Rapid isolation and collection of microbial RNA from biological samples

Abstract

Liquid chromatography is used as a bulk filtration process to rapidly separate and collect microbial RNA from biological samples for the detection and identification of microbial species. In various embodiments, genetic sequencing of microbial RNA molecules derived from a biological sample is improved by obtaining the biological sample and then preparing it as a test sample for liquid chromatography. Liquid chromatography is used to bulk filter the microbial RNA molecules from a mixture of RNA molecules in the test sample to separate and collect the microbial RNA molecules into two or more fraction outputs, at least one of which is a fraction within the void volume of the liquid chromatography. One or more fraction outputs for genetic sequencing are prepared, including the fraction output within the void volume, and genetic sequencing is performed on the one or more prepared outputs to detect microbial RNA derived from the biological sample.

Description

[Technical Field]

[0001] This disclosure relates to the isolation of nucleic acids from biological samples. More specifically, this disclosure relates to more efficient and effective detection and identification of microbial species by using liquid chromatography as a bulk filtration process to isolate and collect microbial RNA from biological samples. [Background technology]

[0002] The detection and identification of microbial species is important for diagnosing and treating disease and identifying sources of contamination in clinical, environmental, or production contexts. Microbial species can include bacteria, viruses, protozoa, fungi, algae, amoebas, and slime molds.

[0003] The detection and identification of specific microbial species can be useful not only in the evaluation and treatment of patients and products, but also in facilities and environments such as healthcare facilities (hospitals, clinics, etc.) and food production environments (factories, kitchens, etc.). For example, detection of specific microbial species is used in healthcare facilities to identify the presence and identity of pathogens that cause infections in patients, allowing for appropriate treatment and rescue. Similar methods are also used in healthcare, the food industry, long-term care, hospitality, homeland security, aerospace, and the private sector.

[0004] Unfortunately, detecting microbial species in biospecimens obtained from such clinical, environmental, or production contexts differs from detecting microbial species in controlled biospecimens used in theoretical or research contexts. Raw biospecimens from such clinical, environmental, or production contexts are uncontrolled and unpurified, and the time and expense required to obtain useful results from biospecimens in theoretical or research contexts will significantly exceed that required to obtain results from biospecimens in clinical, environmental, or production contexts.

[0005] Biospecimens are uniquely complex and can contain a variety of biological materials, ranging from urine and feces to whole blood, serum, and intact tissues. Biospecimens can contain lipids, proteins, nuclear and mitochondrial DNA, and RNA (i.e., tRNA, rRNA, and mRNA), and will contain all of the above macromolecules associated with both the mammalian source specimen and any unicellular organisms (i.e., microbial species) present in the specimen and infecting the host organism. Generally, pathogen biomarkers (e.g., DNA and RNA) are present at significantly lower levels than biomarkers in the source specimen, making them difficult to isolate and detect. Current techniques for detecting microorganisms in theoretical and research contexts rely on cultivation and purification techniques applied to the specimen to form a relevant, usable biospecimen suitable for genetic analysis.

[0006] Culture techniques can take up to 30 hours before a sample can be analyzed, thereby delaying the ability to act on the detection of specific microbial species and obtain timely results. Examples of such culture techniques used to amplify gene sequences are described, for example, in U.S. Patent Nos. 8,313,931 (20-hour incubation) and 9,435,739 (18-24 hours incubation), as well as the 3M® Molecular Detection System (available at https: / / multimedia.3m.com / mws / media / 1353351O / 3m-molecular-detection-assay-2-l-monocytogenes-update.pdf and described in U.S. Patent Application Publication No. 2017 / 0219577A1, with up to 30 hours of incubation).

[0007] Purification techniques for isolating and detecting biomarkers for microbial species using filtration, elution, or binding techniques, such as those described in U.S. Patents 9,062,303 and 8,383,340, have been used. Chaotropic agents such as DNase have been used to degrade non-RNA proteins in clinical samples (Tan et al., J Biomed and Biotech, 2009; Turbo DNase, available from ThermoFisher, U.S. Patent 10,077,439). However, these methods do not allow for the separation of microbial RNA from non-microbial RNA. Similarly, methods for extracting Escherichia coli from human blood by co-purification using different lysis buffers have been described (Brennecke et al., J Med Micro, 2017, 66:301). However, these methods rely on first extracting target RNA from the biological sample and then degrading and removing residual DNA. In research contexts, isolation of specific bacterial strains using specially prepared sterile plates has been utilized to obtain relevant biological samples of each bacterial strain suitable for genetic analysis. See materials available at the following links (https: / / www.wrightlabs.org / metatranscriptomics_2, https: / / aac.asm.org / content / 60 / 8 / 4722, https: / / www.nature.com / articles / s41598-018-21841-9). Unfortunately, these types of purification techniques require further processing and manipulation of the biological sample, which can result in degradation of the genetic biomarkers of interest.

[0008] Various methods and systems for detecting microbial DNA, particularly in theoretical or research contexts, are known in the art. However, these techniques rely on the detection of microbial DNA and cannot distinguish between the presence of live and dead microbial species. Furthermore, because other types of DNA in a biological sample typically overwhelm the relative amount of microbial DNA in that biological sample, detecting microbial DNA using traditional DNA identification requires the cultivation or preparation of a biological sample in which sufficient numbers of the microbial species of interest have been grown to reliably detect and identify those microorganisms.

[0009] To more effectively, efficiently, and rapidly identify microbial species in such biological samples for evaluation of patient treatments and products, as well as for facility and environmental assessment, technologies are needed that can rapidly isolate and detect biomarkers of microbial species from biological samples without significant and deliberate cultivation or purification. Summary of the Invention

[0010] One aspect of the present disclosure is a method for rapidly converting a biological sample into a microbial RNA filtration sample by bulk filtration without degrading microbial RNA, which is a biomarker for pathogenic organisms, by utilizing void volume in the liquid chromatography step to increase the efficiency with which the presence of such pathogenic organisms can be detected by subsequent gene sequencing techniques.

[0011] Another aspect of the present disclosure relates to a method for rapidly filtering a test sample obtained from a biological sample to isolate and collect microbial RNA, a biomarker for live or viable microbial species, which method is substantially free of steps designed to intentionally amplify genetic material. In various embodiments, the present disclosure includes obtaining a biological sample, digesting or preparing the biological sample to enable liquid chromatography to generate a test sample, and isolating and collecting microbial RNA molecules from the test sample using bulk filtration with liquid chromatography.

[0012] In accordance with an aspect of the present invention, a method for rapidly filtering a test sample obtained from a biological specimen to isolate and collect microbial RNA can be achieved by bulk filtering microbial RNA molecules from a mixture of RNA molecules, including host RNA molecules, using only the void volume output of a liquid chromatograph to effectively amplify microbial RNA molecules from the test sample relative to host RNA molecules, thereby eliminating the steps of incubating and purifying genetic material.

[0013] In one embodiment, a method for improving genetic sequencing of microbial RNA molecules derived from a biological sample includes the steps of: (a) obtaining the biological sample; (b) digesting the biological sample by interacting the biological sample with a reagent to generate a test sample; and (c) bulk filtering the microbial RNA molecules from a mixture of RNA molecules in the test sample using liquid chromatography to separate and collect the microbial RNA molecules from the test sample.

[0014] In one embodiment, a method for filtering microbial single-stranded nucleic acid sequences from a mixture of mammalian single-stranded nucleic acid sequences and / or microbial single-stranded nucleic acid sequences by recovering microbial RNA molecules from the void volume of a liquid chromatography process, wherein the microbial single-stranded nucleic acid sequences are catalysts for protein synthesis.

[0015] In one embodiment, a method for isolating microbial RNA by filtering microbial RNA molecules from a biological sample includes obtaining a biological sample, preparing the biological sample by interacting the sample with a reagent, and bulk filtering the microbial RNA molecules from a mixture of RNA molecules in a test sample using a liquid chromatography device to separate and collect the microbial RNA molecules in the void volume of the liquid chromatography device from the test sample.

[0016] In some embodiments, the flow rate of the mobile phase is about 0.5 mL / min to about 3.5 mL / min, in some embodiments, about 550 μL / min to about 2 mL / min, in some embodiments, about 600 μL / min to about 1 mL / min, and in other embodiments, about 650 μL / min to about 850 μL / min.

[0017] In some embodiments, the void volume has a retention time of greater than or equal to 0 seconds and less than about 6 minutes, in some embodiments less than about 5 minutes, in some embodiments less than about 4 minutes, in some embodiments less than about 3 minutes, and in other embodiments less than about 2 minutes.

[0018] In some embodiments, multiple fractions of mobile phase containing the filtered sample material are eluted using liquid chromatography and collected over a time period between about 5 seconds and about 1 minute, in some embodiments, between about 10 seconds and about 45 seconds, and in some embodiments, between about 15 seconds and about 30 seconds, wherein each aliquot contains between about 100 μL and about 1 mL, in some embodiments, between about 125 μL and about 750 μL, in some embodiments, between about 150 μL and about 500 μL, and in some embodiments, between about 175 μL and about 250 μL.

[0019] In one embodiment, the microbial RNA is detected from one or more fractions eluted using liquid chromatography, and the microbial RNA is detected using genetic sequencing for one or more of the fractions. In some embodiments, the one or more fractions include one or more fractions of the void volume eluted using liquid chromatography, and the microbial RNA is detected using genetic sequencing for one or more fractions including the void volume.

[0020] In some embodiments, each fraction is subjected to dehydration prior to gene sequencing, where each aliquot is dehydrated to a volume of between about 15 μL and about 500 μL, in some embodiments, between about 20 μL and about 100 μL, in some embodiments, between about 25 μL and about 75 μL, and in some preferred embodiments, between about 35 μL and about 65 μL.

[0021] The above summary is not intended to describe each illustrated embodiment or every implementation of the subject matter herein. The figures and the detailed description that follow more particularly exemplify various embodiments. [Brief explanation of the drawings]

[0022] A more complete understanding of the subject matter of the present disclosure can be obtained by considering the following detailed description of various embodiments in conjunction with the accompanying drawings, in which:

[0023] [Figure 1] FIG. 1 shows a representative workflow diagram of one embodiment of the bulk filtration method according to the present invention. [Figure 2] Figure 2 shows a representative chromatogram of the isolation of microbial RNA. [Figure 3] Figure 3 shows a representative chromatogram of the isolation of microbial RNA. [Figure 4] FIG. 4 shows a representative block diagram of one embodiment of an overall workflow incorporating bulk filtration as part of the overall process for identifying microbial RNA in a biological sample. [Figure 5A] FIG. 5A shows a representative chromatogram of the separation of a sample having E. coli RNA combined with Homo sapiens RNA, with fractions collected every 15 seconds and retention times overlaid on the 15-second fractions, showing peaks within each fraction that include void volume, according to certain embodiments of the present invention. [Figure 5B] FIG. 5B is the chromatogram of FIG. 5A without the retention times overlaid on each of the 15 second fractions. [Figure 6A] Figure 6A shows another representative chromatogram of the separation of a sample in which E. coli RNA was reproducibly combined with Homo sapiens RNA, such that fractions collected every 15 seconds, according to certain embodiments of the present invention, have very similar peaks within each fraction, including the void volume. [Figure 6B]FIG. 6B is the chromatogram of FIG. 6A without the retention times overlaid on each of the 15 second fractions. [Figure 7] FIG. 7 is a bar graph diagram of raw Homo sapiens read counts for the original sample and the sample filtered into fractions 1-23 using liquid chromatography and then genetically sequenced, according to certain embodiments of the present invention. [Figure 8] FIG. 8 is a bar graph depiction of raw internal standard ERCC counts for original samples and samples filtered into fractions 1-23 using liquid chromatography and then gene sequenced, according to certain embodiments of the present invention. [Figure 9] FIG. 9 is a bar graph depiction of the log-transformed ERCC-normalized Enterobacteriaceae / Homo sapiens ratios for original samples and samples filtered using liquid chromatography into fractions 1-23 and subsequently gene sequenced, according to certain embodiments of the present invention. [Figure 10] Figure 10 is a bar graph depicting the relative abundance of microbial and Homo sapiens reads in an original sample and a sample that was filtered into fractions 1-23 using liquid chromatography and then genetically sequenced, according to certain embodiments of the present invention.

[0024] While the various embodiments are susceptible to various modifications and alternative forms, specifics thereof have been shown by way of example in the drawings and will be described in detail. It is, of course, to be understood that the intention is not to limit the claimed invention to the particular embodiments described. On the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the subject matter defined by the claims. DETAILED DESCRIPTION OF THE INVENTION

[0025] In describing the various embodiments set forth in this disclosure, the following definitions and conventions will be used in understanding the disclosure of the various embodiments. In understanding this disclosure, it should be recognized that this disclosure describes various embodiments that integrate or combine specific techniques and equipment used by microbiologists with those used by analytical chemists. The following definitions and conventions are intended to provide a common set of terms that are useful and helpful in understanding this disclosure, as the two disciplines of microbiologists and analytical chemists may not have a consistent understanding or usage of terms.

[0026] The terms "mammal," "non-microbial species," or "non-microbial population" are understood to include all species that are not bacterial, viral, fungal, or yeast populations, or combinations thereof, or any mixture thereof, in a laboratory or natural environment, and that are comprised of multicellular organisms.

[0027] The terms "microorganism," "microbiome," or "microbial species" are understood to encompass a population of bacteria, viruses, fungi, or yeasts, or a combination thereof, or any mixture thereof, in a laboratory or natural environment, and are comprised of single-celled organisms.

[0028] The term "biological sample" refers to a raw biological sample that is unprocessed and originates from a mammalian species, including whole blood, plasma, mucus, serum, urine, feces, intact tissue, cerebrospinal fluid, synovial fluid, environmental swabs, food sources, and unknown powders obtained from surfaces. Thus, reference to a biological sample may include reference to one or more of the foregoing sources.

[0029] The term "test sample" refers to a biological sample after preparation or pretreatment for introduction into a liquid chromatography device, which may include performing steps to remove proteins, deoxyribonucleic acid (DNA), lipids, and other macromolecules.

[0030] The term "double-stranded nucleic acid sequence" refers to DNA.

[0031] The term "single-stranded nucleic acid sequence" refers to "ribonucleic acid" or "RNA" and is understood to encompass transfer RNA (tRNA), ribosomal RNA (rRNA), messenger RNA (mRNA), large RNA, small RNA, modified and non-modified RNA, fragmented RNA, and intact RNA.

[0032] The terms "liquid chromatography," "HPLC," "HPLC system," and the like are synonyms for the machines, devices, and techniques used to separate and collect microbial RNA from non-microbial RNA, according to various embodiments.

[0033] The terms percent (%), weight percent (%w / w), weight-volume percent (%w / v), weight %, and volume % are synonyms for the concentration of a substance when the weight of the substance is divided by the total weight or volume at standard room temperature and pressure and multiplied by 100.

[0034] The term "about" modifying the amount of an ingredient in a composition of the present disclosure or employed in a method of the present disclosure refers to variations in quantity that may occur due to unintentional procedural errors, differences in the manufacture, source, or purity of ingredients employed to make these compositions and methods, differences in equipment (machine) settings, for example, in typical measuring and handling procedures for making concentrates or solutions in the real world.

[0035] The term "about" also encompasses different amounts due to differences in equilibrium conditions of compositions resulting from a particular initial mixture. Whether modified by the term "about," the claims include equivalents to the amounts. All numbers expressing ingredient quantities or reaction conditions used herein, other than in the operating examples or where otherwise indicated, are understood to be modified in all instances by the term "about."

[0036] The central dogma of molecular biology explains that information may be passed from nucleic acid to nucleic acid or from nucleic acid to protein, but not from protein to protein or from protein to nucleic acid. Within both mammalian and microbial systems, RNA is a nucleic acid sequence involved in either protein synthesis, transport of amino acids for synthesis, or catalysis of protein synthesis. The present disclosure takes advantage of the fact that RNA is a transitional molecule between DNA and protein, and utilizes bulk filtration of microbial RNA as a means to identify species and protein functions of interest within a biological sample.

[0037] There are three "types" of RNA, called mRNA, tRNA, and rRNA. mRNA is responsible for protein synthesis, tRNA is responsible for transporting amino acids for synthesis, and rRNA is responsible for catalyzing or initiating protein synthesis. mRNA is relatively larger than the rRNA fragments and is more commonly studied because it is directly involved in coding for proteins. From an information standpoint, mRNA has historically been considered the most meaningful for scientific research.

[0038] Despite the interest in RNA, this class of molecule is difficult to analyze. Unlike DNA, which can survive for millions of years under ideal conditions, RNA typically degrades within minutes of an organism's death. However, because of its short half-life, its detection usually indicates the presence of an organism that was alive at the time of analysis.

[0039] Traditional molecular biology and biochemistry techniques rely on strategies, including but not limited to enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR), acid guanidinium thiocyanate-phenol-chloroform extraction (AGPC), and liquid-liquid extraction, to isolate RNA molecules from complex biological systems (Molecular Cell Biology, 4th ed., Lodish et al., W.H. Freeman, 1999). However, a key limitation of these techniques is the potential for RNAases present in the sample to degrade the RNA molecules of interest. Furthermore, both ELISA and PCR require prior knowledge of the structure of the target RNA to be useful (Bioanalytical Chemistry, 2nd ed., Manz et al., Imperial College Press, 2015). While AGPC and liquid-liquid extraction can isolate RNA molecules, these techniques isolate all RNA, regardless of the type or species of interest.

[0040] In contrast to traditional molecular biology and biochemistry techniques, traditional analytical chemistry techniques were developed to be applicable to the analysis of small molecules and have traditionally not been suitable for the analysis of biological macromolecules such as nucleic acids (Bioanalytical Chemistry, 2nd ed., Manz et al., Imperial College Press, 2015).

[0041] A hybrid technology field, bioanalytical chemistry, has developed to address the unique challenges of analyzing large, complex biomolecules, including RNA. The use of two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) gels, matrix-assisted laser desorption / ionization-time of flight (MALDI-TOF), capillary electrophoresis, and biosensors has enabled the quantification, structural elucidation, qualitative analysis, and separation of biomolecules of interest with greater specificity and speed than traditional molecular biology and biochemistry techniques.

[0042] Along with these biological analytical methods, high-performance liquid chromatography (HPLC) was adapted, enabling the analysis of large biomolecules (Principles of Instrumental Analysis, 5th ed., Skoog and Holler, Harcourt Brace, 1998). HPLC relies on the chemical separation of a mixture based on the interaction between a column (called the stationary phase) and a liquid (called the mobile phase). A typical HPLC column has a steel shell packed with polystyrene or silanol microbeads. When the mobile phase containing the target analyte is forced through the column, the components of the sample (mixture) are partitioned into and removed from the stationary phase. Components that "adhere" to the stationary phase are retained more easily, and the longer it takes for the component to exit the column, or elute. Once detected, each component is assigned a "retention time" on a plot called a "chromatogram." Each column has a "t0," which refers to all molecules not retained by the column, called the "void volume." This void volume is widely considered informationally unimportant among chromatographic analytical experts. Because this region of the chromatogram contains a mixture of molecules that have not been chemically separated, this region of the chromatogram has not been previously utilized, and the molecules found in this region have not previously been studied or of interest.

[0043] In some aspects of the present invention, the void volume represents a period of up to about 6 minutes from the time a sample is introduced into a column using liquid chromatography, in some embodiments, up to about 5 minutes, in some embodiments, up to about 4 minutes, in some embodiments, up to about 3 minutes, and in other embodiments, up to about 2 minutes, and the flow rate of the mobile phase is from about 0.5 mL / min to about 3.5 mL / min, in some embodiments, from about 550 μL / min to about 2 mL / min, in some embodiments, from about 600 μL / min to about 1 mL / min, and in other embodiments, from about 650 μL / min to about 850 μL / min.

[0044] Two types of HPLC are common in bioanalysis: reversed-phase liquid chromatography (RPLC) and ion-exchange liquid chromatography (IE-LC). RPLC has a column packed with a hydrophobic stationary phase, and molecules elute from the column according to both size and polarity. Hydrophobic molecules are retained for a long time, while hydrophilic molecules are hardly retained. Most hydrophobic molecules elute within the void volume of the column. The void volume in RPLC is made up of a mixture of one or more hydrophilic molecules of various sizes that fail to interact with the column stationary phase.

[0045] Unlike RPLC, IE-LC chemically separates molecules based on their net, overall charge. The column stationary phase is made of microspheres with a net positive charge. Molecules are adsorbed to the column stationary phase and desorbed by increasing the salt concentration or pH of the mobile phase. Molecules then elute from the column and are detected. Using IE-LC, molecules with larger size or more negative charge are retained the longest and take longer to elute from the column. Meanwhile, neutral and positively charged molecules elute in the void volume. While this method has previously been used to chemically separate mRNA from a mixture of total RNA, the void volume has not previously been explored or utilized as a means of RNA collection.

[0046] Traditionally, mRNA has been the most widely studied because mRNA is relatively larger than rRNA fragments, allowing scientists to obtain information more accurately and quickly. Traditional methods for analyzing such mRNA have removed and discarded other types of RNA, such as rRNA and tRNA, rather than retaining them for further analysis. Unlike these traditional approaches, various embodiments of the present disclosure utilize the central dogma of molecular biology to rapidly filter and isolate microbial RNA by collecting all RNA fragments found in the void volume of a liquid chromatography process, rather than chemically isolating the microbial RNA, with a particular focus on rRNA for further bioinformatics studies.

[0047] The present disclosure relates to a method for rapidly filtering a test sample obtained from a biological sample to isolate and collect microbial RNA, a biomarker for live or viable microbial species, which method is substantially free of steps designed to intentionally amplify genetic material. In various embodiments, the disclosure includes obtaining a biological sample, digesting or preparing the biological sample to generate a test sample that is amenable to chromatography, and isolating and collecting microbial RNA molecules from the test sample using bulk filtration with liquid chromatography.

[0048] In some embodiments of the invention, the methods of the invention can eliminate the need to incubate genetic material or purify genetic material by bulk filtering microbial RNA molecules from a mixture of RNA molecules, such as RNA molecules derived from the host from which the biological sample was obtained, to amplify microbial RNA molecules from a test sample. Thus, in some embodiments, the methods of the invention eliminate the step of incubating the test sample to grow genetic material by using only the void volume output of liquid chromatography to effectively amplify microbial RNA molecules. In some embodiments, the methods of the invention eliminate the step of purifying the test sample by using only the void volume output of liquid chromatography to effectively amplify microbial RNA molecules.

[0049] As illustrated in Figure 1, a clinical sample 100 is obtained. In one embodiment, the clinical sample is an environmental swab from a hard surface, which is extracted into a buffer solution in a test vial. The buffer solution may be 0.1X to 2.5X phosphate buffered saline (PBS), 0.05M to 1.5M peptone water, or 30% to 50% guanidine hydrochloride and 0.1% to 1% maleic acid.

[0050] In another embodiment, the biological sample is tissue or excrement obtained from mammalian species, including, but not limited to, humans and livestock species. Biological samples include, but are not limited to, whole blood, plasma, mucus, serum, urine, feces, cerebrospinal fluid, synovial fluid, and intact tissue. Clinical samples may be collected by blood draw, punch biopsy, stool culture, nasal swab, saliva sample, urinalysis, dermatological scraping, as well as any other established protocols for collecting specific forms of biological samples. Biological samples may include large RNA molecules, small RNA molecules, tRNA molecules, rRNA molecules, mRNA molecules, denatured and non-denatured RNA molecules, microbial RNA molecules, non-microbial RNA molecules, genomic DNA molecules, protein molecules, and other macromolecules.

[0051] The biological sample may be optionally mechanically homogenized, nitrogen cavitated, or sonicated using methods known in the art to prepare the sample for the subsequent digestion step and generate a test sample. The biological sample then undergoes a digestion step 200. Digestion of clinical samples may include enzymes, chaotropes, surfactants, detergents, and other additives known in the art. The process of digesting the biological sample removes genomic DNA molecules, protein molecules, and non-RNA macromolecules, and may be performed at low temperatures to prevent degradation of the biological sample, and may optionally include additives known to protect target molecules, such as those listed in Table 1.

[0052] Table 1: Additives to protect target molecules during digestion of clinical samples TIFF0007725453000001.tif103167

[0053] In one embodiment, digestion of a biological sample begins with dissolving the clinical sample by interacting the biological sample with guanidinium thiocyanate, N-lauroyl sarcosine, and ethanol. In one embodiment, a buffer solution of 55% to 85% guanidinium thiocyanate and 1% to 20% N-lauroyl sarcosine is mixed in equal volumes with a 70% to 100% ethanol solution. In one embodiment, a buffer solution of 65% to 75% guanidinium thiocyanate and 1% to 10% N-lauroyl sarcosine is mixed in equal volumes with a 70% to 100% ethanol solution. In yet another embodiment, a buffer solution of 65% to 75% guanidinium thiocyanate and 1% to 10% N-lauroyl sarcosine is mixed in equal volumes with a 90% to 100% ethanol solution.

[0054] In one embodiment, digestion of the biological sample by lysing the sample is continued by adding 1 to 3 volumes of a buffer and ethanol mixture to 1 volume of clinical sample. In one embodiment, 2 volumes of the buffer and ethanol mixture are added to 1 volume of clinical sample. In another embodiment, 200 μL to 400 μL of the buffer and ethanol mixture are added to 1 volume of clinical sample. In one embodiment, 250 μL to 350 μL of the buffer and ethanol mixture are added to 1 volume of clinical sample.

[0055] The mixture of biological samples was mixed by manual inversion, vortexing, or other means known in the art, and then transferred through a silica or polypropylene filter by centrifugation at 500-2000 g for 15-90 seconds. In one embodiment, centrifugation times of 15-45 seconds were used. The material that passed through the filter was discarded, while the DNA and RNA retained on the filter were subjected to further preparation steps.

[0056] Once the biological sample is digested, the next step in biological sample preparation and cleaning can involve interacting the clinical sample with at least one of guanidinium chloride, ethanol, 2-amino-2-(hydroxymethyl)-propane-1,3-dihydrochloride, and edetate disodium. In one embodiment, a mixture of 25%-55% guanidinium chloride in 70%-100% ethanol is added to the same silica or polypropylene filter in a volume of 300 μL-500 μL and centrifuged at 500 g-2000 g for 15-90 seconds. Material that passes through the filter is discarded. In another embodiment, 12 mg / m 3 ~790mg / m 3 2-amino-2-(hydroxymethyl)propane-1,3-dihydrochloride and 20 mg / m 3 ~2000mg / m 3 A mixture of 12 mg / m edetate disodium is prepared and added to the same silica or polypropylene filter in a volume of 400 μL to 900 μL, and centrifuged at 500 g to 2000 g for 15 to 60 seconds. The material that passes through the filter is discarded. In yet another embodiment, a 12 mg / m edetate disodium mixture is prepared. 3 ~790mg / m 3 2-amino-2-(hydroxymethyl)propane-1,3-dihydrochloride and 20 mg / m 3 ~2000mg / m 3 A second mixture of 100% EDTA and 100% EDTA is prepared and added to the same silica or polypropylene filter in a volume of 400 μL to 900 μL and centrifuged at 500 g to 2000 g for 30 to 180 seconds. In another embodiment, water is added to the same silica or polypropylene filter and centrifuged at 50 g to 2000 g for 15 to 60 seconds. The material that passes through the filter is the biological sample and is retained.

[0057] In one embodiment, a mixture of 30-47% guanidinium chloride in 80-100% ethanol is added to the same silica or polypropylene filter and centrifuged at 500-2000 g for 15-60 seconds. In yet another embodiment, a mixture of 35-45% guanidinium chloride in 90-100% ethanol is added to the same silica or polypropylene filter and centrifuged at 500-2000 g for 15-45 seconds. The material that passes through the filter is the biological sample and is retained.

[0058] In another embodiment, 25 mg / m 3 ~600mg / m 3 2-amino-2-(hydroxymethyl)propane-1,3-dihydrochloride and 50 mg / m 3 ~1000mg / m 3 In yet another embodiment, a mixture of 25 mg / m with edetate disodium is prepared and added to the same silica or polypropylene filter in a volume of 200 μL to 500 μL, and centrifuged at 500 g to 2000 g for 15 to 60 seconds. 3 ~600mg / m 3 2-amino-2-(hydroxymethyl)propane-1,3-dihydrochloride and 50 mg / m 3 ~1000mg / m 3 In yet another embodiment, a mixture of 25 mg / m with edetate disodium is prepared and added to the same silica or polypropylene filter in a volume of 200 μL to 500 μL, and centrifuged at 500 g to 2000 g for 15 to 60 seconds. 3 ~600mg / m 3 2-amino-2-(hydroxymethyl)propane-1,3-dihydrochloride and 50 mg / m 3 ~1000mg / m 3 Prepare a mixture of 100 µL of EDTA with disodium edetate and add it to the same silica or polypropylene filter in a volume of 200 µL to 500 µL, then centrifuge at 500 g to 2000 g for 15 to 45 seconds.

[0059] In another embodiment, 25 mg / m 3 ~600mg / m 32-amino-2-(hydroxymethyl)propane-1,3-dihydrochloride and 50 mg / m 3 ~1000mg / m 3 In yet another embodiment, a second mixture of 25 mg / m edetate disodium is prepared and added to the same silica or polypropylene filter in a volume of 400 μL to 900 μL, and centrifuged at 500 g to 2000 g for 30 seconds to 180 seconds. 3 ~600mg / m 3 2-amino-2-(hydroxymethyl)propane-1,3-dihydrochloride and 50 mg / m 3 ~1000mg / m 3 In yet another embodiment, a second mixture of 25 mg / m edetate disodium is prepared and added to the same silica or polypropylene filter in a volume of 600 μL to 800 μL, and centrifuged at 500 g to 2000 g for 30 seconds to 180 seconds. 3 ~600mg / m 3 2-amino-2-(hydroxymethyl)propane-1,3-dihydrochloride and 50 mg / m 3 ~1000mg / m 3 Prepare a second mixture with edetate disodium and add it to the same silica or polypropylene filter in a volume of 600 µL–800 µL and centrifuge it at 500 g–2000 g for 90 s–150 s.

[0060] In another embodiment, water is added to the same silica or polypropylene filter and centrifuged at 50g to 2000g for 15 to 45 seconds. In yet another embodiment, water is added to the same silica or polypropylene filter and centrifuged at 1500g to 2000g for 15 to 45 seconds. In yet another embodiment, the water is DNase / RNase-free.

[0061] The biological sample digestion proceeds to a step of washing the biological sample by interacting the biological sample with at least one of proteinase K, guanidinium thiocyanate, N-lauroylsarcosine, ethanol, 2-amino-2-(hydroxymethyl)propane-1,3-dihydrochloride, and edetate disodium. In one embodiment, 4 U to 12 U of proteinase K is added to the washed biological sample and the sample is maintained at 45°C to 65°C for 15 to 60 minutes. In one embodiment, 4 U to 8 U of proteinase K is added to the washed biological sample and the sample is maintained at 45°C to 65°C for 15 to 60 minutes. In yet another embodiment, 4 U to 8 U of proteinase K is added to the washed biological sample and the sample is maintained at 50°C to 60°C for 15 to 60 minutes. In yet another embodiment, 4 U to 8 U of proteinase K is added to the washed biological sample and the sample is maintained at 50°C to 60°C for 20 to 40 minutes. In another embodiment, for solid tissues or composite matrices, incubation lasts from 1 to 3 hours. In another embodiment, 1 to 3 volumes of a mixture of 55% to 85% guanidinium thiocyanate and 1% to 20% N-lauroyl sarcosine are added to the retained biological sample. In a preferred embodiment, 1 to 2 volumes of 55% to 85% guanidinium thiocyanate and 1% to 20% N-lauroyl sarcosine are added to the retained biological sample. In yet another preferred embodiment, 1 to 2 volumes of 65% to 75% guanidinium thiocyanate and 1% to 10% N-lauroyl sarcosine are added to the retained biological sample.

[0062] In one embodiment, a buffer solution of 55% to 85% guanidinium thiocyanate and 1% to 20% N-lauroyl sarcosine is mixed with an equal volume of a 70% to 100% ethanol solution. In one embodiment, a buffer solution of 65% to 75% guanidinium thiocyanate and 1% to 10% N-lauroyl sarcosine is mixed with an equal volume of a 70% to 100% ethanol solution. In yet another embodiment, a buffer solution of 65% to 75% guanidinium thiocyanate and 1% to 10% N-lauroyl sarcosine is mixed with an equal volume of a 90% to 100% ethanol solution. In this embodiment, the biological sample is now washed and ready for digestion and separation of a test sample from the biological sample.

[0063] The digestion of the biological sample proceeds to a step of separating the test sample from the biological sample by interacting the biological sample with at least one of 55%-85% guanidinium thiocyanate, 1%-20% N-lauroylsarcosine, 70%-100% ethanol, DNase I, 2-amino-2-(hydroxymethyl)-propane-1,3-dihydrochloride, and edetate disodium. In one embodiment, the biological sample is first centrifuged at ≥10,000 g for 1 minute. In another embodiment, the supernatant from the centrifuged biological sample is transferred to a new silica or polypropylene filter and centrifuged at ≥10,000 g for 1 minute. The material that passes through the filter is the biological sample and is retained.

[0064] In another embodiment, 1-3 volumes of 70-100% ethanol are added to a biological sample in a mixture of 25%-85% guanidinium thiocyanate and 1%-20% N-lauroyl sarcosine, and the resulting solution is mixed thoroughly by manual inversion, vortexing, or other methods known in the art. In a preferred embodiment, 1-2 volumes of 90-100% ethanol are added to a clinical sample in a mixture of 65%-76% guanidinium thiocyanate and 1%-10% N-lauroyl sarcosine, and the resulting solution is mixed thoroughly.

[0065] In another embodiment, the resulting solution is transferred to a silica or polypropylene filter and centrifuged at 10,000 g to 16,000 g for 15 to 60 seconds. In a preferred embodiment, the resulting solution is centrifuged for 15 to 45 seconds. The material that passes through the filter is discarded.

[0066] In another embodiment, 200 μL to 600 μL of a mixture of 25% to 85% guanidinium thiocyanate and 1% to 20% N-lauroyl sarcosine is added to a silica or polypropylene filter, and the filter is centrifuged at 10,000 g to 16,000 g for 15 to 60 seconds. In a preferred embodiment, 65% to 75% guanidinium thiocyanate and 1% to 10% N-lauroyl sarcosine are added to a silica or polypropylene filter, and the filter is centrifuged at 10,000 g to 16,000 g for 15 to 45 seconds. The material that passes through the filter is discarded.

[0067] In another embodiment, 1 U to 15 U of DNase I is added to a silica or polypropylene filter and incubated at room temperature for 10 to 25 minutes. In a preferred embodiment, 3 U to 8 U of DNase I is added to a silica or polypropylene filter and incubated at room temperature for 10 to 25 minutes. In yet another preferred embodiment, 3 U to 8 U of DNase I is added to a silica or polypropylene filter and incubated at room temperature for 12 to 20 minutes. In another embodiment, 200 μL to 700 μL of a mixture of 25% to 55% guanidinium chloride and 70% to 99% ethanol is added to a silica or polypropylene filter and centrifuged at 10,000 g to 16,000 g for 15 to 60 seconds. In a preferred embodiment, 300 μL to 500 μL of a mixture of 35% to 45% guanidinium chloride and 95% to 99% ethanol is added to a silica or polypropylene filter and centrifuged at 10,000 g to 16,000 g for 15 to 45 seconds. In another embodiment, 12 mg / m 3 ~790mg / m 3 of 2-amino-2-(hydroxymethyl)propane-1,3-dihydrochloride and 20 mg / m 3 ~2,000mg / m 3400 μL to 900 μL of the mixture with edetate disodium is added to a silica or polypropylene filter and centrifuged at 10,000 g to 16,000 g for 15 to 60 seconds. 3 ~600mg / m 3 of 2-amino-2-(hydroxymethyl)propane-1,3-dihydrochloride and 50 mg / m 3 ~1,000mg / m 3 In another embodiment, 600 μL to 800 μL of the mixture of 12 mg / m edetate disodium is added to a silica or polypropylene filter and centrifuged at 10,000 g to 16,000 g for 15 to 45 seconds. 3 ~790mg / m 3 of 2-amino-2-(hydroxymethyl)propane-1,3-dihydrochloride and 20 mg / m 3 ~2,000mg / m 3 200 μL to 700 μL of the mixture with edetate disodium is added to a silica or polypropylene filter and centrifuged at 10,000 g to 16,000 g for 60 to 180 seconds. 3 ~600mg / m 3 of 2-amino-2-(hydroxymethyl)propane-1,3-dihydrochloride and 50 mg / m 3 ~1,000mg / m 3 300 μL to 500 μL of the mixture of edetate disodium is added to a silica or polypropylene filter and centrifuged at 10,000 g to 16,000 g for 90 to 150 seconds. In another embodiment, water is added to the same silica or polypropylene filter and centrifuged at 10,000 g to 16,000 g for 15 to 450 seconds. The material that passes through the filter is the test sample and is retained.

[0068] Test samples derived from biological samples may be further completed, as needed, by boiling, treatment with a denaturant, or storage at -70°C. In one embodiment, the test sample may be completed by boiling at 100°C to 120°C for 10 to 30 minutes. In another embodiment, the test sample may be completed by boiling at 100°C to 105°C for 10 to 20 minutes. In another embodiment, the test sample may be completed by treating the test sample with a denaturant comprising 25% to 50% of at least one of polysorbate 20, polysorbate 80, (1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol, polyethylene glycol tert-octylphenyl ether, 4-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol, t-octylphenoxypolyethoxyethanol, and polyethylene glycol tert-octylphenyl ether.

[0069] In another embodiment, the test sample may be completed by storing it at −70° C. The test sample contains large RNA molecules, small RNA molecules, tRNA molecules, rRNA molecules, mRNA molecules, modified and non-modified RNA molecules, microbial RNA molecules, and non-microbial RNA molecules and is now ready to undergo a step of isolating and collecting the microbial RNA molecules from the test sample using liquid chromatography 300.

[0070] The process of bulk filtering microbial RNA molecules from a test sample using liquid chromatography begins by injecting the test sample into the sample port of the liquid chromatography device 310. In one embodiment, the injection volume is 0.1 mL to 2 mL. In one embodiment, the injection volume is 0.5 to 1.5 mL. Within the liquid chromatography device 310, the test sample is pumped at a predetermined flow rate across a porous stationary phase column using a liquid mixture called the mobile phase. This process is controlled by a computer 320. Conventionally, the test sample is processed by the liquid chromatography device 310 to separate components with similar properties from the component of interest. Typically, all components of the test sample pass through the liquid chromatography device 310 to a waste line. However, in various embodiments of the present disclosure, the region containing the component of interest 330 is diverted to a collection vessel for further identification, either automatically or manually. In various embodiments, the component of interest 330 in the form of microbial RNA is detected using either a diode array, UV-Vis, or differential refractive index detector. Areas without the target component 340 are detected as flat lines ("baselines").

[0071] Liquid chromatography instruments filter microbial RNA molecules from non-microbial RNA molecules by decreasing and then increasing the amount of organic buffer in the mobile phase relative to the aqueous buffer in the mobile phase, separating and collecting these molecules. In one embodiment, the amount of organic buffer in the mobile phase varies between 20% and 100%, as shown in Table 2.

[0072] Table 2: Mobile phase composition for microbial RNA isolation and collection TIFF0007725453000002.tif80166

[0073] In one embodiment, the amount of organic buffer in the mobile phase varies between 30% and 100%, as shown in Table 3.

[0074] Table 3: Mobile phase composition for microbial RNA isolation and collection TIFF0007725453000003.tif81168

[0075] In some embodiments, the void volume elutes in the liquid chromatography mobile phase when the aqueous buffer percentage is greater than about 40%, greater than about 45%, greater than about 50%, greater than about 55%, and greater than about 60%. In some embodiments, the void volume elutes in the liquid chromatography mobile phase when the organic buffer percentage is less than about 60%, less than about 55%, less than about 50%, less than about 45%, and less than about 40%.

[0076] In one embodiment, the flow rate of the liquid chromatography mobile phase delivered by the pump is 0.5 mL / min to 3.5 mL / min. In one embodiment, the flow rate of the liquid chromatography mobile phase delivered by the pump is 1 mL / min to 2.5 mL / min. In yet another embodiment, the flow rate of the liquid chromatography mobile phase delivered by the pump is 1 mL / min to 1.5 mL / min.

[0077] In some embodiments, the flow rate of the mobile phase in the liquid is about 0.5 mL / min to about 3.5 mL / min, in some embodiments, about 550 μL / min to about 2 mL / min, in some embodiments, about 600 μL / min to about 1 mL / min, and in some embodiments, about 650 μL / min to about 850 μL / min.

[0078] The mobile phase is delivered as a mixture of two buffers: an organic buffer and an aqueous buffer. In one embodiment, the aqueous buffer contains 0.05-0.9 M triethylammonium acetate, phosphoric acid, citric acid, ammonium bicarbonate, formic acid, lactic acid, 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid, maleic acid, diethanolamine, piperidine, ethanolamine, and triethanolamine. In particular, a 0.05-0.5 M buffer containing at least one of triethylammonium acetate, formic acid, lactic acid, 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid, maleic acid, triethanolamine, and piperidine is used. More preferably, a 0.05-0.2 M solution of at least one of triethylammonium acetate, formic acid, 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid, maleic acid, and triethanolamine is used.

[0079] In another embodiment, the organic buffer comprises 5% to 60% of at least one of acetonitrile, methanol, ethanol, 1-propanol, 2-propanol, acetone, and tetrahydrofuran, mixed with a 0.05M to 0.9M aqueous buffer. Specifically, the organic buffer comprises 7% to 50% of at least one of acetonitrile, methanol, ethanol, 1-propanol, and acetone, mixed with a 0.05M to 0.5M aqueous buffer. In one embodiment, the organic buffer comprises 10% to 40% of at least one of acetonitrile, methanol, and acetone, mixed with a 0.05M to 0.5M aqueous buffer.

[0080] In various embodiments, a nonpolar compound, either in the form of polymer beads, polymer microspheres, or polymerized blocks, functions as a porous stationary phase column. Regardless of its exact form, the polymeric stationary phase column is inherently porous, meaning that it is characterized by pores. Stationary phase column materials are commercially available and are either uncoated or coated with special polymer compounds designed to cover the pores on the bead or microsphere surface, preventing microbial RNA from irreversibly interacting with the stationary phase column material. Within the outer structure of the stationary phase column are polymer microspheres, preferably composed of alkylated nonporous polystyrene-divinylbenzene copolymer. The stationary phase column is equipped with microspheres with a particle size of 8.0 μm to 50 μm, preferably 8.0 μm to 25 μm. As a result, the pore size of the stationary phase column is 1000 A to 5000 A, particularly 1000 A to 4000 A, more preferably 2000 A to 4000 A or 2500 A to 4000 A. The stationary phase column may have dimensions of 4 mm to 30 mm width x 40 mm to 150 mm length, preferably 5 mm to 10 mm width x 40 mm to 100 mm length, and more preferably 7 mm to 9 mm width x 40 mm to 60 mm length. According to one embodiment, the stationary phase column may be operated at ambient temperature, more preferably controlled at 20°C to 27°C.

[0081] Microbial RNA is filtered and separated from non-microbial RNA within the stationary phase column due to selective interaction of the mobile phase with the microspheres and pores of the stationary phase column. After filtration and separation, the microbial RNA and non-microbial RNA exit the stationary phase column at different times, i.e., elute, and are detected by a detector along with the microbial RNA eluted in the void volume of the column. In one embodiment, the separated microbial RNA is detected by a UV-Vis or diode array detector coupled to a liquid chromatography system at the outlet of the stationary phase column. The detection wavelength is 200 nm to 220 nm, particularly 203 nm to 217 nm, and more preferably 205 nm to 215 nm. In another embodiment, the separated microbial RNA is detected by a UV-Vis or diode array detector coupled to a liquid chromatography system at the outlet of the stationary phase column. The detection wavelength is 250 nm to 270 nm, particularly 257 nm to 267 nm, and more preferably 255 nm to 265 nm. In another embodiment, the isolated microbial RNA is detected using a differential refractive index detector connected to a liquid chromatography device at the outlet of the stationary phase column, with the default refractive index range set to 0.75RI to 2.00RI, particularly 0.9RI to 1.90RI, and more preferably 1.00RI to 1.75RI.

[0082] The separated microbial RNA is detected in a data trace called a chromatogram. Exemplary chromatograms of separated microbial RNA are shown in Figures 2 and 3. The peak observed in the window between 0 and 3 minutes corresponds to the separated microbial RNA. Non-microbial RNA will be detected in the window between 7 and 15 minutes, as disclosed by Ketterer et al. (see US Pat. No. 8,383,340). If microbial RNA is detected, the mobile phase containing the compound of interest (e.g., microbial RNA) is diverted from the waste line to a sample collection vial for future identification or experimentation.

[0083] In some embodiments, the sample collection of the mobile phase exiting the liquid chromatography includes the void volume and at least a portion of the mobile phase corresponding to the peak associated with the non-microbial RNA. In some embodiments, the mobile phase containing the compound of interest (e.g., microbial RNA) may be collected in one or more fractions of the eluted sample. In some embodiments, multiple fractions of the eluted sample are collected based on time, volume, or both, as the mobile phase containing any compound of interest elutes from the column.

[0084] In some embodiments, each fraction is collected from the column over a period of time between about 5 seconds and about 1 minute, between about 10 seconds and about 45 seconds, and between about 15 seconds and about 30 seconds. In some embodiments, each collected fraction has a volume between about 100 μL and about 1 mL, between about 125 μL and about 750 μL, between about 150 μL and about 500 μL, and between about 175 μL and about 250 μL.

[0085] In some embodiments, at least a portion of the void volume may be collected in a desired number of fractions, from at least 1 fraction to up to about 72 fractions, in some embodiments from at least 1 fraction to up to about 36 fractions, and in other embodiments from at least 1 fraction to up to about 24 fractions.

[0086] After the sample volume is collected into one or more fractions from the liquid chromatography, each of the one or more fractions can be subjected to gene sequencing.

[0087] In one embodiment, the microbial RNA is detected from one or more fractions eluted from the void volume using liquid chromatography, wherein the microbial RNA is detected from at least one of the one or more fractions eluted from the void volume using gene sequencing.

[0088] In some embodiments, each fraction is dehydrated prior to gene sequencing, where each fraction is dehydrated to a volume of between about 15 μL and about 500 μL, in some embodiments between about 20 μL and about 100 μL, in some embodiments between about 25 μL and about 75 μL, and in some preferred embodiments between about 35 μL and about 65 μL.

[0089] In some embodiments, a control may be introduced into a biological sample to normalize or monitor microbial RNA relative to non-microbial RNA. In some embodiments, the control may be introduced into the biological sample before digesting the biological sample, or after digestion, along with the test sample being introduced into liquid chromatography, or when the desired sample is subjected to genetic sequencing. The control is preferably selected to elute from the column in both the void volume and the normal sample separation volume. The control may be selected from any desired source that does not interfere with microbial or sample RNA. In some preferred embodiments, the control is synthetically derived RNA, such as ERCC RNA control or ERCC RNA control Ambion®, available from Thermo Fisher Scientific.

[0090] Various embodiments according to the present disclosure allow for the simultaneous isolation and collection of all microbial RNAs of interest, thereby enabling future identification of all viable (living) microbial species within a biological sample, or subsequent structural elucidation, quantification, or qualitative analysis of the microbial RNA. Various embodiments of the present disclosure allow for the simultaneous isolation and collection of microbial RNA from Gram-positive bacteria, Gram-negative bacteria, bacterial spores, enveloped viruses, non-enveloped viruses, RNA viruses, fungi, yeast, and protozoa. Exemplary microbial species within a test sample that may be filtered, isolated, and collected as microbial RNA using the methods of the present disclosure are found in Table 4.

[0091] Table 4. Microbial species detected by RNA isolation and collection from clinical samples TIFF0007725453000004.tif156168

[0092] FIG. 4 is a representative block diagram of one embodiment of an overall workflow incorporating bulk filtration as part of an overall process for identifying microbial RNA in a biological sample. At 410, a biological sample 402 is sampled or collected from a clinical, production, or environmental setting. The biological sample may be processed according to various embodiments at a facility immediately adjacent to the location where it was sampled, or in other embodiments, the biological sample 402 may be transported to a different facility at 420 for filtration according to various embodiments. Through various embodiments, the biological sample is converted into a test sample, allowing for the isolation and collection of microbial RNA using liquid chromatography. At 430, a test sample 404 containing the isolated and collected microbial RNA is generated in the void volume of an HPLC according to various embodiments. The test sample 404, containing only microbial RNA, is then sequenced at 440 to identify all viable (living) microbial species in the test sample. In other embodiments, 440 can include structural elucidation, quantitative, or qualitative analysis of the microbial RNA. Identification of viable microbial species from the biological sample may be collected in a database 450 and presented or reported via a user interface 460 accessible via a secure network interface.

[0093] Example The study involved using liquid chromatography to filter a mixture of microbial and human RNA and prepare libraries for subsequent gene sequencing. The materials used were Universal Human Reference (Thermofisher QS0639; Lot 244273) for human RNA, E. coli total RNA (Thermofisher AM7940; Lot 2219538) for microbial RNA, RoboSep columns, Wave Optimized® A, Wave Optimized® B, Wave Optimized® C, and nuclease-free ultrapure water (NFW).

[0094] The HPLC (Agilent 1260 Infinity II) was set to a constant flow rate of 0.75 mL / min, the column temperature was maintained at approximately 75 °C throughout the run, the DAD was set to detect at 260 nm (1 nm bandwidth), the fraction collector was set to collect 15-second fractions from 0:30 to 12 min, and 50 μL of sample was injected. The HPLC was set to have the mobile phase gradient shown in Table 5.

[0095] Table 5. Mobile phase elution gradient (flow rate 0.75mL / min) TIFF0007725453000005.tif45167

[0096] Human RNA was diluted in nuclease-free ultrapure water from 1,000 ng / μL to 50 ng / μL by diluting the entire 10 μL stock with 190 μL of chilled NFW. E. coli RNA was diluted to 10 ng / μL in two steps with NFW: (i) 10 μL of the 1,000 ng / μL E. coli RNA stock was diluted with 90 μL of chilled NFW to obtain a 100 ng / μL aliquot; and (ii) 10 μL of the 100 ng / μL diluted concentration was diluted with 90 μL of chilled NFW to obtain a 10 ng / μL aliquot. 19 μL of 50 ng / μL human RNA (950 ng) was mixed with 5 μL of 10 ng / μL E. coli RNA (50 ng) to generate a sample containing 5% E. coli RNA relative to human RNA. This mixture was then brought to 100 μL by adding 76 μL of chilled NFW.

[0097] The mixed RNA sample was then loaded onto the HPLC, with fraction collection performed by plating it onto position A1 of a new sterile PCR plate and holding it at 4°C. The method was initiated, and 50 μL (~500 ng) of sample was injected onto the column. Fractions from 0:30 to 12 min were collected onto a new sterile PCR plate at 15-second intervals (i.e., two fractions per 30-second collection). Each collected fraction was approximately 0.188 mL every 15 seconds. This time corresponds to the size range of 0 to 6000 nt based on the RiboRubler High Range Ladder run in this protocol. The chromatograms shown in Figures 5A-5B were obtained for that sample. As shown in the chromatographs of 15-second fractions in Figures 5A-5B, minor peaks eluted from the column within the void volume at retention times of approximately 1.3, 2.1, and 2.7 minutes, while the major peak eluted from the column after 8 minutes until approximately 12 minutes.

[0098] The peaks from 0:30 seconds to 6 minutes had low RNA absorbance. Without being bound by theory, these fractions within the void volume are likely composed of small RNA molecules, RNA fragments, RNA molecules with secondary and tertiary features, and mRNA. After the void volume (e.g., retention time 8 to 12 minutes), there are two large peaks, which, without being bound by theory, are likely composed of larger mRNA or rRNA transcripts.

[0099] The method was repeated with an additional 50 μL of sample (~500 ng) injected onto the column. The chromatogram in Figure 6 was obtained for this replicate. The chromatograms in Figures 5 and 6 are nearly identical, confirming the reproducibility of the method.

[0100] The collected fractions were then concentrated using ZymoClean and the Concentrate-5 kit before RNA quantification. Two of the 15-second collected fractions between 0:30 and 12 min (e.g., 0:30–0:45 and 0:45–1 min, 1–1:15 and 1:15–1:30, etc.) were combined into a sterile, nuclease-free 5 mL tube to provide a combined fraction of approximately 375 μL. 750 μL (2X fraction volume) of RNA binding buffer was added to each fraction and then shaken to mix thoroughly. 1.125 μL of 200-proof ethanol (1X fraction + buffer volume) was added to the mixture and shaken to mix thoroughly. The mixture was then filtered to 800 μL and spun at 10,000 × g for 30 seconds to allow binding. This was a three-step process. 400 μL of RNA Prep Buffer was added to the filter after binding the total RNA, followed by washing at 10,000 × g for 30 seconds. Then, 700 μL of RNA Wash Buffer was added to the filter after removing the previous flow-through, followed by washing at 10,000 × g for 30 seconds. After discarding the flow-through, 400 μL of RNA Wash Buffer was added to the filter, followed by washing at 10,000 × g for 1 minute. The column was then carefully transferred to a new, sterile, nuclease-free microcentrifuge tube. Once in the new tube, 15 μL of nuclease-free water from a similarly labeled tube was added to the filter, and the total RNA was eluted at 10,000 × g for 1 minute.

[0101] The fraction concentrations corresponding to the fractions obtained for gene sequencing are shown in Table 6.

[0102] Table 6. Fractionation table between HPLC and assay fractions TIFF0007725453000006.tif255160

[0103] The eluted RNA fractions were then quantified using the Quanti-T RNA HS assay. This was set up by adding 6766 μL of Quant-iT RNA buffer to a 15 mL conical tube, followed by 34 μL of Quant-iT RNA reagent, and vortexing for 10 seconds. A 3 μL aliquot of each assay fraction, shown in Table 6, was plated along with the standards in column 12 (3 μL RNA input). After 30 seconds of orbital shaking and a 2-minute incubation, the plate was read on a Tecan Infinite Pro 200 plate reader. The quantification results are shown in Table 7.

[0104] Table 7. RNA quantification results TIFF0007725453000007.tif151162

[0105] As shown in Table 7, the concentrations of the fractions ranged from 0.1 to 69.1 ng / µL. These concentrations were used as input for library preparation to prepare RNA for sequencing. Seven µL of purified RNA, along with 1 µL (15 pg) of synthetic ERCC internal control spike, was input into the NEBBEXT single-cell / low-input RNA library preparation protocol along with NEBNext multiplex oligos (96 unique dual-index primer pairs) for Illumina. This protocol, in summary, includes the following: (i) 7 μL of original 5% E. coli RNA was prepared in the subsequent comparison position; (ii) a no-template control (NTC) was added to a fixed position along with 3.5 μL of pre-run HPLC flow-through and 3.5 μL of NFW from the Zymo concentrator kit; (iii) fractions 18 and 19 each had 1 μL of RNA input, and because their concentrations were much higher than those of the other fractions, they were adjusted to 7 μL with NFW; and (iv) these two fractions, 18 and 19, coincided with a large peak in the chromatogram. RT primers were annealed to the RNA after adding 1 μL of primer to each RNA sample. Primed RNA was then reverse transcribed to cDNA using 15 cycles of cDNA amplification and 6 cycles of library PCR. The amplified cDNA was then purified twice with Ampure XP beads to increase purity. The purified cDNA was then quantified using the Quant-iT dsDNA 1X assay and had the concentrations shown in Table 8.

[0106] Table 8. Concentration of amplified cDNA TIFF0007725453000008.tif156126

[0107] After quantification, cDNA was normalized to 40 ng input for each sample (all volumes were added according to the manufacturer's protocol, except for fractions 1, 15, and 23, which were too concentrated for this input). The normalized cDNA was then fragmented and end-repaired. The end-repaired cDNA was ligated to Illumina adapters. The ligated cDNA was cleaned up with Ampure XP beads to remove unligated adapters. Once purified, the cDNA was subjected to six cycles of library PCR for cDNA inputs of 20 to 100 ng, followed by PCR enrichment. After PCR, the amplified libraries were purified with Ampure XP beads and then quantified using the Quant-iT dsDNA 1X assay at the normalized concentrations shown in Table 8.

[0108] Each sample was pooled, with 25 ng of each sample (except fraction 23, which had insufficient input and was added in its entirety). The pooled sample was further purified using 0.9x volume of AmpureXP beads to remove remaining impurities and concentrate the sample. The concentration of the pooled pure sample was 9.88 ng / uL (expected size 300 bp, ~50 nM). This purified sequencing library was diluted and denatured to the loading concentration according to the Illumina protocol and sequenced on a NextSeq550 with V2 high-output chemistry for 150 cycles using the indexes listed in Table 9.

[0109] Table 9. Sequence indexes TIFF0007725453000009.tif161145

[0110] Single-end FASTQ files were generated for each HPLC fraction and for the original sample before HPLC fractionation. All FASTQ files were then processed by performing quality filtering and sequence annotation in parallel. The filtered reads were annotated using a manually curated database. All database sequences were interrogated for common contaminating reads in the reference genome nucleic acid sequence and filtered for any contaminating reads using a platform that masks such regions to prevent systematic misannotation. Once complete, a data frame consisting of annotation counts for microbial species, Homo sapiens, and ERCC internal controls was collated as shown in Table 10.

[0111] Table 10. Sequence Read Counts TIFF0007725453000010.tif164170

[0112] The raw H. sapiens read counts for the original sample and fractions 1–23 are shown in the bar graph provided in Figure 7, which shows the relatively low H. sapiens read counts below fraction 11. The relative absence of H. sapiens read counts below fraction 11 enhances the presence of microbial RNA in these fractions, which correspond to void volume.

[0113] The raw internal standard ERCC counts for the original sample and fractions 1-23 are shown in the bar graph provided in Figure 8, which demonstrates that fractionation of the liquid chromatography eluent selects for specific RNAs. By fraction 14, the Homo sapiens fraction is relatively depleted.

[0114] The observed microbial and H. sapiens annotation counts were normalized by the observed ERCC sequence counts within each sample and then multiplied by a factor of 1,000,000. The calculated Enterobacteriaceae and H. sapiens ERCC-normalized counts were extracted and the Enterobacteriaceae to H. sapiens ratios were calculated, which are the normalized counts within the original sample and each of fractions 1-23, as shown in Figure 9. The bar graph in Figure 9 shows that the ERCC to H. sapiens ratios across each fraction are filtered for microbial content, so that the liquid chromatograph does not exclude samples based on microbial content.

[0115] The relative abundance of microbial reads (top of each bar) and Homo sapiens reads (bottom of each bar) in the original sample and fractions 1-23 is shown in the bar graph provided in Figure 10. The distribution of annotated reads demonstrates the relative amplification of microbial reads in the void volume fractions. Specifically, approximately 5% of the reads in fraction 2 and nearly 8% of the reads in fraction 3 are attributable to microbial reads (E. coli RNA), compared to approximately 0.03% of the microbial reads in the original sample. The void volume output of liquid chromatography effectively amplifies microbial RNA molecules from the test sample relative to host RNA molecules by approximately 20- to 10-fold in the void volume, compared to 1,000,000-fold in the original sample. The effective amplification of certain embodiments of the present method enables RNA detection in genetic material in the pictogram to nanogram range.

[0116] Embodiment The following embodiments are offered by way of example only and are not intended to limit the scope of the claimed invention as defined in the appended claims.

[0117] I. A method for isolating microbial RNA by filtering microbial RNA molecules from a biological sample, comprising: (a) obtaining a biological sample; (b) digesting the biological sample by interacting a reagent with the sample to generate a test sample; (c) bulk filtering the microbial RNA molecules from the mixture of RNA molecules in the test sample using liquid chromatography to separate and recover the microbial RNA molecules from the test sample.

[0118] II. The method of embodiment I, wherein the biological sample is a mammalian sample.

[0119] III. The method of embodiment I, wherein the biological sample comprises at least one of the following: large RNA molecules, small RNA molecules, tRNA molecules, rRNA molecules, mRNA molecules, denatured and non-denatured RNA molecules, microbial RNA molecules, non-microbial RNA molecules, genomic DNA molecules, protein molecules, and other macromolecules.

[0120] IV. The method of embodiment III, wherein digesting the biological sample removes genomic DNA molecules, protein molecules, and other macromolecules.

[0121] V. The method of embodiment I, wherein the microbial RNA molecule comprises fungal, viral, protozoan, amoeba, or bacterial RNA.

[0122] VI. The method of embodiment I, wherein obtaining the biological sample is performed in a clinical setting using at least one of sterile or aseptic techniques.

[0123] VII. The method of embodiment I, wherein the step of digesting the biological sample uses a lysis reagent as the reagent.

[0124] VIII. The method of embodiment I, wherein the step of using liquid chromatography comprises injecting the test sample into a sample port of the liquid chromatography instrument.

[0125] IX. The method of embodiment VIII, wherein the liquid chromatography device separates the microbial RNA molecules by decreasing and then increasing the amount of organic buffer in the mobile phase relative to the aqueous buffer in the mobile phase.

[0126] X. The method of embodiment IX, wherein the amount of organic buffer in the mobile phase is between 30% and 100%.

[0127] XI. The method of embodiment VIII, wherein the microbial RNA molecules are detected in a liquid chromatography device at wavelengths between 205 nm and 215 nm.

[0128] XII. The method of embodiment VIII, wherein the microbial RNA molecules are detected in a liquid chromatography device at wavelengths between 255 nm and 265 nm.

[0129] XIII. The method of embodiment VIII, wherein the microbial RNA molecules separated by the liquid chromatography device are collected by diverting the mobile phase from a waste line.

[0130] XIV. The method of embodiment VIII, wherein the flow rate of the liquid chromatography apparatus is between 1 mL / min and 2.5 mL / min.

[0131] XV. The method of embodiment IX, wherein the aqueous buffer comprises at least one of a 0.05M to 0.2M solution of triethylammonium acetate, phosphoric acid, citric acid, ammonium bicarbonate, formic acid, lactic acid, 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid, maleic acid, diethanolamine, piperidine, ethanolamine, and triethanolamine.

[0132] XVI. The method of embodiment IX, wherein the organic buffer comprises a mixture of 0.05M to 0.2M aqueous buffer in at least one of 10% to 40% acetonitrile, methanol, ethanol, 1-propanol, 2-propanol, acetone, and tetrahydrofuran.

[0133] XVII. The method of embodiment VII, wherein digesting the biological sample comprises the steps of: (a) lysing the biological sample by interacting the biological sample with at least one of guanidinium thiocyanate, N-lauroyl sarcosine, and ethanol; (b) washing the biological sample by interacting the biological sample with at least one of guanidinium chloride, ethanol, 2-amino-2-(hydroxymethyl)propane-1,3-dihydrochloride, and edetate disodium; (c) cleaning the biological sample by interacting it with at least one of proteinase K, guanidinium thiocyanate, N-lauroylsarcosine, ethanol, 2-amino-2-(hydroxymethyl)propane-1,3-dihydrochloride, and edetate disodium; (d) separating the test sample from the biological sample by interacting the biological sample with at least one of 55% to 85% guanidinium thiocyanate, 1% to 20% N-lauroyl sarcosine, 70% to 100% ethanol, DNase I, 2-amino-2-(hydroxymethyl)propane-1,3-dihydrochloride, and edetate disodium.

[0134] XVIII. The method of embodiment XVII, wherein the step of lysing the biological sample is completed by interacting the biological sample with at least one of 55% to 85% guanidinium thiocyanate, 1% to 20% N-lauroyl sarcosine, and 70% to 100% ethanol.

[0135] XIX. The method of embodiment XVII, wherein the step of washing the biological sample comprises washing the biological sample with a solution of 25% to 55% guanidinium chloride, 70% to 99% ethanol, 12 mg / m 3 ~790mg / m 3 of 2-amino-2-(hydroxymethyl)propane-1,3-dihydrochloride, and 20 mg / m 3 ~2,000mg / m 3 The reaction is completed by interacting with at least one of the following edetate disodium salts:

[0136] XX. The method of embodiment XVII, wherein the step of cleaning the biological sample is completed by interacting the clinical sample with at least one of 4 U to 12 U of proteinase K, 55% to 85% guanidinium thiocyanate, 1% to 20% N-lauroyl sarcosine, and 70% to 100% ethanol.

[0137] XXI. The method of embodiment XVII, wherein the step of separating the test sample from the biological sample comprises separating the biological sample in a solution of 1 U to 15 U of DNase I, 25% to 85% guanidinium thiocyanate, 1% to 20% N-lauroyl sarcosine, 70% to 100% ethanol, DNase I, 12 mg / m 3 ~790mg / m 3 of 2-amino-2-(hydroxymethyl)-propane-1,3-dihydrochloride and 20 mg / m 3 ~2,000mg / m 3 The method is completed by interacting with at least one of the following:

[0138] XXII. The method of embodiment XXI, wherein separating the test sample from the biological sample is completed by at least one of boiling the test sample, treating the test sample with a denaturing agent, or storing the test sample.

[0139] XXIII. The method of embodiment XXI, wherein the step of separating the test sample from the clinical sample is completed by boiling the test sample at 100°C to 120°C for 10 to 30 minutes.

[0140] XXIV. The method of embodiment XXII, wherein the step of treating the test sample with a denaturing agent is completed by interacting the test sample with 25% to 50% of at least one of polysorbate 20, polysorbate 80, (1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol, polyethylene glycol tert-octylphenyl ether, and 4-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol, t-octylphenoxypolyethoxyethanol, polyethylene glycol tert-octylphenyl ether.

[0141] XXV. The method of embodiment VIII, wherein the microbial RNA molecules separated by the liquid chromatography device are detected at an elution time between 0 and 3 minutes.

[0142] XXVI. A method for filtering microbial single-stranded nucleic acid sequences from a mixture of mammalian single-stranded nucleic acid sequences and / or microbial single-stranded nucleic acid sequences by recovering microbial RNA molecules from the void volume of a liquid chromatography apparatus, wherein the microbial single-stranded nucleic acid sequences are catalysts for protein synthesis.

[0143] XXVII. The method of embodiment XXVI, wherein the void volume is represented by an initial time period of the chromatogram.

[0144] XXVIII. The method of embodiment XXVII, wherein the initial time is from 0 minutes to 6 minutes.

[0145] XXIX. The method of embodiment XXVIII, wherein the microbial RNA molecules in the void volume are positively charged.

[0146] XXX. The method of embodiment XXVIII, wherein the microbial RNA molecules within the void volume carry a net zero charge.

[0147] XXXI. The method of embodiment XXVIII, wherein the microbial RNA molecules in the void volume have a size between 1 nucleotide and 280 nucleotides.

[0148] XXXII. A method for isolating microbial RNA by filtering microbial RNA molecules from a biological sample, comprising obtaining a biological sample; preparing the biological sample to generate a test sample by interacting a reagent with the sample; and bulk filtering the microbial RNA molecules from a mixture of RNA molecules in the test sample using a liquid chromatography device to separate and recover the microbial RNA molecules from the test sample in the void volume of the liquid chromatography device.

[0149] XXXIII. The method of embodiment XXXII, wherein the biological sample is a mammalian sample.

[0150] XXXIV. The method of embodiment XXXII, wherein the biological sample comprises at least one of the following: large RNA molecules, small RNA molecules, tRNA molecules, rRNA molecules, mRNA molecules, denatured and non-denatured RNA molecules, microbial RNA molecules, non-microbial RNA molecules, genomic DNA molecules, protein molecules, and other macromolecules.

[0151] XXXV. The method of embodiment XXXIII, wherein the step of preparing the biological sample purifies the sample and alters the relative identity of the genetic material.

[0152] XXXVI. The method of embodiment XXXV, wherein the step of preparing the biological sample does not substantially involve the intentional amplification of genetic material.

[0153] XXXVII. The method of embodiment XXXIV, wherein the step of preparing the biological sample purifies the sample to allow filtration by liquid chromatography.

[0154] XXXVIII. The method of embodiment XXXIV, wherein the biological sample preparation step purifies the sample to remove genomic DNA molecules, protein molecules, and other macromolecules.

[0155] XXXIX. The method of embodiment XXXII, wherein the step of preparing the biological sample does not intentionally amplify genetic material.

[0156] XL. The method of embodiment XXXII, wherein the microbial RNA molecule comprises fungal, viral, protozoan, amoeba, or bacterial RNA.

[0157] XLI. The method of embodiment XXXII, wherein obtaining the biological sample is performed in a clinical setting using at least one of aseptic or sterile techniques.

[0158] XLII. The method of embodiment XXXII, wherein the step of digesting the biological sample uses a lysis reagent as the reagent.

[0159] XLIII. The method of embodiment XXXII, wherein the step of using liquid chromatography comprises injecting the test sample into a sample port of the liquid chromatography instrument.

[0160] XLIV. The method of embodiment XLIII, wherein the liquid chromatography device separates the microbial RNA molecules by decreasing and then increasing the amount of organic buffer in the mobile phase relative to the aqueous buffer in the mobile phase.

[0161] XLV. The method of embodiment XLIV, wherein the amount of organic buffer in the mobile phase is between 30% and 100%.

[0162] XLVI. The method of embodiment XLIII, wherein the microbial RNA molecules are detected in a liquid chromatography device at wavelengths between 205 nm and 215 nm.

[0163] XLVII. The method of embodiment XLIII, wherein the microbial RNA molecules are detected in a liquid chromatography device at wavelengths between 255 nm and 265 nm.

[0164] XLVIII. The method of embodiment XXXII, wherein the microbial RNA molecules separated by the liquid chromatography device are collected in the void volume by diverting the mobile phase from a waste line.

[0165] For background and understanding of certain aspects of the present disclosure, reference is made to the following attachments, which are incorporated herein by reference: 1. Stewart, DB; Write, JR; Fowler, M.; McLimans, CJ; Tokarev, V.; Amaniera, I.; Baker, O.; Wong, H.; Brabec, J.; Drucker, R.; Lamendella, R. difficile infection”, Clinical Science and Epidemiology, 4(4), 1-16. 2. Goswami, K.; Purtill, JJ; Shope, AJ; Wright, J.; Lamendella, R. “Shotgun Metatranscriptomics for PJI diagnosis: ANovel Prospective Investigation, 2019 Abstract submission, AAHKS AnnualMeeting. 3. Goswami, K.; Purtill, JJ; Shope, AJ; Wright, J.; Lamendella, R. “Shotgun Metatranscriptomics for PJI diagnosis: ANovel Prospective Investigation, 2019 Slide deck presentation, AAHKS AnnualMeeting. 4. Slide deck for CSI Compendium, Confidential InvestorPresentation, unpublished work (2019).

[0166] Various embodiments of systems, devices, and methods have been described herein. These embodiments are provided by way of example only and are not intended to limit the scope of the invention as set forth in the claims. Furthermore, it should be understood that various features of the previously described embodiments can be combined in various ways to create numerous additional embodiments. Furthermore, while various materials, dimensions, shapes, configurations, arrangements, etc. have been described for use in the disclosed embodiments, other than those disclosed may be utilized without departing from the scope of the invention as set forth in the claims.

[0167] Those skilled in the relevant art will recognize that the inventive subject matter may include fewer features than those described in the individual embodiments above. The embodiments described herein are not intended to be an exhaustive list of ways in which various features of the inventive subject matter can be combined. Thus, the embodiments are not mutually exclusive combinations of features; rather, as one skilled in the art will understand, various embodiments may include combinations of different individual features selected from different individual embodiments. Furthermore, elements described with respect to one embodiment can be implemented in other embodiments even if not described in such embodiment, unless otherwise specified.

[0168] Although a dependent claim may refer to a specific combination with one or more other claims in the claims, other embodiments may also include combinations of the dependent claim with the subject matter of each other dependent claim, or combinations of one or more features with other dependent or independent claims. Such combinations are presented herein unless it is stated that a specific combination is not intended.

[0169] It should be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, a clinical sample containing "a microbial species" may contain a mixture of two or more microbial species. It should also be noted that the term "or" is generally employed in its sense to include "and / or" unless the context dictates otherwise.

[0170] The incorporation by reference of the above documents is limited so that no subject matter contrary to the express disclosure of this specification is incorporated. The incorporation by reference of the above documents is further limited so that no claims contained in the documents are incorporated herein by reference. The incorporation by reference of the above documents is further limited so that definitions provided within the documents are not incorporated herein by reference, unless expressly included herein.

[0171] In interpreting the claims, it is expressly intended that the provisions of 35 U.S.C. § 112(f) shall not apply unless the specific words "means for" or "step for" appear in the claims.

Claims

1. 1. A method for improving gene sequencing of microbial RNA molecules from a biological sample, comprising: (a) preparing a biological sample from an organism as a test sample for liquid chromatography; (b) bulk filtering the microbial RNA molecules from the mixture of RNA molecules in the test sample using liquid chromatography to separate and collect the microbial RNA molecules into two or more fraction outputs of the liquid chromatography, at least one of the two or more fraction outputs being a fraction output within the void volume of the liquid chromatography; (c) preparing one or more fraction outputs for genetic sequencing, the one or more fraction outputs being fraction outputs within a void volume; (d) performing genetic sequencing on the prepared one or more fraction outputs to detect microbial RNA from the biological sample; In step (b), the liquid chromatography has a non-polar stationary phase column comprising alkylated non-porous polystyrene-divinylbenzene copolymer particles; the non-polar stationary phase column is 4 mm to 30 mm wide by 40 mm to 150 mm long; Separating the microbial RNA molecules by liquid chromatography using a concentration gradient of an aqueous buffer relative to an organic buffer in the mobile phase at a flow rate of 0.550 mL / min to 2 mL / min; the aqueous buffer comprises a 0.05M to 0.2M solution of at least one selected from triethylammonium acetate, phosphoric acid, citric acid, ammonium bicarbonate, formic acid, lactic acid, 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid, maleic acid, diethanolamine, piperidine, ethanolamine, and triethanolamine; The organic buffer solution comprises 10% to 40% of at least one selected from acetonitrile, methanol, ethanol, 1-propanol, 2-propanol, acetone, and tetrahydrofuran mixed with 0.05M to 0.2M of an aqueous buffer solution.

2. 10. The method of claim 1, wherein the biological sample is a mammalian sample.

3. 10. The method of claim 1, wherein preparing the biological sample comprises digesting the biological sample by interacting a reagent with the biological sample to generate a test sample.

4. The method according to claim 3, wherein the step of digesting the biological sample comprises: (i) lysing the biological sample by interacting the biological sample with at least one of guanidinium thiocyanate, N-lauroyl sarcosine, and ethanol; (ii) washing the biological sample by interacting the biological sample with at least one of guanidinium chloride, ethanol, 2-amino-2-(hydroxymethyl)-propane-1,3-dihydrochloride, and edetate disodium; (iii) cleaning the biological sample by interacting the biological sample with at least one of proteinase K, guanidinium thiocyanate, N-lauroylsarcosine, ethanol, 2-amino-2-(hydroxymethyl)propane-1,3-dihydrochloride, and edetate disodium; (iv) separating the test sample from the biological sample by interacting the biological sample with at least one of 55% to 85% guanidinium thiocyanate, 1% to 20% N-lauroylsarcosine, 70% to 100% ethanol, DNase I, 2-amino-2-(hydroxymethyl)-propane-1,3-dihydrochloride, and edetate disodium.

5. A method according to claim 3, wherein genomic DNA molecules, protein molecules, and other macromolecules are removed by digesting a biological sample.

6. The method according to claim 4, The procedure of lysing the biological sample is completed by interacting the biological sample with at least one of 55% to 85% guanidinium thiocyanate, 1% to 20% N-lauroyl sarcosine, and 70% to 100% ethanol; The procedure for washing the biological sample comprises washing the biological sample with 25% to 55% guanidinium chloride, 70% to 99% ethanol, 12 mg / m ３ ~790 mg / m ３ of 2-amino-2-(hydroxymethyl)propane-1,3-dihydrochloride, and 20 mg / m ３ ~2,000mg / m ３ and completing the interaction with at least one of edetate disodium of the step of cleaning the biological sample is completed by interacting the biological sample with at least one of 4 U to 12 U proteinase K, 55% to 85% guanidinium thiocyanate, 1% to 20% N-lauroyl sarcosine, and 70% to 100% ethanol; The procedure for separating the test sample from the biological sample comprises: separating the biological sample into 1 U to 15 U of DNase I, 25% to 85% guanidinium thiocyanate, 1% to 20% N-lauroyl sarcosine, 70% to 100% ethanol, DNase I, 12 mg / m ３ ~790 mg / m ３ 2-amino-2-(hydroxymethyl)-propane-1,3-dihydrochloride, and 20 mg / m ３ ~2,000mg / m ３ and / or by interacting the compound with at least one of the following compounds:

7. 10. The method of claim 1, wherein the microbial RNA molecule comprises RNA from a fungus, a virus, a protozoan, an amoeba, or a bacterium.

8. 10. The method of claim 1, wherein each of the two or more fractions output from the liquid chromatography is collected between 10 seconds and 45 seconds and has a volume between 125 μL and 750 μL.

9. 10. The method of claim 1, comprising filtering microbial RNA molecules from a mixture of RNA molecules in a test sample using liquid chromatography without incubating the biological sample.

10. 10. The method of claim 1, wherein microbial RNA molecules separated in a liquid chromatography device are detected in a void volume having an elution time of less than 6 minutes or less than 4 minutes.

11. 10. The method of claim 1, wherein preparing one or more fraction outputs for gene sequencing comprises: (i) concentrating each of the one or more fraction outputs to provide one or more concentrated fraction outputs; (ii) priming RNA material in each of the one or more enriched fraction outputs to provide one or more primed RNA fraction outputs; (iii) reverse transcribing each of the one or more primed RNA fraction outputs into one or more cDNA fraction outputs.

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