the biological sample and the alkaline reagent). The result of combining the first liquid composition with the pH buffer, surfactant, and target capture reagent is a second liquid composition having a pH below pH 9.5.

[0057] Preferably, the pH buffered surfactant reagent comprising solid support beads or particles that capture nucleic acids additionally comprises one or more immobilizable or immobilized oligonucleotides. Optionally, the solid support displays or has attached (e.g., covalently attached) oligonucleotides internally that are involved in the capture of nucleic acids from the surrounding solution phase. Using a single reagent comprising each of the pH buffer, surfactant, and solid support can simplify the reagent addition step, thereby making the method amenable to automation by using a robotic pipetter or fluid handling device. Excellent results have been obtained using a pH buffered surfactant reagent comprising solid support beads, resulting in a pH buffer concentration in the range of about 200 mM to about 1.0 M, resulting in a final pH buffer concentration in the range of about 200 mM to about 600 mM, more preferably 300 mM to 500 mM, and even more preferably about 400 mM (i.e., after the pH buffered surfactant and capture reagent are combined with the biological sample). Of course, the amount of pH buffer added can be adjusted to bring the final pH of the mixture to one of the ranges specified herein.

[0058] Certain pH ranges are preferred for isolating nucleic acids according to the disclosed technology. Preferably, the liquid composition obtained by combining the biological sample with the alkaline composition (sometimes referred to herein as the "first" liquid composition) has a pH in the range of pH 12.0 to pH 13.5, even more preferably, pH 12.5 to pH 13.2, or even more preferably, pH 12.8 to pH 13.1. The alkaline composition added preferably comprises a strong base in aqueous solution. Those skilled in the art will understand that a strong base is fully ionic and may fully dissociate in aqueous solution to yield hydroxide ions. By mixing the first liquid composition with a pH buffer, a surfactant, and a target capture reagent (e.g., optionally including magnetically attractable beads having polynucleotides immobilized thereon), a liquid composition having a pH lower than pH 9.5 (sometimes referred to herein as the "second" liquid composition) is obtained. More preferably, the second liquid composition has a pH in the range of pH 7.0 to pH 9.5, even more preferably in the range of pH 7.4 to pH 9.0, and even more preferably in the range of pH 7.6 to pH 8.8.

[0059] Treatment method--Alkaline and surfactant conditions In a preferred embodiment, a first liquid composition comprising a combination of a biological sample and an alkaline composition is mixed with each of a pH buffer and a surfactant, and optionally a target capture reagent, resulting in a second liquid composition. The second liquid composition is mixed and preferably incubated with an immobilized capture probe and, optionally, a soluble capture probe capable of forming a crosslink between the immobilized probe and the target nucleic acid of interest. As this method is typically performed, the alkaline composition and the biological sample are first combined in a test tube or other reaction vessel. For example, the alkaline composition can be added to a test tube or other reaction vessel already containing a biological sample. Then, each of the pH buffer, surfactant, and target capture reagent can be mixed with the first liquid composition to create the second liquid composition. In a preferred embodiment, the pH buffer, surfactant, and magnetically attractable beads displaying immobilized polynucleotides are delivered to the first liquid composition by a single reagent addition. In a highly preferred embodiment, the target capture reagent comprises a soluble capture probe that is partially complementary to the immobilized polynucleotide of the magnetically attractable beads.

[0060] The substance that can be used as the alkaline composition to effect cell lysis and nucleic acid denaturation can be any solid, liquid or gaseous agent that forms a strong alkaline solution when dissolved in an aqueous solution. Strong bases are highly preferred for use as the alkaline composition (sometimes referred to herein as "alkaline hydroxides"). Examples of preferred alkaline hydroxides that can be used to carry out sample preparation methods include sodium hydroxide (NaOH), lithium hydroxide (LiOH), potassium hydroxide (KOH), and the like. Although it is contemplated that solid alkaline compositions can be combined with biological samples, preferred alkaline compositions include strong bases in aqueous solutions.

[0061] The alkaline condition of the first liquid composition resulting from combining the biological sample with the alkaline composition can be neutralized by adding a pH buffer to result in a second liquid composition having a pH lower than pH 9.5. The second liquid composition optionally further comprises a surfactant and a target capture reagent (e.g., magnetically attractable beads having polynucleotides immobilized thereon). The pH buffer, surfactant, and target capture reagent can optionally be combined before mixing with the first liquid composition.

[0062] Preferred Surfactants Surfactants that can be used in connection with the disclosed sample preparation techniques include anionic, nonionic, zwitterionic, or cationic surfactants. Of these, anionic and nonionic surfactants are most preferred. The final surfactant concentration in the second liquid composition (i.e., resulting from the aggregated combination of the biological sample, the alkaline composition, and the pH buffer surfactant and capture reagent) is preferably in the concentration range of 0.01% (w / v) to 5.0% (w / v), more preferably in the concentration range of 1.0% (w / v) to 2.0% (w / v), and even more preferably in the concentration range of 1.2% (w / v) to 1.8% (w / v). Strong anionic surfactants, including sulfates of alkyl alcohols and N-acyl amino acids, are highly preferred. Although the exact nature of the surfactant used to carry out the sample preparation procedure is not believed to be critical, examples of particularly preferred surfactants include lithium lauryl sulfate (LLS) and sodium dodecyl sulfate (SDS).

[0063] Processing period In a preferred embodiment, the biological sample is combined with an alkaline composition (e.g., a strong base in an aqueous solution) in a test tube or reaction vessel to provide a first liquid composition. Optionally, the first liquid composition can be agitated to ensure homogeneity. Advantageously, the denatured DNA is stable under the strong alkaline conditions of the first liquid composition, which denatures nucleases that may be present in the first liquid composition. After a period of about 1 second to about 1 hour, the first liquid composition is mixed with each of a pH buffer, a surfactant, and a reagent for target capture of DNA (e.g., magnetic beads, optionally including polynucleotides immobilized thereon) to provide a second liquid composition. Optionally, the pH buffer and surfactant are first combined with each other before being added to the first liquid composition. Optionally, the pH buffer, surfactant, and target capture reagent are combined before being added to the first liquid composition. After a period of 1 second to 1 hour, the DNA can be isolated from the second liquid composition. This may include separating or isolating the solid support having the DNA captured thereon. Optionally, this isolation step may include washing the solid support with a wash buffer that allows the captured DNA to remain immobilized on the solid support while still removing non-immobilized components of the second liquid composition. To facilitate laboratory productivity, the length of time that the target capture step is performed is desirably no longer than necessary. However, since the DNA released from the biological sample is stable in the second liquid composition (e.g., due to the presence of a surfactant), leaving the mixture for at least several hours is not expected to be detrimental to the target DNA.

[0064] Plastic containers discarded by automated analysis equipment The sample preparation method is preferably carried out in a disposable reaction vessel, such as a plastic tube, or a disposable unit comprising a plurality of spaced apart tubes. For example, the disposable reaction vessel containing the biological sample is preferably placed in an automated instrument or analytical device when an alkaline composition (e.g., an alkaline hydroxide solution) is added to form the first liquid composition. The adding step is preferably carried out by an automated or robotic pipetting device. The alkaline composition added to the biological sample is sufficient to dissolve or destroy biological membranes, such as bacterial (e.g., gram-positive) cell walls, cell membranes, and viral envelopes, even when no surfactant is added. In a highly preferred embodiment, the disposable reaction vessel is loaded into the analytical device, and an automated or robotic pipetting device adds an aliquot of the biological sample and an aliquot of the alkaline composition to the vessel. The automated or robotic pipetting device can then add each of a pH buffer, a surfactant, and a target capture reagent (e.g., magnetically attractable beads) to the first liquid composition contained in the disposable reaction vessel. Optionally, the pH buffer, surfactant, and target capture reagent can all be added to the first liquid composition in a single reagent addition to create the second liquid composition. The contents of the tube are then vortexed to ensure complete mixing, and the mixed sample is incubated at a temperature and for a period of time sufficient to allow capture of the liberated polynucleotides. This approach allows DNA to be isolated even from bacterial cells that are difficult to lyse. However, RNA is significantly degraded. Thus, DNA that is substantially free of RNA can be isolated by this sample preparation technique. Given the chemical stability of DNA, and the fact that nuclease enzymes are substantially inactivated by the harsh alkaline and surfactant conditions of the first and second liquid compositions, there is no substantial chemical degradation that is known to occur with long or variable standing periods, as may occur when different analytical protocols are run on an automated analyzer in a daily cycle of clinical testing. Finally, the plastic material of the disposable reaction vessel must be chemically resistant to each mixture contained therein.

[0065] Target Capture--Methods and Oligonucleotides The disclosed sample preparation method has particular value when it includes a target capture procedure that enriches sample for nucleic acid. Separate preferred embodiments rely on non-specific target capture (i.e., when nucleic acid is captured in a manner that is substantially independent of the base sequence of nucleic acid), and sequence-specific target capture. Either or both of these methods can use immobilizable or immobilized capture oligonucleotides, and each method can capture single-stranded (e.g., "denatured") DNA.

[0066] A preferred capture oligonucleotide comprises a first sequence that is complementary to a polynucleotide containing a target sequence to be amplified and is covalently linked to a second sequence (e.g., a "tail" sequence) that serves as a target for immobilization to a solid support. Any backbone for linking the base sequences of the capture oligonucleotide can be used. In certain preferred embodiments, the capture oligonucleotide comprises a DNA backbone. In other preferred embodiments, the capture oligonucleotide comprises at least one sugar phosphate backbone analog. For example, the backbone may have at least one methoxy linkage. The tail sequence, preferably at the 3' end of the capture oligonucleotide, is used to hybridize to the complementary base sequence and provide a means for capturing target nucleic acids that are preferentially hybridized to other components in the biological sample.

[0067] While any base sequence that hybridizes to a complementary base sequence can be used for the tail sequence, it is preferred that the hybridizing sequence spans a length of about 5 to 50 nucleotide residues. Particularly preferred tail sequences are substantially homopolymeric and contain about 10 to about 40 nucleotide residues, or more preferably about 14 to about 30 residues. The capture oligonucleotide optionally comprises a first sequence that specifically binds to a target polynucleotide and a second sequence that specifically binds to an oligo(dT) stretch immobilized on a solid support.

[0068] In some embodiments, the assay for detecting a nucleic acid sequence in a biological sample comprises the steps of capturing a target nucleic acid using a capture oligonucleotide, amplifying the captured target region using at least one, preferably at least two, amplification oligonucleotides, or at least two primers, and first detecting the amplified nucleic acid by hybridizing an oligonucleotide probe to a sequence contained in the amplified nucleic acid. The signal resulting from the bound probe is preferably detected. Optionally, the probe has a detectable label (e.g., a fluorescent label) therein or contains a stretch of nucleotides at its 5' end (e.g., a probe involved in an invasive cleavage assay).

[0069] The capture step preferably uses a capture oligonucleotide, where under hybridization conditions, a portion of the capture oligonucleotide specifically hybridizes to a sequence in the target nucleic acid, and the tail portion serves as one component of a binding pair, such as a ligand (e.g., a biotin-avidin binding pair), that allows the target region to be separated from other components of the sample. Preferably, the tail portion of the capture oligonucleotide is a sequence that hybridizes to a complementary sequence immobilized on a solid support particle. Preferably, initially, the capture oligonucleotide and the target nucleic acid are in solution to utilize solution-phase hybridization kinetics. Hybridization produces a capture oligonucleotide:target nucleic acid complex that can bind to the immobilized probe via hybridization of the tail portion of the capture oligonucleotide with the complementary immobilized sequence. Thus, a complex comprising the target nucleic acid, the capture oligonucleotide and the immobilized probe is formed under hybridization conditions. Preferably, the immobilized probe is a repetitive sequence, more preferably a homopolymeric sequence (e.g., polyA, polyT, polyC or polyG) that is complementary to the tail sequence and attached to the solid support. For example, if the tail portion of the capture oligonucleotide contains a polyA sequence, the immobilized probe will contain a polyT sequence, although any combination of complementary sequences may be used. The capture oligonucleotide may also contain a "spacer" residue, which is one or more bases located between the base sequence that hybridizes to the target and the base sequence of the tail that hybridizes to the immobilized probe. Any solid support can be used to bind the target nucleic acid:capture oligonucleotide complex. Useful supports can be either matrices, beads, or particles that are free in solution (e.g., nitrocellulose, nylon, glass, polyacrylate, mixed polymers, polystyrene, silane polypropylene, and preferably magnetically attracting beads or particles). Methods for attaching immobilized probes to solid supports are well known. The support is preferably a particle that can be retrieved from solution using standard methods (e.g., centrifugation, magnetic force of magnetic particles, etc.).Preferred supports are paramagnetic monodisperse particles (ie, uniform in size to within about ±5%).

[0070] By recovering the target nucleic acid: capture oligonucleotide: immobilized probe complex, the target nucleic acid is effectively concentrated compared to its concentration in the biological sample, and the target nucleic acid is purified from amplification inhibitors that may be present in the biological sample. The captured target nucleic acid can be washed one or more times, for example, by resuspending the particles with the attached target nucleic acid: capture oligonucleotide: immobilized probe complex in a washing solution, and then recovering the particles with the attached complex from the washing solution, thereby further purifying the target. In a preferred embodiment, the capture step is performed consecutively by hybridizing the capture oligonucleotide with the target nucleic acid, and then adjusting the hybridization conditions to allow hybridization of the tail portion of the capture oligonucleotide with the immobilized complementary sequence (e.g., as described in PCT No. WO98 / 50583). After the capture step and any washing step are completed, the target nucleic acid can be amplified. To limit the number of processing steps, the target nucleic acid can be optionally amplified without being released from the capture oligonucleotide.

[0071] Useful capture oligonucleotides can contain mismatches to the above sequences, so long as the mismatch sequence hybridizes to a nucleic acid containing the sequence to be amplified.

[0072] Previous testing has shown that poor target capture results can occur when the pH of the second liquid composition exceeds pH 9.5. Thus, target capture can occur in liquid compositions having a pH lower than pH 9.5.

[0073] Useful amplification methods Amplification methods useful in conjunction with sample preparation techniques include transcription-mediated amplification (TMA), nucleic acid sequence-based amplification (NASBA), polymerase chain reaction (PCR), strand displacement amplification (SDA), and amplification methods using self-replicating polynucleotide molecules and replicating enzymes (such as MDV-1 RNA and Q-beta enzyme). Methods for carrying out these various amplification techniques can be found in U.S. Patent No. 5,399,491, published European patent application EP0525882, U.S. Patent Nos. 4,965,188, 5,455,166, and 5,472,840, respectively, and Lizardi, et al., BioTechnology 6:1197 (1988). The disclosures of these documents that describe methods for carrying out nucleic acid amplification reactions are incorporated herein by reference.

[0074] In some embodiments, the target nucleic acid sequence is amplified using the TMA protocol. According to this protocol, the reverse transcriptase that provides the DNA polymerase activity also has an intrinsic ribonuclease activity. One of the primers used in this procedure contains a promoter sequence located upstream of a sequence complementary to one strand of the target nucleic acid to be amplified. In the first step of amplification, the promoter-primer hybridizes to the target RNA at a defined site. The reverse transcriptase forms a complementary DNA copy of the target RNA by extension from the 3' end of the promoter-primer. Following the interaction of the opposite strand primer with the newly synthesized DNA strand, a second strand of DNA is synthesized from the end of the primer by the reverse transcriptase, thereby forming a double-stranded DNA molecule. The RNA polymerase recognizes the promoter sequence of this double-stranded DNA template and initiates transcription. Each newly synthesized RNA amplification product re-enters the TMA process and serves as a template for a new round of replication, thereby resulting in the exponential expansion of the RNA amplification products. Because each DNA template can generate 100-1000 copies of the RNA amplification product, this expansion can generate 10 billion amplification products within an hour. The entire process is autocatalytic and takes place at a constant temperature.

[0075] In some embodiments, the target nucleic acid sequence is amplified by PCR. Optionally, detection of the amplification product can be performed as the reaction is occurring (i.e., so-called "real-time PCR"). Generally, a fluorescent signal that increases with time or cycle number indicates the presence of an amplification product in the reaction. The procedure used in the examples described below employed PCR technology.

[0076] kit The present disclosure also encompasses kits that can be used to carry out the disclosed sample preparation procedures. The kit typically includes an alkaline composition (e.g., a strong base), a pH buffer, a surfactant, and a target capture reagent in separate vials or containers. In certain embodiments, one or more of these reagents are dry, lyophilized, or semi-solid compositions that can be reconstituted with a liquid component, such as water, before use. In certain embodiments, the alkaline composition needs to be reconstituted with a liquid agent before use. In other embodiments, the alkaline composition is packaged in the kit as a liquid composition.

[0077] The following examples describe the development of sample preparation techniques useful for isolating nucleic acids from a variety of bacteria and provide reasons why certain methods were deemed superior to others. Workflows employing initial contact of the biological sample under test with a lysis reagent containing detergent and either a strong base at high pH or a pH-buffered detergent solution at substantially neutral pH, unexpectedly, did not yield the best results when isolating nucleic acids from organisms that were considered difficult to lyse. Most of the tests described below were performed using automated equipment that performed sample processing and real-time nucleic acid amplification and detection steps. In some cases, the automated equipment was used only for sample processing (e.g., isolation of DNA from biological samples) and nucleic acid amplification was performed on a separate instrument. In all cases, detection of the target organism using a nucleic acid amplification assay was evidence of efficient lysis of the target organism in the biological sample.

[0078] Example 1 shows that a lysis protocol using a combination of strong base and detergent produced successful results in a prototype real-time multiplex assay for the detection of methicillin-resistant Staphylococcus aureus (MRSA). Results showed 100% detection of the target organism at an input level of 1,000 CFU / ml.

[0079] Example 1 Demonstration of lysis of Methicillin-resistant Staphylococcus aureus (MRSA) The efficiency of bacterial cell lysis and nucleic acid isolation was determined using a prototype real-time assay for the detection of S. aureus or MRSA. The assay amplified four target nucleic acid sequences in a multiplex PCR format, with one of the targets being an internal control (IC) that was independent of the presence of the target organism. The three nucleic acid targets used to detect MRSA were the S. aureus-specific target; mecA or mecC (i.e., indicative of drug resistance); and a "junction" sequence, indicative of the insertion of a mobile drug resistance element into the S. aureus chromosome. A positive MRSA call was made when the S. aureus marker, the junction marker, and the mecA or mecC sequence were all detected.

[0080] The procedure for isolating nucleic acids from target organisms was divided into three parts: sample lysis; target capture; and a step for washing the captured nucleic acids. A tube containing a sample of MRSA bacteria (ATCC BAA-41 strain) at 100 CFU / ml to 10,000 CFU / ml in modified Liquid Amies transport medium (Copan Diagnostics Inc.; Corona, CA). This detergent-free transport medium, which maintains the viability of bacterial cells for subsequent culture or growth, contains NaCl, KCl, CaCl2, MgCl2, monopotassium phosphate, disodium phosphate, and sodium thioglycolate in an aqueous solution. Sample lysis was performed by combining 126 μl of lysis reagent ("LR-A") with 300 μl of liquid containing the biological sample to be tested, thereby resulting in a first liquid composition. The LR-A used in the procedure was a detergent-free aqueous solution containing 0.4 N LiOH, 8% LLS, 2% TRITON® X-100, and 0.05% antifoam. TRITON® X-100 was included in the LR-A reagent to aid in processing samples containing added mucin (e.g., to model nasal swab samples). The resulting alkaline mixture was incubated at 43° C. for 10 minutes to model a workflow on an automated sample processing instrument. The incubation step is optional and can be omitted. Capture of the nucleic acid target to the solid support involved mixing 450 μl of the target capture reagent with the first liquid composition to give a second liquid composition. The target capture reagent contained LiOH, HEPES buffer, lithium lauryl sulfate, succinic acid, antifoam, poly(dT) 14 Magnetic beads and their 3' ends are poly(dA) 30The solution was an aqueous solution containing a target capture oligonucleotide with a tail. The non-specific capture oligonucleotide used in the procedure was as described in U.S. Pat. No. 9,051,601, the disclosure of which is incorporated herein by reference. The beads containing the captured nucleic acid were pulled to the side of the tube under the influence of a magnetic field, the liquid was aspirated, and the beads were washed using a wash buffer containing HEPES (pH 7.5), EDTA, NaCl, and sodium lauryl sulfate. After isolating and washing the magnetic beads to remove non-immobilized material, the nucleic acid captured on the beads served as a template for a real-time PCR multiplex assay.

[0081] Table 1 shows results supporting that MRSA bacteria were readily detected when samples contained at least 1,000 CFU / ml of the target organism. Indeed, evidence showed that the limit of detection (LoD) of the assay was between 300 CFU / ml and 1,000 CFU / ml. [Table 1]

[0082] Example 2 describes the use of the lysis reagents and conditions from Example 1 to isolate nucleic acid from a different organism. As shown below, C. difficile bacteria were not efficiently lysed in this manner to produce amplifiable nucleic acid.

[0083] Example 2 Reagents that lyse Staphylococcus aureus do not lyse C. difficile efficiently Next, nucleic acid was isolated from C. difficile bacteria using the lysis and target capture reagents and conditions from Example 1. The isolated nucleic acid served as a template for a prototype real-time amplification assay to detect C. difficile bacteria, and the results of the assay indicated the success of the sample processing procedure. The "pre-" sample processing was tested by comparing the results of a prototype C. difficile assay, the variable being the composition of the liquid reagent used to transport the biological sample. In one example, the sample transport tube contained 2.9 ml of a phosphate buffer (pH 6.6 to pH 6.8) solution containing 3% (w / v) lithium lauryl sulfate. In addition to promoting cell lysis, this solution (specimen transport medium, or "STM") protects the released nucleic acids by inhibiting the activity of nuclease enzymes that may be present in the sample. Alternatively, STM was replaced with a lysis reagent that was 0.4 N LiOH and 10% LLS.

[0084] The results of the amplification reactions confirmed that both sample processing procedures gave good final results from the amplification and detection assays. Regardless of whether the C. difficile samples were introduced into tubes containing STM or lysis reagent, each condition correctly yielded 3 / 3 positive results. However, there were important differences in the nature of the measurements that led to these conclusions. Samples processed using lysis buffer showed a Ct value of approximately 22.2, whereas samples processed using STM showed a Ct value of approximately 26.7. This substantial difference in appearance times during real-time amplification indicated that at least 10 times more DNA was liberated when the biological sample was contained in the alkaline detergent lysis reagent rather than in STM. Practical considerations made it undesirable to include strong alkali in the same tubes used for sample collection. This prompted efforts to develop a universal sample preparation technique that could be used to isolate nucleic acids from S. aureus, C. difficile, and other bacteria considered difficult to lyse.

[0085] Example 3 describes the procedure used to investigate the benefits of increasing the concentration of strong base in the lysis reagent. The experiments described below specifically compared the effect of including or omitting a detergent in the alkaline lysis reagent. Surprisingly, the presence of detergent actually reduced assay sensitivity as the concentration of strong base was increased.

[0086] Example 3 Detergents in the lysis reagent reduce assay sensitivity The effect of increasing the concentration of strong base in the lysis buffer was evaluated using a modified version of the procedure described in Example 1. The effect of including or omitting detergent in the lysis reagent was also evaluated using the same procedure. All samples containing 300 or 1,000 CFU / ml of MRSA bacteria (ATCC BAA-41 strain) in LiquidAmies transport medium further contained 0.25% mucin to simulate nasal swab specimens. Again, sample processing involved combining 300 μl of liquid containing the biological sample with 126 μl of lysis reagent. The lysis reagent used in this procedure was either 1.35 N LiOH or a combination of 1.35 N LiOH and 2% TRITON® X-100. After a short incubation, 450 μl of target capture reagent (e.g., pH-buffered detergent solution containing target capture reagent) was added to neutralize the alkaline mixture and promote immobilization or capture of nucleic acids to the solid support beads. All tests were performed in replicates of 20.

[0087] Table 2 shows results that support the observation that samples containing MRSA at levels of at least 1,000 CFU / ml resulted in positive detection of all three targets required to make an identification of MRSA. The table further includes results for positive detection of the internal control (IC), which is not dependent on successful bacterial lysis to generate a positive result. Indeed, the IC template nucleic acid amplified and detected in the MRSA assay was always added as a component of the pH-buffered detergent solution containing the target capture reagent. Importantly, increasing the strong base concentration from 0.4N to 1.35N increased the efficiency of bacterial lysis until each target could be detected in the amplification assay at an MRSA input level of 300 CFU / ml. Thus, increasing the concentration of strong base promoted bacterial lysis even in the absence of detergent. Surprisingly, the results in Table 2 also show that the presence of detergent in the alkaline lysis reagent had a clear detrimental effect as evidenced by the reduced positivity of all targets at an MRSA input level of 300 CFU / ml. Additional investigations conducted using increasing concentrations of strong base and LLS as surfactants revealed the formation of very thick mixtures containing precipitates, rendering the composition unusable as a lysis reagent when the concentration of strong base was increased to 2.5N. [Table 2]

[0088] The findings presented in Example 3 indicated that high concentrations of strong base were not suitable for use in combination with detergent. More specifically, the lysis reagent containing a combination of 1.35N LiOH and 2% TRITON® X-100 did not provide the same high level of sensitivity as when detergent was omitted. In fact, the use of strong base as the lysis reagent in the absence of pH buffer and detergent allowed 100% detection of 300 CFU / ml, which is a better sensitivity level than when LR-A of Example 1 was used. In addition, further increasing the concentration of strong base to 2.5N undesirably formed precipitates at a level of at least 8% in the lysis reagent containing LLS.

[0089] Example 4 describes a procedure demonstrating useful pH conditions for isolating nucleic acids from C. difficile bacteria. As described elsewhere herein, the pH conditions shown to be useful in this example were within the range of desired pH conditions for lysing cells and capturing nucleic acids on a solid support.

[0090] Example 4 Demonstration of pH conditions for bacterial cell lysis and nucleic acid capture pH readings were determined at each of the three steps where sample transport, bacterial lysis, and target capture occurred. As expected, the pH of the STM biological sample was determined to be 6.6. Combining 300 μl of biological sample with 126 μl of LiOH solution with a concentration ranging from 1.325 N to 2.5 N resulted in a pH of approximately pH 12.95 to approximately pH 12.99. Mixing this alkaline sample with 450 μl of target capture reagent resulted in a pH ranging from pH 7.91 to pH 8.75. The captured and washed nucleic acid was subjected to nucleic acid amplification and detection using a prototype amplification assay for detecting C. difficile bacteria. The efficiency of lysis and target capture was evaluated using the results of the amplification assay.

[0091] The results of the procedure showed that bacterial cell lysis and target capture were efficient in almost all cases. Target capture was inefficient only when the pH of the second liquid composition resulting from mixing the base-treated biological sample with the target capture reagent (e.g., a pH-buffered detergent solution) was pH 11.81 (e.g., resulting from the use of a poorly buffered target capture reagent). All these results are consistent with the range of pH 12.0 to pH 13.5 favorable for cell lysis and DNA denaturation, and below pH 9.5 (e.g., pH 7.9 to pH 9.2) when allowing hybridization-based target capture.

[0092] Example 5 describes results showing that a surfactant may be required for best results in sample preparation procedures that use certain concentrations of strong base.

[0093] Example 5 Reducing detergent concentration during the lysis step will adversely affect results The procedure described in Example 1 was essentially followed, except that the concentration of detergent in the lysis reagent was varied from 0% (w / v) to 10% (w / v) instead of being fixed at 8% (w / v). All lysis reagents tested contained 0.4 N LiOH. All tests were performed using a fixed MRSA level of 1,000 CFU / ml, and 0.25% mucin in modified Liquid Amies transport medium to simulate samples containing nasal fluid. Positive detection of each of the four amplified targets was evaluated and compared at each detergent level.

[0094] The results shown in Table 3 unexpectedly indicate that the various amplified targets had different sensitivities to the amount of detergent used during the sample lysis procedure. Also, the complete elimination of detergent resulted in very poor results. These findings suggested that a combination of detergent and alkali was necessary to efficiently lyse MRSA bacteria at the test levels of strong base. As shown elsewhere herein, conditions that efficiently lysed one type of bacteria did not efficiently lyse all other types of bacteria. Therefore, there was interest in developing a single lysis reagent that could be used in an automated sample preparation platform to lyse all types of bacteria. [Table 3]

[0095] Example 6 demonstrates how a sample preparation technique using contacting a biological sample with an alkaline composition lacking detergent and pH buffer, followed by neutralization in the presence of detergent and target capture reagent, can improve the results of a prototype amplification assay for detecting Group B Streptococcus (GBS). GBS bacteria are another group of bacteria known to be difficult to lyse, such as Staphylococcus aureus and C. difficile. Streptococcus agalactiae served as a representative of GBS bacteria in this example. The comparative results shown below confirmed that superior results can be obtained using the techniques disclosed herein.

[0096] Example 6 Sample preparation techniques dramatically improve detection of GBS bacteria A prototype assay for detecting nucleic acids of GBS bacteria was used to compare the effectiveness of two different sample preparation techniques. The two methods used substantially identical reagents, but different procedural steps were used to process the samples, resulting in widely different results. The first method involved combining a biological sample with a pH-buffered detergent solution containing a target capture reagent (e.g., magnetic beads displaying immobilized oligonucleotides, etc.) to create a first liquid composition having a pH that was in the range of pH 6.5 to pH 8.0. An aliquot of a strong base solution (1.68 N LiOH) was then mixed with the first liquid composition to create a second liquid composition that had a pH below pH 9.5 (e.g., pH 8.2 to pH 9.2) and provided suitable conditions for capture of nucleic acids by immobilization to a solid support. The captured nucleic acids were purified by washing the solid support with a wash buffer. The second method involved combining a biological sample with an aliquot of an alkaline composition (1.68 N LiOH), which lyses cells and denatures nucleic acids, to create a first liquid composition. The alkaline composition mixed with the biological sample was free of surfactant and pH buffer, and the pH of the first liquid composition was greater than pH 12.5 (e.g., pH 12.5-pH 13.2). An aliquot of a pH-buffered surfactant solution containing a target capture reagent (e.g., magnetic beads displaying immobilized oligonucleotides, etc.) was then mixed with the first liquid composition to create a second liquid composition having a pH less than pH 9.5 (e.g., pH 7.8-pH 8.9) and providing suitable conditions for capture of nucleic acids by immobilization to a solid support. The captured nucleic acids were purified by washing the solid support with a wash buffer.

[0097] The first method described in this example was useful for isolating both RNA and DNA, while the second method was useful for isolating DNA and substantially eliminating RNA from the final purified nucleic acid composition. Tests conducted using known input levels of RNA virus targets prepared by two different methods demonstrate this fact. More specifically, quantitative real-time PCR amplification and detection revealed that the difference in appearance time of the two running curves was approximately 24 Ct intervals. This meant that the copy number of the RNA target was reduced by approximately 16 million times using the second sample method.

[0098] The results shown in Table 4 confirmed that the sample preparation technique using an alkaline composition (strong base without pH buffer or surfactant) to lyse the bacteria yielded superior results. More specifically, with this technique, the detection limit was determined to be approximately 39 CFU / ml. Conversely, the use of an alkaline shock technique to lyse the GBS bacteria gave a detection limit of 1,416 CFU / ml. Again, initial treatment with a strong base followed by neutralization in the presence of a pH buffer, surfactant, and target capture reagent yielded superior results in an automated sample preparation workflow. [Table 4]

[0099] Importantly, the second of the two sample preparation methods used in Example 6 was also used to obtain efficient lysis and capture of nucleic acids from biological samples containing either S. aureus or C. difficile bacteria.

[0100] As used herein, a first liquid composition can be said to be "substantially detergent-free" if the detergent concentration is less than 0.001% (w / v). Biological samples stored in modified Liquid Amies transport medium and combined or mixed with a detergent-free alkaline composition resulted in a first liquid composition that was completely detergent-free and provided excellent results in DNA isolation procedures.

[0101] Advantageously, bacteria transported in detergent-free medium remained viable and could be cultured for species verification or typing using conventional microbiological techniques. Thus, a single biological sample can be used for both molecular and microbiological analysis. However, apparently the presence of detergent in the first liquid composition did not impair the ability to achieve good results in the DNA isolation method. In fact, the first liquid composition obtained by combining the biological sample (stored in STM) and the alkaline composition of the previous example, having a detergent concentration of about 2% (w / v), also provided excellent results as shown by the efficient detection of GBS sequences in the PCR protocol.

[0102] The present invention has been described with reference to certain specific examples and embodiments thereof. Of course, several different embodiments of the present invention will be apparent to those of ordinary skill in the art upon review of the foregoing detailed description. Thus, the true scope of the present invention should be determined with reference to the appended claims. The present invention provides, for example, the following items. (Item 1) 1. A method for processing a biological sample, comprising the steps of: (a) mixing the biological sample with an alkaline composition that lyses cells and denatures DNA to produce a first liquid composition, The first liquid composition has a pH in the range of pH 12.0 to pH 13.5; (b) mixing the first liquid composition with a pH buffered surfactant reagent to produce a second liquid composition having a pH less than pH 9.5; the pH buffered detergent reagent comprises a pH buffer, a detergent, and solid support particles that capture DNA; (c) isolating said solid support particles and any DNA captured thereon from said second liquid composition. (Item 2) 2. The method of claim 1, wherein the first liquid composition is substantially free of a surfactant. (Item 3) 3. The method of claim 1 or 2, wherein the biological sample contains viable bacterial cells, and the method further comprises culturing the viable bacterial cells to increase the number of bacterial cells. (Item 4) 4. The method according to any one of items 1 to 3, wherein the second liquid composition has a pH in the range of pH 8.0 to pH 9.2. (Item 5) 5. The method of any one of items 1 to 4, wherein each of steps (a) to (c) is performed on an automated instrument comprising a robotic fluid transfer device. (Item 6) (d) performing an in vitro nucleic acid amplification reaction using the DNA isolated in step (c) as a template, and detecting a product of the in vitro nucleic acid amplification reaction. The method according to any one of items 1 to 5. (Item 7) 7. The method of claim 6, wherein detecting a product of the in vitro nucleic acid amplification reaction indicates the presence of a species of Gram-positive bacteria in the biological sample. (Item 8) 7. The method of claim 6, wherein step (d) is carried out on an automated device. (Item 9) 9. The method of any one of items 5 to 8, wherein step (a) comprises combining the biological sample and the alkaline composition in a reaction vessel to create the first liquid composition, and step (b) comprises adding the pH buffered surfactant reagent to the reaction vessel containing the first liquid composition to create the second liquid composition, and each of steps (a) and (b) is performed using the robotic fluid transfer device of the automated instrument. (Item 10) The method according to any one of items 5 to 9, wherein the automated device further comprises a transport mechanism for moving the reaction vessel from one position within the automated device to a different position within the automated device. (Item 11) 11. The method according to any one of items 1 to 10, wherein after step (b) and before step (c), there is a step of incubating the first liquid composition for a period of from 1 minute to 10 minutes. (Item 12) 12. The method of claim 11, wherein the incubating step comprises heating the first liquid composition. (Item 13) 12. The method of claim 11, wherein the incubating step comprises transporting the reaction vessel from a first location of the automated instrument to a second location of the automated instrument, the second location of the automated instrument being at a higher temperature than the temperature of the first location of the automated instrument. (Item 14) 14. The method of any one of items 1 to 13, wherein the solid support particles comprise magnetically attractable particles. (Item 15) 15. The method according to any one of items 1 to 14, wherein the solid support particles in step (b) are solid support particles that capture DNA regardless of base sequence, and step (c) comprises washing the solid support particles to remove any material not immobilized thereon, and then retaining the washed solid support particles, thereby isolating the captured DNA. (Item 16) 16. The method according to any one of items 1 to 15, wherein the alkaline composition comprises a strong base in aqueous solution at a concentration of 0.1N to 2.2N. (Item 17) 17. The method according to any one of items 1 to 16, wherein the pH of the first liquid composition is in the range of pH 12.5 to pH 13.2. (Item 18) 18. The method according to any one of items 1 to 17, wherein the pH of the second liquid composition is in the range of pH 7.6 to pH 8.8. (Item 19) 19. The method according to any one of items 1 to 18, wherein the alkaline composition comprises a strong base in a concentration of 1.0N to 1.7N. (Item 20) 20. The method of claim 16 or 19, wherein the strong base is selected from the group consisting of NaOH, KOH, and LiOH. (Item 21) 21. The method according to any one of items 1 to 20, wherein all steps are carried out under automated process control.

Claims

1. 1. A method for processing a biological sample, comprising the steps of: (a) mixing the biological sample with an alkaline composition that lyses cells and denatures DNA in the biological sample to prepare a first liquid composition, the alkaline composition comprising a strong base at a concentration of 1.0 N to 2.2 N; the first liquid composition having a pH in the range of pH 12.0 to pH 13.5 and less than 0.001% (w / v) surfactant; (b) mixing the first liquid composition with a pH buffering surfactant reagent to produce a second liquid composition having a pH less than pH 9.5; the pH buffered detergent reagent comprises a pH buffer, a detergent, and solid support particles that capture DNA; (c) isolating said solid support particles and any DNA captured thereon from said second liquid composition.

2. 10. The method of claim 1, wherein the biological sample contains viable bacterial cells, the method further comprising culturing the viable bacterial cells to increase the number of bacterial cells.

3. The method of claim 1 or 2, wherein the second liquid composition has a pH in the range of pH 8.0 to pH 9.

2.

4. The method of any one of claims 1 to 3, wherein each of steps (a) to (c) is performed on an automated instrument comprising a robotic fluid transfer device.

5. 5. The method of any one of claims 1 to 4, further comprising the step of: (d) performing an in vitro nucleic acid amplification reaction using the DNA isolated in step (c) as a template, and detecting a product of the in vitro nucleic acid amplification reaction.

6. 6. The method of claim 5, wherein detecting a product of the in vitro nucleic acid amplification reaction indicates the presence of a species of Gram-positive bacteria in a biological sample.

7. The method of claim 5 , wherein step (d) is performed on an automated machine.

8. 8. The method of any one of claims 4 to 7, wherein step (a) comprises combining the biological sample and the alkaline composition in a reaction vessel to create the first liquid composition, and step (b) comprises adding the pH buffered detergent reagent to the reaction vessel containing the first liquid composition to create the second liquid composition, and each of steps (a) and (b) is performed using the robotic fluid transfer device of the automated instrument.

9. 9. The method of claim 8, wherein the automated instrument further comprises a transport mechanism for moving the reaction vessel from one location within the automated instrument to a different location within the automated instrument.

10. The method of any one of claims 1 to 9, wherein after step (b) and before step (c) there is a step of incubating said first liquid composition for a period of from 1 minute to 10 minutes.

11. The method of claim 10 , wherein the incubating step comprises heating the first liquid composition.

12. The method of claim 8 or claim 9, wherein after step (b) and before step (c), there is a step of incubating the first liquid composition for a period of from 1 minute to 10 minutes, the incubating step including transporting the reaction vessel from a first location of the automated equipment to a second location of the automated equipment, the second location of the automated equipment being at a higher temperature than the temperature of the first location of the automated equipment.

13. The method of any one of claims 1 to 12, wherein the solid support particles comprise magnetically attractable particles.

14. 14. The method according to any one of claims 1 to 13, wherein the solid support particles in step (b) are solid support particles that capture DNA regardless of base sequence, and step (c) comprises washing the solid support particles to remove any material that is not immobilized on the solid support particles, and then retaining the solid support particles after washing, thereby isolating the captured DNA.

15. The method according to any one of claims 1 to 14, wherein the alkaline composition comprises a strong base in aqueous solution at a concentration of from 0.1N to 2.2N.

16. The method according to any one of claims 1 to 15, wherein the pH of the first liquid composition is in the range of pH 12.5 to pH 13.

2.

17. The method of any one of claims 1 to 16, wherein the pH of the second liquid composition is in the range of pH 7.6 to pH 8.

8.

18. The method according to any one of claims 1 to 17, wherein the alkaline composition comprises a strong base in a concentration of from 1.0N to 1.7N.

19. 19. The method of claim 15 or claim 18, wherein the strong base is selected from the group consisting of NaOH, KOH, and LiOH.

20. The method of any one of claims 1 to 19, wherein all steps are performed under automated process control.

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