WO2019116234A1

WO2019116234A1 - Methods for determining microbiome ribosomal rna composition changes

Info

Publication number: WO2019116234A1
Application number: PCT/IB2018/059889
Authority: WO
Inventors: Larry Mimms; Jordan STACHELSKI
Original assignee: Nestec S.A.
Priority date: 2017-12-12
Filing date: 2018-12-11
Publication date: 2019-06-20

Abstract

Provided herein are methods for analyzing ribosomal RNA populations in a biological sample. Methods for detecting human and microbiome ribosomal RNA changes associated with gastrointestinal-related or liver-related conditions are also provided.

Description

METHODS FOR DETERMINING MICROBIOME RIBOSOMAL RNA

COMPOSITION CHANGES

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 62/597,728, filed December 12, 2017, the disclosure of which is hereby incorporated by reference in its entirety for all purposes.

BACKGROUND OF THE INVENTION

[0002] The microbiome is the aggregate of microorganisms that reside within or on human tissues and bodily fluids, such as skin, lungs, oral mucosa, saliva, urine and gastrointestinal tract. It includes bacteria, archaea, protozoa, fungi and viruses. Some microbiota that colonize humans are commensal, others have a mutualistic relationship with their host, others are pathogenic or produce pathogenic metabolites. Gastrointestinal microbiota is a vast, complex community of microorganisms that live in the digestive tracts of humans and other animals, including insects. It represents approximately more than 1000 bacterial species, comprising 2 million genes, present in the largest quantity (-10-100 trillion organisms) as compared to other microbiota of the body (Quigley, Gastroenterology and Heptology , 9, 560- 569 (2013)). At birth, the intestinal tract is sterile and becomes colonized by maternal and environmental bacteria during birth and feeding. By the age of 2.5 years, the gut microbiota fully resembles the gut microbiota of an adult in terms of composition. Microbiome studies typically extract DNA from a sample obtained from a subject and quantify representatives of distinct populations present in the sample.

[0003] Hypervariable regions of individual highly conserved genes, such as the small ribosomal subunit in non-eukaryotes, has served as a proxy for species identification since the late l970’s (Woese and Fox, PNAS, 74(11):5088-5090 (1977)). Ribosomal RNA (rRNA) genes are both highly conserved and present in multiple copies, therefore making them suitable targets for gene amplification and molecular analysis (Sandhu et al. , J. Clin. Micro., 33:2913-19 (1995)). For instance, E. coll contains -10,000 copies of rRNA per cell. The 16S ribosomal RNA (16S rRNA) gene is present in all bacteria and archaea and was observed to have a slow rate of evolution (Id). Additionally, 16S rRNA genes from hundreds of thousands of organisms have been sequenced and classified (Cole et al, Nucleic Acids Res., 33:D294-296 (2005) and Cole et al., Nucleic Acids Res., 35:Dl69-72 (2007)). The 18S ribosomal RNA (18S rRNA) gene is found in all eukaryotes and fungi (Zhou et al, Mol. Cell. Probes, 14:339-348 (2000)).

[0004] Crohn’s Disease (CD) is a chronic inflammatory bowel disease (IBD) that may affect any part of the gastrointestinal tract from mouth to anus. The age of onset is generally between 15-30 years and it is equally prevalent in women and men. The highest prevalence is found in Europe and North America with just over 300 per 100,000 persons (Molodecky et al, Gastroenterology, 142(1):46-54 (2012)). CD generally leads to abdominal pain, severe diarrhea and weight disorders. Crohn’s Disease is of unknown etiology and multifactorial: environmental factors, host genetics and gut microbiome have all been shown to impact the risk of disease and its severity (Cho & Brant, Gastroenterology, 140(6): 1704-12 (2011)).

[0005] Clinical diagnosis of CD is supported by serologic, radiologic, endoscopic, and histologic findings. There are few standalone laboratory tests that allow the diagnosis of CD. Serum C-Reactive Protein (CRP), serum anti -Saccharomyces cerevisiae antibodies (ASCA), fecal calprotectin and lactoferrin are the most widely used biomarkers of CD, but these biomarkers are not specific to CD. Crohn’s Disease activity can be measured, for example, by the Crohn’s Disease Activity index (CDAI), a score resulting from the combination of multiple parameters or the Harvey-Bradshaw index (HBI), which consists of only clinical parameters (Bennebroek et al., J. Clin. Gastroenterol., 47(l0):850-6 (2014)).

[0006] Ulcerative Colitis (UC) is a chronic IBD that results in inflammation and ulcers of the colon and rectum. The age of onset is generally between 15-30 years and it is equally prevalent in women and men. The highest prevalence is found in Europe and North America with 5 to 500 people per 100,000 individuals being affected (Ford et al, BMJ., 346:432 (2013)). Ulcerative Colitis generally leads to abdominal pain and diarrhea, and may also include anemia and weight disorders. Ulcerative Colitis has unknown etiology, and while not directly affecting life expectancy, places those afflicted with increased risk for colon cancer. Similar to Crohn’s Disease, it is a condition that has active (flares) and inactive (remission) stages of disease.

[0007] The clinical diagnosis of Ulcerative Colitis is primarily supported by endoscopic findings ( e.g ., sigmoidoscopy). Disease activity can be measured for example using the Simple Clinical Colitis Activity Index (SCCAI) (Walmsley et al., Gut, 43(l):29-32(l998)), a score resulting from the combination of five clinical parameters used to assess the severity of symptoms.

[0008] IBS is collectively a group of symptoms including abdominal pain and change in bowel movements without any evidence of underlying damage. It is often classified based on the manifestation of diarrhea or constipation. IBS has unknown etiology but onset may be triggered by intestinal infections and food sensitivities (Grundmann and Yoon, Journal of Gastroenterology and Hepatology , 25:691-699 (2010)). Approximately 10% to 15% of adults in the U.S. are believed to be affected with IBS, while only half this number receives a corresponding diagnosis. It is more prevalent in South America and less common in Southeast Asia. IBS affects twice as many women as compared to men, and age of onset is often before 45.

[0009] Obesity, metabolic syndromes, liver diseases, nonalcoholic fatty liver (NAFL) disease and nonalcoholic steatohepatitis (NASH) have all been linked with mechanisms involving the microbiota {see, e.g., Ley et al., Proc Natl Acad Sci USA, 102(31): 11070-11075 (2005); Henao-Mejia et al., Nature , 482(7384): 179-185 (2012); Abu-Shanab and Quigley, Nat Rev Gastroenterology Hepatology, 7(l2):69l-70l (2010); and Rak and Rader, Nature, 472(734l):40-4l (2011)).

[0010] The importance of microbiota-host interactions in gastrointestinal-related and liver- related conditions is further supported by the many genetic studies that have identified a host of changes in genes that code for molecules involved in bacterial recognition, host-bacteria engagement, and the resultant inflammatory cascade (Van Limbergen et al, Gastroenterology, 141(5): 1566-1571 (2011). About 70% of the liver’s blood supply is supplied by the portal vein and can therefore be greatly affected by changes in gut microbiota due to entry of gut bacteria or their metabolites into the liver through the portal vein (Minemura and Shimizu, World J. Gastroenterol, 21(6)1691-1702 (2015)).

[0011] Moreover, for subjects diagnosed with gastrointestinal -related or liver-related conditions, monitoring clinical symptoms alone is not reliable enough to assess disease activity. For example, patients self-reporting low CD disease activity often present with intestinal lesions during an endoscopic exam. Biological markers, such as fecal calprotectin are useful, but are nonspecific and their increase is typically associated with mucosal inflammation at the late onset of a CD flare. Endoscopy allows for detection of mucosal healing, which is considered a robust and reliable sign of disease remission; however, repeated endoscopic monitoring is not feasible because of the required bowel preparation, general anesthesia, and risk of colon perforation. New imaging tools, such as MRI, have been shown in some cases to be effective, but are expensive, time-consuming, and its limited access preclude routine use.

[0012] Individuals with, or suspected of having, a gastrointestinal-related or liver-related condition, and their healthcare providers, would benefit from non-invasive tools enabling evaluation of disease activity ( e.g ., active versus inactive), monitoring patient care (e.g., predicting flare or remission), and adjustment of medical treatment (e.g., modulation of drug/probiotic regime). Accordingly, there remains a need in the art to identify reliable, specific and sensitive biomarkers of gastrointestinal-related or liver-related conditions that would allow the detection and diagnosis of gastrointestinal-related or liver-related conditions in a non-invasive and accurate manner. There is also a need for identifying biomarkers that can distinguish between subjects suffering from an active versus inactive (remission) disease state. This information could assist healthcare providers in diagnosing disease (e.g., CD, UC versus NASH), predicting the occurrence of ribosomal RNA changes in the subject based on the host rRNA or bacterial/fungal rRNA content, in order to choose from different treatment options (intensive or conventional), without having to perform an endoscopic or other invasive analyses.

[0013] The abundance of host rRNA in stool samples from subjects with gastrointestinal- related or liver-related conditions has not been assessed to date. We analyzed human (host) rRNA abundance in a number of stool samples from healthy controls (i.e., stool samples obtained from subjects not having or suspected of having a gastrointestinal -related or liver- related condition) and subjects with gastrointestinal-related or liver-related conditions. We observed that the abundance of human rRNA as compared to other populations of rRNA in the stool sample can act as a biomarker of CD and UC disease, and that patients suffering from active CD or UC disease versus remission states can be distinguished using these hiomarkers. Furthermore, we observed that ratios of human rRNA to other populations of rRNA can act as biomarkers of CD and UC disease and that patients suffering from active CD or UC disease versus remission states can be distinguished using these ratios. Accordingly, the presence of host rRNA, bacterial rRNA and/or fungal rRNA in the feces of gastrointestinal-related or liver-related patients can he used as a biomarker of gastrointestinal - related or liver-related conditions (e.g., CD and UC) and of its severity. BRIEF SUMMARY OF THE INVENTION

[0014] In one aspect, the present invention provides an in vitro method for analyzing a sample from a subject to determine the relative abundance of the subject’s 18S rRNA in the sample, the method comprising:

(a) determining the abundance of the subject’s 18S rRNA in the sample; and;

(b) comparing the abundance of the subject’s 18S rRNA in the sample to a reference value, thereby determining the relative abundance of the subject’s 18S rRNA in the sample. In one embodiment, the subject has or is suspected of having a gastrointestinal- related or liver-related condition. In some embodiments, the gastrointestinal-related or liver- related condition is selected from the group consisting of Crohn’s Disease, Ulcerative Colitis, irritable bowel syndrome, liver cancer, liver cirrhosis, autoimmune hepatitis, non-alcoholic steatohepatitis (NASH) or non-alcoholic fatty liver disease (NAFLD), graft versus host disorder (GvHD), thymoma-associated multiorgan autoimmunity (TAMA), and Celiac disease.

[0015] In some embodiments, the method further comprises determining the abundance of bacterial 16S rRNA in the sample, and comparing the abundance of the bacterial 16S rRNA in the sample to the reference value, thereby determining the relative abundance of the bacterial 16S rRNA in the sample.

[0016] In some embodiments, the method further comprises determining the abundance of fungal 18S rRNA in the sample, and comparing the abundance of the fungal 18S rRNA in the sample to the reference value, thereby determining the relative abundance of the fungal 18S rRNA in the sample.

[0017] In some embodiments, the method further comprises determining an amount of calprotectin in the subject’s sample.

[0018] In some embodiments, the abundance of the subject’s 18S rRNA in the sample is determined by hybridization protection assay (HP A).

[0019] In some embodiments, the sample comprises a stool lysate or ribonucleic acids isolated from a subject’s sample.

[0020] In some embodiments, the determining step is performed directly on the sample in the absence of a purification procedure. [0021] In some embodiments, the method does not include amplifying any portion of the rRNA present in the subject’s sample prior to the determining step.

[0022] In some embodiments, the method further comprises classifying the subject as having an active or inactive inflammatory bowel disease based on the relative abundance of the subject’s 18S rRNA in the sample.

[0023] In some embodiments, the method further comprises classifying the subject as having an active or inactive inflammatory bowel disease based on the relative abundance of the bacterial 16S rRNA in the sample.

[0024] In some embodiments, the subject is a human. In one embodiment, the subject has been diagnosed with Crohn’s Disease or Ulcerative Colitis. In another embodiment, the subject has active Crohn’s Disease or active Ulcerative Colitis. In some embodiments, the subject has inactive Crohn’s Disease or inactive Ulcerative Colitis. In another embodiment, the subject has non-alcoholic steatohepatitis, liver cancer, liver cirrhosis, autoimmune hepatitis or non-alcoholic fatty liver disease. In yet another embodiment, the subject has been diagnosed with a bacterial infection, viral infection, fungal infection or intestinal parasites. In another embodiment, the subject has been diagnosed with celiac disease, graft versus host disorder or thymoma-associated multiorgan autoimmunity.

[0025] In some embodiments, the reference value comprises a median or mean value for the abundance of host 18S rRNA, bacterial 16S rRNA or fungal 18S rRNA from a cohort of normal, healthy samples (i.e., samples not having or suspected of having a gastrointestinal- related or liver-related condition).

[0026] In some embodiments, the reference value comprises a median or mean value selected from the group consisting of the abundance of human 18S rRNA from a cohort of healthy, normal samples (i.e., a stool sample from a subject who is not diagnosed with, or experiencing symptoms related to a gastrointestinal-related or liver-related condition), abundance of bacterial 16S rRNA from a cohort of healthy, normal samples, abundance of fungal 18S rRNA from a cohort of healthy, normal samples, abundance of human 18S rRNA from a cohort of samples from subjects diagnosed with a gastrointestinal-related or liver- related condition, abundance of bacterial 16S rRNA from a cohort of samples from subjects diagnosed with a gastrointestinal-related or liver-related condition, and abundance of fungal 18S rRNA from a cohort of samples from subjects diagnosed with a gastrointestinal -related or liver-related condition. In one embodiment, the reference value can comprise the abundance of human 18S rRNA from a cohort of samples from subjects diagnosed with Crohn’s Disease, abundance of bacterial 16S rRNA from a cohort of samples from subjects diagnosed with Crohn’s Disease, and abundance of fungal 18S rRNA from a cohort of samples from subjects diagnosed with Crohn’s Disease. In another embodiment, the reference value can comprise the abundance of human 18S rRNA from a cohort of samples from subjects diagnosed with UC, abundance of bacterial 16S rRNA from a cohort of samples from subjects diagnosed with UC, and abundance of fungal 18S rRNA from a cohort of samples from subjects diagnosed with UC.

[0027] In one aspect, the present invention provides methods for detecting human and microbiome ribosomal RNA changes associated with a gastrointestinal-related or liver- related condition, the method comprising:

(a) performing a first assay to determine the abundance of a human subject’s 18S rRNA in a sample from the subject to generate a first dataset, wherein the first dataset is optionally compared to a reference value;

(b) performing a second assay to determine the abundance of bacterial 16S rRNA in the sample from the subject to generate a second dataset, wherein the second dataset is optionally compared to the reference value; and

(c) optionally, performing a third assay to determine the abundance of fungal 18S rRNA in the sample from the subject to generate a third dataset, wherein the third dataset is optionally compared to the reference value.

[0028] In some embodiments, the reference value comprises a median or mean value selected from the group consisting of the abundance of human 18S rRNA from a cohort of healthy, normal samples, abundance of bacterial 16S rRNA from a cohort of healthy, normal samples, abundance of fungal 18S rRNA from a cohort of healthy, normal samples, abundance of human 18S rRNA from a cohort of samples from subjects diagnosed with a gastrointestinal -related or liver-related condition, abundance of bacterial 16S rRNA from a cohort of samples from subjects diagnosed with a gastrointestinal-related or liver-related condition, and abundance of fungal 18S rRNA from a cohort of samples from subjects diagnosed with a gastrointestinal-related or liver-related condition. In one embodiment, the reference value can comprise the abundance of human 18S rRNA from a cohort of samples from subjects diagnosed with Crohn’s Disease, abundance of bacterial 16S rRNA from a cohort of samples from subjects diagnosed with Crohn’s Disease, and abundance of fungal 18S rRNA from a cohort of samples from subjects diagnosed with Crohn’s Disease. In another embodiment, the reference value can comprise the abundance of human 18S rRNA from a cohort of samples from subjects diagnosed with UC, abundance of bacterial 16S rRNA from a cohort of samples from subjects diagnosed with UC, and abundance of fungal 18S rRNA from a cohort of samples from subjects diagnosed with UC. [0029] In some embodiments, the reference value comprises a median or mean value for the abundance of host 18S rRNA, bacterial 16S rRNA or fungal 18S rRNA from a cohort of normal, healthy samples (i.e., samples not suspected of, or having, a gastrointestinal-related or liver-related condition).

[0030] In some embodiments, the first and second datasets are applied to calculate a human 18S rRNA:bacterial 16S rRNA ratio.

[0031] In some embodiments, the first and third datasets are applied to calculate a human 18S rRNA:fungal 18S rRNA ratio.

[0032] In some embodiments, the human 18S rRNA:bacterial 16S rRNA ratio and/or the human 18S rRNA:fungal 18S rRNA ratio is indicative of the subject having or developing a gastrointestinal -related or liver-related condition.

[0033] In some embodiments, the gastrointestinal-related or liver-related condition is selected from the group consisting of Crohn’s Disease (CD), Ulcerative Colitis (UC), liver cancer, liver cirrhosis, autoimmune hepatitis, non-alcoholic steatohepatitis (NASH), non alcoholic fatty liver disease (NAFLD), Celiac disease and graft versus host disorder (GvHD). [0034] In some embodiments, the sample is a stool sample. In another embodiment, the sample is a peripheral (whole) blood sample, serum sample, tissue biopsy, tissue sample, intestinal mucosal sample ( e.g intestinal mucosal cells) or liver sample.

[0035] In some embodiments, the method distinguishes between active or inactive inflammatory bowel disease based on the abundance of the human 18S rRNA in the sample. [0036] In some embodiments, the method distinguishes between active or inactive inflammatory bowel disease based on the abundance of the bacterial 16S rRNA in the sample.

[0037] In some embodiments, the method distinguishes between active or inactive inflammatory bowel disease based on the abundance of the fungal 18S rRNA in the sample. [0038] In some embodiments, the method identifies active inflammatory bowel disease by detecting an increase in the abundance of human 18S rRNA in the subject’s sample as compared to the abundance of human 18S rRNA in a normal, healthy sample (/.<?., a sample not suspected of, or having, a gastrointestinal-related or liver-related condition) tested under the same conditions.

[0039] In some embodiments, the method distinguishes between an active or inactive inflammatory bowel disease based on the human 18S rRNA:bacterial 16S rRNA ratio.

[0040] In some embodiments, the method distinguishes between an active inflammatory bowel disease and normal, healthy sample based on the human 18S rRNA:bacterial 16S rRNA ratio.

[0041] In some embodiments, the first, second, and third assays each comprise a hybridization protection assay (HP A).

[0042] In some embodiments, the first and second assays comprise a hybridization protection assay (HP A) and the third assay comprises a qPCR assay.

[0043] In some embodiments, the first, second or third assay comprises amplification of at least a portion of rRNA in the sample prior to performing the first, second or third assay.

[0044] In another embodiment, the first, second and third assay each comprise amplifying at least a portion of rRNA in the sample prior to performing the first, second and third assay.

[0045] In some embodiments, the third assay comprises amplifying at least a portion of the fungal 18S rRNA prior to performing the third assay.

[0046] In some embodiments, the first assay comprises:

(a) contacting the subject’s sample or ribonucleic acids isolated from the subject’s sample with a first acridinium ester labeled-DNA probe;

(b) hybridizing the first acridinium ester labeled-DNA probe to a target region present in the subject’s sample or ribonucleic acids isolated from the subject’s sample;

(c) separating the hybridized first acridinium ester labeled-DNA probe from unhybridized acridinium ester labeled-DNA probe; and

(d) detecting a chemiluminescent signal from the hybridized first acridinium ester labeled-DNA probe under chemiluminescent conditions.

[0047] In some embodiments, the second assay comprises: (a) contacting the subject’s sample or ribonucleic acids isolated from the subject’s sample with a second acridinium ester labeled-DNA probe;

(b) hybridizing the second acridinium ester labeled-DNA probe to a target region present in the subject’s sample or ribonucleic acids isolated from the subject’s sample;

(c) separating the hybridized second acridinium ester labeled-DNA probe from unhybridized acridinium ester labeled-DNA probe; and

(d) detecting a chemiluminescent signal from the hybridized second acridinium ester labeled-DNA probe under chemiluminescent conditions.

[0048] In some embodiments, the third assay comprises:

(a) contacting the subject’s sample or ribonucleic acids isolated from the subject’s sample with a third acridinium ester labeled-DNA probe;

(b) hybridizing the third acridinium ester labeled-DNA probe to a target region present in the subject’s sample or ribonucleic acids isolated from the subject’s sample;

(c) separating the hybridized third acridinium ester labeled-DNA probe from unhybridized acridinium ester labeled-DNA probe; and

(d) detecting a chemiluminescent signal from the hybridized third acridinium ester labeled-DNA probe under chemiluminescent conditions.

[0049] In some embodiments, the first, second and third assays are performed sequentially, simultaneously, or independently on the subject’s sample. [0050] In some embodiments, the first acridinium ester labeled-DNA probe comprises a human 18S rRNA probe.

[0051] In some embodiments, the second acridinium ester labeled-DNA probe comprises a pan bacterial 16S rRNA probe.

[0052] In some embodiments, the third acridinium ester labeled-DNA probe comprises a pan fungal 18S rRNA probe.

[0053] In some embodiments, the first acridinium ester labeled-DNA probe comprises the sequence set forth in SEQ ID NO: 1.

[0054] In some embodiments, the second acridinium ester labeled-DNA probe comprises the sequence set forth in SEQ ID NO:2. [0055] In some embodiments, the third acridinium ester labeled-DNA probe comprises the sequence set forth in SEQ ID NO:3.

[0056] In some embodiments, the separating comprises degrading the unhybridized acridinium ester-labeled DNA probe. In one embodiment, the separating comprises hydrolysis of the unhybridized acridinium ester-labeled DNA probe.

[0057] In some embodiments, the method detects or predicts inflammatory bowel disease based on the abundance of human 18S rRNA or the abundance of bacterial 16S rRNA in the subject’s sample.

[0058] In another aspect, the present invention provides methods for testing the efficacy of a clinical treatment in a human subject having a gastrointestinal -related or liver-related condition ( e.g ., active inflammatory bowel disease), the method comprising:

(a) measuring the abundance of human 18S rRNA, bacterial 16S rRNA and optionally, fungal 18S rRNA in a pre-clinical treatment sample and a post-clinical treatment sample from the human subject;

(b) calculating a human 18S rRNA:bacterial 16S rRNA ratio and/or a human 18S rRNA:fungal 18S rRNA ratio based on the abundance of human 18S rRNA, bacterial 16S rRNA and fungal 18S rRNA present in the pre-clinical treatment and post-clinical treatment samples; and

(c) determining if the clinical treatment provides efficacy for the gastrointestinal-related or liver-related condition (e.g., active inflammatory bowel disease) in the human subject based on the human 18S rRNA:bacterial 16S rRNA ratio and/or human 18S rRNA:fungal 18S rRNA ratio.

[0059] In some embodiments, the clinical treatment provides efficacy for a gastrointestinal- related or liver-related condition (e.g., active inflammatory bowel disease) in the human subject if the post-clinical treatment human 18S rRNA:bacterial 16S rRNA ratio is lower than the pre-clinical treatment human 18S rRNA:bacterial 16S rRNA ratio.

[0060] In some embodiments, the clinical treatment is an antibiotic, aminosalicylate, corticosteroid, immunosuppressant and/or monoclonal antibody-based therapy.

[0061] In another aspect, the present invention provides methods for predicting or identifying a gastrointestinal-related or liver-related condition in a human subject, the method comprising: (a) performing a first hybridization protection assay to detect human 18S rRNA in a sample from the human subject or ribonucleic acids isolated from the human subject’s sample;

(b) calculating the relative abundance of human 18S rRNA in the subject’s sample based on the assay of step (a);

(c) performing a second hybridization protection assay to detect bacterial 16S rRNA in the subject’s sample or ribonucleic acids isolated from the subject’s sample;

(d) calculating the relative abundance of bacterial 16S rRNA in the subject’s sample based on the assay of step (c);

(e) determining a ratio of human 18S rRNA:bacterial 16S rRNA based on the values obtained in steps (b) and (d); and

(f) identifying the subject as having a gastrointestinal-related or liver-related condition or predicting the subject to develop a gastrointestinal -related or liver-related condition based on the ratio of step (e). [0062] In some embodiments, the method further comprises performing a third hybridization protection assay to detect fungal 18S rRNA in the subject’s sample or ribonucleic acids isolated from the subject’s sample; calculating the relative abundance of fungal 18S rRNA in the subject’s sample; determining a ratio of human 18S rRNA:fungal 18S rRNA based on the relative abundance values obtained; and identifying the subject as having a gastrointestinal-related condition or liver-related or predicting the subject to develop a gastrointestinal-related or liver-related condition based on the ratio obtained.

[0063] In some embodiments, the first, second and third hybridization protection assays are performed sequentially, simultaneously, or independently on the subject’s sample.

[0064] In some embodiments, the first, second and third hybridization protection assays comprise acridinium ester labeled-DNA probes.

[0065] In some embodiments, the first hybridization protection assay comprises an acridinium ester labeled-DNA probe capable of hybridizing to human 18S rRNA.

[0066] In some embodiments, the second hybridization protection assay comprises an acridinium ester labeled-DNA probe capable of hybridizing to bacterial 16S rRNA. [0067] In some embodiments, the third hybridization protection assay comprises an acridinium ester labeled-DNA probe capable of hybridizing to fungal 18S rRNA or an amplified product derived from fungal 18S rRNA.

[0068] In some embodiments, each of the first, second and third hybridization protection assays comprises a different acridinium ester-labeled DNA probe capable of hybridizing to one of a fungal 18S rRNA, bacterial 16S rRNA or human 18S rRNA.

[0069] In some embodiments, the acridinium ester labeled-DNA probe comprises the sequence set forth in SEQ ID NO: 1.

[0070] In some embodiments, the acridinium ester labeled-DNA probe comprises the sequence set forth in SEQ ID NO:2.

[0071] In some embodiments, the acridinium ester labeled-DNA probe comprises the sequence set forth in SEQ ID NO:3.

[0072] In some embodiments, the gastrointestinal-related or liver-related condition is identified or predicted when the human 18S rRNA:bacterial 16S rRNA ratio in the subject’s sample is equal to, or greater than, two standard deviations above a human 18S rRNA:bacterial 16S rRNA mean ratio obtained from a population of normal, healthy samples (/.<?., samples obtained from subjects not suspected of, or having, a gastrointestinal-related or liver-related condition) under the same conditions.

[0073] In some embodiments, the gastrointestinal-related or liver-related condition is identified or predicted when the human 18S rRNA:bacterial 16S rRNA ratio in the subject’s stool sample is about 3.5-fold greater than a human 18S rRNA:bacterial 16S rRNA mean ratio obtained from a population of normal, healthy samples (/.<?., samples obtained from subjects not suspected of, or having, a gastrointestinal -related or liver-related condition) under the same conditions.

[0074] In another aspect, the present invention provides a kit comprising:

(a) a first oligonucleotide having a nucleic acid sequence that is complementary to a region of human 18S rRNA;

(b) a second oligonucleotide having a nucleic acid sequence that is complementary to a region of bacterial 16S rRNA; and optionally

(c) a third oligonucleotide having a nucleic acid sequence that is complementary to a region of fungal 18S rRNA. [0075] In one embodiment, the first, second and/or third oligonucleotide each comprise an acridinium ester.

[0076] In another embodiment, the kit comprises:

(a) an oligonucleotide comprising SEQ ID NO: l;

(b) an oligonucleotide comprising SEQ ID NO:2; and optionally,

(c) an oligonucleotide comprising SEQ ID NO:3.

[0077] In one embodiment, the kit further comprises a cell lysis buffer and/or a rRNA purification reagent.

BRIEF DESCRIPTION OF THE DRAWINGS

[0078] FIG. l is a schematic illustrating the specificity of the hybridization protection assay (HP A). Depiction of HPA in which AE labeled probe (DNA or RNA or Hybridizing molecule) binding to complementary target rRNA or DNA is protected against hydrolysis. Unhybridized probe or if a mismatch occurs near the site of AE labeling, the AE in the mismatched hybridized probe will not be protected from hydrolysis.

[0079] FIGS. 2A-2C show standard curves obtained for 5 separate runs. Standards were generated by spiking known concentrations of rRNA into fecal lysate. FIG. 2A is a standard curve for detection of bacterial 16S rRNA. FIG. 2B is a standard curve for detection of human 18S rRNA. FIG. 2C is a standard curve for detection of fungal 18S rRNA using exemplary methods of the instant application.

[0080] FIGS. 3A-3C show abundance of bacterial 16S rRNA from 100 human stool samples as detected using HPA. 100 human stool samples (normal and diseased samples sets) in sets of twenty samples, measured by relative lights units (RLUs) and as a function of the log of rRNA molecules detected using HPA. Samples were quantified by comparison of RLU to known standard curve shown in FIG. 2A. FIG. 3A illustrates the abundance of bacterial 16S rRNA in normal healthy individuals (stool samples obtained from subjects not having nor suspected of having a gastrointestinal-related or liver-related condition) and diseased sample sets (Crohn’s Disease and Ulcerative Colitis) (CD, p=0.0044 and UC, p= <0.0001). FIG. 3B illustrates the abundance of bacterial 16S rRNA in normal (stool samples obtained from subjects not having nor suspected of having a gastrointestinal -related or liver- related condition) and active or inactive Crohn’s Disease stool samples. FIG. 3C illustrates the abundance of bacterial 16S rRNA in normal (stool samples obtained from subjects not having nor suspected of having a gastrointestinal-related or liver-related condition) and active or inactive Ulcerative Colitis stool samples.

[0081] FIGS. 4A-4C show abundance of human 18S rRNA from 100 human stool samples (fecal lysates) as detected using HP A. FIG. 4 A illustrates the abundance of human 18S rRNA in normal (stool samples obtained from subjects not having nor suspected of having a gastrointestinal -related or liver-related condition) and diseased sample sets (Crohn’s Disease and Ulcerative Colitis). FIG. 4B illustrates the abundance of human 18S rRNA in normal and active or inactive Crohn’s Disease stool samples as fecal lysates (stool samples obtained from subjects not having nor suspected of having a gastrointestinal-related or liver-related condition). FIG. 4C illustrates the abundance of human 18S rRNA in normal and active or inactive Ulcerative Colitis stool samples as fecal lysates (stool samples obtained from subjects not having nor suspected of having a gastrointestinal-related or liver-related condition).

[0082] FIGS. 5A-5C are plots illustrating the ratio of human 18S rRNA to bacterial 16S rRNA from 100 human stool samples as detected using HP A. FIG. 5 A illustrates the ratio of human 18S rRNA to bacterial 16S rRNA in normal (stool samples obtained from subjects not having nor suspected of having a gastrointestinal-related or liver-related condition) and diseased sample sets (Crohn’s Disease and Ulcerative Colitis). FIG. 5B illustrates the ratio of human 18S rRNA to bacterial 16S rRNA in normal (stool samples obtained from subjects not having nor suspected of having a gastrointestinal-related or liver-related condition) and active or inactive Crohn’s Disease stool samples. FIG. 5C illustrates the ratio of human 18S rRNA to bacterial 16S rRNA in normal (stool samples obtained from subjects not having nor suspected of having a gastrointestinal -related or liver-related condition) and active or inactive Ulcerative Colitis stool samples.

[0083] FIGS. 6A and 6B are comparisons of bacterial rRNA detection from direct fecal lysate samples or fecal lysate samples having undergone a subsequent nucleic acid extraction or purification procedure prior to HPA. FIG. 6A is a plot showing the level of detection of bacterial 16S rRNA from fecal lysate samples having undergone a nucleic acid extraction step. FIG. 6B is a plot showing the level of detection of bacterial 16S rRNA from direct fecal lysate samples.

[0084] FIGS. 7A-7C show abundance of bacterial 16S rRNA from 100 human stool samples as detected using RT-qPCR. FIG. 7A illustrates the abundance of bacterial 16S rRNA in normal (stool samples obtained from subjects not having nor suspected of having a gastrointestinal -related or liver-related condition) and diseased sample sets (Crohn’s Disease and Ulcerative Colitis). FIG. 7B illustrates the abundance of bacterial 16S rRNA in normal (stool samples obtained from subjects not having nor suspected of having a gastrointestinal- related or liver-related condition) and active or inactive Crohn’s Disease stool samples. FIG. 7C illustrates the abundance of bacterial 16S rRNA in normal (stool samples obtained from subjects not having nor suspected of having a gastrointestinal-related or liver-related condition) and active or inactive Ulcerative Colitis stool samples.

[0085] FIGS. 8A-8C show abundance of human 18S rRNA from 100 human stool samples as detected using RT-qPCR. FIG. 8 A illustrates the abundance of human 18S rRNA in normal (stool samples obtained from subjects not having nor suspected of having a gastrointestinal -related or liver-related condition) and diseased sample sets (Crohn’s Disease and Ulcerative Colitis). FIG. 8B illustrates the abundance of human 18S rRNA in normal (stool samples obtained from subjects not having nor suspected of having a gastrointestinal- related or liver-related condition) and active or inactive Crohn’s Disease stool samples. FIG. 8C illustrates the abundance of human 18S rRNA in normal (stool samples obtained from subjects not having nor suspected of having a gastrointestinal-related or liver-related condition) and active or inactive Ulcerative Colitis stool samples.

[0086] FIGS. 9A-9C show abundance of fungal 18S rRNA from 100 human stool samples as detected using RT-qPCR. FIG. 9 A illustrates the abundance of fungal 18S rRNA in normal (stool samples obtained from subjects not having nor suspected of having a gastrointestinal -related or liver-related condition) and diseased sample sets (Crohn’s Disease and Ulcerative Colitis). FIG. 9B illustrates the abundance of fungal 18S rRNA in normal (stool samples obtained from subjects not having nor suspected of having, a gastrointestinal- related or liver-related condition) and active or inactive Crohn’s Disease stool samples. FIG. 9C illustrates the abundance of fungal 18S rRNA in normal (stool samples obtained from subjects not having nor suspected of having a gastrointestinal-related or liver-related condition) and active or inactive Ulcerative Colitis stool samples.

[0087] FIGS. 10A-10C are plots illustrating the ratio of human 18S rRNA to bacterial 16S rRNA from 100 human stool samples as detected using RT-qPCR. FIG. 10A illustrates the ratio of human 18S rRNA to bacterial 16S rRNA in normal (stool samples obtained from subjects not having nor suspected of having a gastrointestinal-related or liver-related condition) and diseased sample sets (Crohn’s Disease and Ulcerative Colitis). FIG. 10B illustrates the ratio of human 18S rRNA to bacterial 16S rRNA in normal (stool samples obtained from subjects not having nor suspected of having a gastrointestinal -related or liver- related condition) and active or inactive Crohn’s Disease stool samples. FIG. 10C illustrates the ratio of human 18S rRNA to bacterial 16S rRNA in normal (stool samples obtained from subjects not having nor suspected of having a gastrointestinal-related or liver-related condition) and active or inactive Ulcerative Colitis stool samples.

[0088] FIG. 11 is a plot illustrating the quantification of calprotectin (mg/kg) from 100 human stool samples as detected using RT-qPCR. FIG. 11 illustrates the amount of calprotectin identified in normal (stool samples obtained from subjects not having nor suspected of having a gastrointestinal-related or liver-related condition) and diseased sample sets (Crohn’s Disease and Ulcerative Colitis).

DETAILED DESCRIPTION OF THE INVENTION

I. INTRODUCTION

[0089] Gastrointestinal microbiota is a vast, complex community of microorganisms that live in the digestive tracts of humans and other animals. It represents >1000 bacteria species, comprising 2 million genes, and is present in large quantities (-10-100 trillion organisms). Ribosomal RNA (rRNA) genes are both highly conserved and present in multiple copies in cells, therefore making them suitable targets for gene amplification and molecular analysis (Sandhu et al. , . Clin. Micro., 33, 2913-19 (1995)). The 16S ribosomal RNA (16S rRNA) gene is present in bacteria and archaea and is observed to have a slow rate of evolution, while the 18S ribosomal RNA (18S rRNA) gene is found in all eukaryotes and fungi (Zhou et al, Mol. Cell. Probes, 14, 339-348 (2000).

[0090] The importance of microbiota-host interactions in gastrointestinal-related conditions is supported by many genetic studies that have identified a host of changes in genes that code for molecules involved in bacterial recognition, host-bacteria engagement, and the resultant inflammatory cascade (Van Limbergen et al, Gastroenterology, 141 (5): 1566- 1571 (2011)). About 70% of the liver’s blood supply is supplied by the portal vein and can therefore be greatly affected by changes in gut microbiota due to entry of gut bacteria or their metabolites into the liver through the portal vein (Minemura and Shimizu, World J. Gastroenterol, 21(6)1691-1702 (2015). [0091] The present invention provides methods for analyzing samples from subjects having or suspected of having a gastrointestinal-related or liver-related condition to determine the relative abundance of ribosomal RNA populations in the sample, which can be used as an indicator of disease and/or disease severity. Non-invasive samples, such as stool samples are particularly useful in the methods described herein. Other samples, such as whole blood, serum, peripheral blood, intestinal biopsy samples, intestinal mucosal samples, and liver tissue samples are particularly useful in the methods described herein. In some embodiments, the methods are homogeneous assays meaning that the measurement of rRNA in the sample does not require a physical separation process, such as a purification or amplification step prior to analysis, for example, by nucleic acid hybridization assays. In some embodiments, the method described herein is a hybridization protection assay (HPA) utilizing chemiluminescent molecules, such as acridinium esters and derivatives thereof. FIG. 1 illustrates an exemplary HPA of the present invention in which DNA probes complementary to the target RNA sequence of interest are bound, and unbound or mismatched DNA/RNA hybrids are hydrolyzed by basic conditions.

[0092] As explained in greater detail herein, target ribosomal RNA sequences can be detected, quantified, and manipulated such that specific ratios for populations of rRNA can be determined and used as an indicator of disease and/or disease severity.

II. DEFINITIONS

[0093] It is to be understood that terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. As used in this specification and the appended claims, the singular forms“a,”“an” and“the” include pluralities unless the context clearly dictates otherwise. Thus, for example, reference to“an oligonucleotide” includes one or more oligonucleotides, and reference to“a stool sample” includes one or more stool samples.

[0094] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although other methods and materials similar, or equivalent, to those described herein can be useful in the present invention, preferred materials and methods are described herein. [0095] In view of the teachings of the present specification, one of ordinary skill in the art can employ conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant polynucleotides, as taught, for example, by the following standard texts: PCR 2: A Practical Approach, M. J. McPherson, et al., IRL Press, ISBN 978-0199634248 (1995); Methods in Molecular Biology (Series), J. M. Walker, ISSN 1064-3745, Humana Press; RNA: A Laboratory Manual, D. C. Rio, et al. , Cold Spring Harbor Laboratory Press, ISBN 978-0879698911 (2010); Methods in Enzymology (Series), Academic Press; Molecular Cloning: A Laboratory Manual (Fourth Edition), M. R. Green, et al. , Cold Spring Harbor Laboratory Press, ISBN 978-1605500560 (2012); and Bioconjugate Techniques, Third Edition, G. T. Hermanson, Academic Press, ISBN 978-0123822390 (2013).

[0096] As used herein, the following terms have the meanings ascribed to them unless specified otherwise.

[0097] The terms“microbiota,”“microflora,” and“microbiome” refer to the community of living microorganisms that typically inhabit a bodily organ or part of the body ( e.g ., skin). Members of the gastrointestinal (GI) microbiota include, but are not limited to, microorganisms of the phyla of Firmicutes, Bacteroidetes, Proteobacteria and Actinobacteria ; microorganisms of the Bacteroides, Prevotella or Ruminococcus genera; microorganisms of the Bifidobacteria , Enterobacteraceae , Lactobacillus , Veillonella , Bacteoides , Streptococcus, Actinomycinaea, Helicobacter, Peptostreptococcus , Collin sella, Clostridium , Enterococcus , Coprococcus , Coprobacillus, Proteobacteria, Lactobacillus, Ruminococus, Faecalibcaterium, Eubacterium, Dorea, Acinetobacter, and Escherichia coll species; microorganisms of the Ruminococcus torques , R. torques- like, Collinsella aerofaciens-\ ke, Clostridium cocleatum, Eubacterium rectale, Clostridium coccoides , Rhinobatos productus types. In some instances, the gastrointestinal microbiota includes mucosa-associated microbiota, which is located at the surface or apical end of the gastrointestinal tract, and luminal-associated microbiota, which is found in the lumen of the gastrointestinal tract.

[0098] The terms“irritable bowel syndrome” and“IBS” include a group of bowel disorders characterized by one or more symptoms including, but not limited to, abdominal pain, abdominal discomfort, change in bowel pattern, loose or more frequent bowel movements, diarrhea, and constipation, without any evidence of underlying damage (e.g., ulcers in the GI tract). There are at least four forms of IBS, depending on which symptom predominates: (1) diarrhea-predominant (IBS-D); (2) constipation-predominant (IBS-C); (3) IBS with a mixture of diarrhea and constipation (IBS-M); and (4) IBS without frequent constipation or diarrhea (IBS-U). There are also various clinical subtypes of IBS, such as post-infectious IBS (IBS- PI).

[0099] The terms“inflammatory bowel disease” and“IBD” include a group of immune- mediated chronic gastrointestinal conditions characterized by one or more symptoms including, but not limited to, inflammation in the gastrointestinal tract, abdominal pain, abdominal discomfort, change in bowel pattern, loose or more frequent bowel movements, diarrhea, bloody stool, and constipation. Most commonly, IBD includes but is not limited to, Crohn’s Disease and Ulcerative Colitis.

[0100] The term“active” as used herein refers to a measure of disease activity. In one embodiment, active refers to a physiological state in which the subject is experiencing, or the GI tract is reflecting, acute or sporadic flare-ups associated with a gastrointestinal-related or liver-related condition. For example, a subject may experience symptoms such as diarrhea, abdominal pain and constipation. The symptoms will generally mirror those associated with the gastrointestinal-related or liver-related condition. For example, Ulcerative Colitis can present as the development of ulcers in the colon or rectum, or when the subject experiences diarrhea, abdominal pain or constipation. Determining whether a subject is manifesting an active disease state can be performed by any methods known in the art, such as patient self- reporting, endoscopy, or through application of one or more of the methods disclosed herein.

[0101] The term“inactive,”“quiescent,” or“remission” as used herein refers to a measure of disease activity. In one embodiment, inactive refers to a physiological state in which the subject is not experiencing symptoms, or the GI tract is reflecting an absence of a gastrointestinal-related or liver-related condition. For example, remission of Crohn’s Disease occurs when the disease is no longer active, inflammation has subsided and mucosal healing may occur. In some embodiments, inactive includes one or more periods of time where the subject is no longer experiencing symptoms such as abdominal pain, diarrhea and/or constipation. Determining whether a subject is manifesting an inactive disease state can be performed by any methods known in the art, such as patient self-reporting, endoscopy, or through application of one or more of the methods disclosed herein. [0102] The term“biomarker” or“marker” includes any marker such as a genetic marker, microbial marker or other clinical characteristic that can be used to classify a sample from a subject having a gastrointestinal-related or liver-related condition, such as IBS, IBD, NASH, liver cancer, liver cirrhosis, autoimmune hepatitis or NAFLD, or to rule out one or more gastrointestinal-related or liver-related conditions in a sample from a subject. Non-limiting examples of suitable markers for use in the present invention include ribosomal RNA molecules. In preferred embodiments, a biomarker for use in the present invention includes, without limitation, bacterial 16S rRNA, human 18S rRNA or fungal 18S rRNA. In some embodiments, the biomarker can be used to classify or diagnose a gastrointestinal-related or liver-related condition into a specific disease ( e.g ., CD or UC) or one of its various forms or clinical subtypes (e.g., IBS-C or IBS-M). In other embodiments, the biomarker can be used to classify a sample as an IBS sample, CD sample, UC sample, NASH sample, liver cancer sample, liver cirrhosis sample, autoimmune hepatitis sample, NAFLD sample, or to rule out one or more of the above gastrointestinal-related or liver-related conditions. In some embodiments, the biomarker can include a pan bacterial, pan fungal, or human probe (e.g., an oligonucleotide sequence). In some aspects, the biomarker is a DNA probe that is complementary to a bacterial rRNA sequence, fungal rRNA sequence or human rRNA sequence. In certain embodiments, the biomarker is a single-stranded DNA oligonucleotide that is complementary along its length (or at least 95% complementary) to a bacterial 16S rRNA sequence. In another embodiment, the biomarker is a single-stranded DNA oligonucleotide that is complementary along its length (or at least 95% complementary) to a fungal 18S rRNA sequence. In yet another embodiment, the biomarker is a single-stranded DNA oligonucleotide that is complementary along its length (or at least 95% complementary) to a human 16S rRNA sequence. In a preferred embodiment, a pan bacterial biomarker comprises or consists of SEQ ID NO:2. In a preferred embodiment, a human biomarker comprises or consists of SEQ ID NO: l. In a preferred embodiment, a pan fungal biomarker comprises or consists of SEQ ID NO:3.

[0103] The term“gastrointestinal-related or liver-related condition” as used herein refers to inflammatory conditions associated with the gut, GI tract, or liver. Gastrointestinal-related or liver-related conditions can include: (1) infectious agents including, but not limited to, bacterial infections, viral infections, fungal infections, and intestinal parasites (e.g., helminths and protozoa); (2) autoimmune responses including, but not limited to, celiac disease, graft versus host disorder (GvHD) and thymoma-associated multiorgan autoimmunity (TAMA); and (3) gastrointestinal or liver diseases including, but not limited to, irritable bowel syndrome (IBS), inflammatory bowel disease (IBD) ( e.g ., Crohn’s Disease, Ulcerative Colitis, diversion colitis), non-alcoholic fatty liver disease (NAFLD), liver cancer, liver cirrhosis, autoimmune hepatitis and nonalcoholic steatohepatitis (NASH). Typically, gastrointestinal-related or liver-related conditions modulate the gut microbiome in an manner that detrimentally impacts the health of the host subject. In some embodiments, a gastrointestinal-related or liver-related condition includes the manifestation of symptoms in the host such as diarrhea, bloody stool, constipation, anemia, weight loss, and loss of appetite.

[0104] The term“sample” includes any biological specimen obtained from an individual. Suitable samples for use in the present invention include, without limitation, whole blood, plasma, serum, saliva, urine, stool (i.e., feces or fecal swab), any other bodily fluid, or a tissue sample (i.e., biopsy) such as a small intestine or colon sample (e.g., intestinal biopsy or mucosal layer sample). In one embodiment, the sample is a stool sample or fecal swab. In another embodiment, the sample is a whole blood, plasma or serum sample. In yet another embodiment, the sample is a biopsy or mucosal layer sample from the GI tract or liver. In certain embodiments, the sample is a sample from which ribosomal RNA has been subsequently extracted or purified from non-rRNA components of the sample (i.e., indirect detection of ribosomal RNA in the sample). In certain instances, the term“sample” includes, but is not limited to a sample from which ribosomal RNA has not been amplified or purified prior to analyses by any of the methods set forth herein (i.e., direct detection of ribosomal RNA in the sample).

[0105] The term“target” or“target of interest” for detection and/or quantitation as set forth in the methods disclosed herein are rRNA molecules. Ribosomal RNA is subject to a process termed“biological amplification” and each cell contains numerous copies of rRNA. For example, one bacterial cell may contain up to 10,000 copies of rRNA per cell. Accordingly, an advantageous aspect of the methods disclosed herein is that rRNA molecules present in a sample can be detected without the need for amplification, as is typically required by DNA detection assays (which rely on one or only a few copies of DNA per cell), or physical separation (e.g., purification) prior to analysis. In a preferred embodiment, the methods disclosed herein relate to homogeneous assays. The use of samples such as stool samples is well known in the art (see, e.g., Brim et ah, PLoS One, 8(12) e8l352 (2013)). Furthermore, extracting 16S rRNA from samples is well known in the art (QIAcube ® RNA isolation from stool sample using RNeasy^® PowerMicrobiome^® Kit, Application Note, Qiagen GmbH, QIAGEN Strasse 1, 40724, Hilden Germany). One skilled in the art will appreciate that the samples can be diluted or aliquoted prior to use in any of the methods described herein.

[0106] As used herein, the term“bacterial 16S rRNA” refers to a bacterial ribosomal RNA sequence that shares substantial (i.e., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) or identical {i.e., 100%) sequence homology or sequence identity to one or more bacterial species or genus. In particular aspects of the invention, a bacterial 16S rRNA is a synthetic oligonucleotide having substantial or identical sequence homology or sequence identity to a bacterial 16S rRNA sequence of at least 2, 5, 10, 15, 20, 30, 40, 50, or more, bacterial species. As set forth in more detail herein, the bacterial 16S rRNA oligonucleotide is typically up to 60 nucleotides in length, more preferably, 15-40, 15-30, or 15-25 nucleotides in length. It will be apparent that the bacterial 16S rRNA oligonucleotide can comprise a nucleic acid sequence having substantial or total complementarity to a 16S rRNA sequence found in two or more bacterial genus ( e.g ., Actinomyces, Aquabacterium, Spartobacteria, Desulfitobacterium ). It will also be apparent that design of a bacterial 16S rRNA synthetic oligonucleotide can be modulated based on the bacterial genus or species of particular interest. Various public databases provide ribosomal RNA gene sequences (rRNA sequences) for example, the Ribosomal Database Project (RDP) (htt : //rdp cme.ni sn . edu/) houses bacterial rRNA sequences from the International Nucleotide Sequence Databases (INSD: GenBank/EMBL/DDBJ) on a monthly basis. These sequences are aligned against a general bacterial rRNA model, which incorporates the conserved bacterial sequence information. Thus, it is contemplated that a bacterial 16S rRNA sequence having the properties set forth herein can be readily obtained from such databases for use in the present invention.

[0107] As used herein, the term“human 18S rRNA” refers to a human {Homo sapiens) 18S rRNA sequence that shares substantial {i.e., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) or identical {i.e., 100%) sequence homology or sequence identity to a Homo sapiens 18S rRNA sequence. The 18S rRNAs in Homo sapiens are processed from a 45S pre-RNA. The 45S rRNA genes are present in clusters on Chromosomes 13, 14, 15, 21 and 22 and encode a 45S rRNA precursor, which is processed to form the 18S rRNAs along with 5.8S and 28 S rRNAs. There are at least five recognized 18S RNAs, one per chromosome location {i.e., RNA18S1 (GenBank Gene ID: 106632259); RNA18S2 (GenBank Gene ID: 106632258); RNA18S3 (GenBank Gene ID: 106631782); RNA18S4 (GenBank Gene ID: 110255165/Accession No.: NR_l45820.l); and RNA18S5 (GenBank Gene ID: 110255169/ Accession No.: NR_l46l 19.1)). In particular aspects of the invention, a human 18S rRNA is a synthetic oligonucleotide that is typically up to 60 nucleotides in length, more preferably, 15-40, 15-30, or 15-25 nucleotides in length. As shown above, the GenBank database, among others, provides ribosomal RNA gene sequences (rRNA sequences) for Homo Sapiens 18S rRNA. Thus, it is contemplated that a human 18S rRNA sequence can be readily obtained from such a database for use in the present invention.

[0108] As used herein, the term“fungal 18S rRNA” refers to a fungal ribosomal RNA sequence that shares substantial (i.e., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) or identical (i.e., 100%) sequence homology or sequence identity to one or more fungal species or genus. In particular aspects of the invention, a fungal 18S rRNA is a synthetic oligonucleotide having substantial or identical sequence homology or sequence identity to a fungal 18S rRNA sequence of at least 2, 5, 10, 15, 20, 30, 40, 50, or more, fungal species. As set forth in more detail herein, the fungal 18S rRNA oligonucleotide is typically up to 60 nucleotides in length, more preferably, 15-40, 15-30, or 15-25 nucleotides in length. It will be apparent that the fungal 18S rRNA oligonucleotide can comprise a nucleic acid sequence having substantial or total complementarity to a 18S rRNA sequence found in two or more fungal genus ( e.g ., Saccharomyces, Candida, Aspergillus, and Neurospora). It will also be apparent that design of a fungal 18S rRNA synthetic oligonucleotide can be modulated based on the fungal genus or species of particular interest. Various public databases provide ribosomal RNA gene sequences (rRNA sequences), for example, the Saccharomyces Genome Database (https.v/www.yeastgenome.org) and SILVA (htps://wyvw.arb-siiva.de)_· These fungal sequences can be aligned (1) against a proposed fungal 18S rRNA synthetic oligonucleotide of the present invention or (2) assessed to determine conserved nucleotide sequences that may be used to prepare fungal 18S rRNA synthetic oligonucleotides. Thus, it is contemplated that a fungal 18S rRNA sequence having the properties set forth herein can be readily obtained from such databases for use in the present invention.

[0109] The term“subject” or“patient” refers to any member of the phylum Chordata, more preferably any member of the subphylum vertebrata, or most preferably, any member of the class Mammalia, including, without limitation, humans and other primates, including non human primates such as rhesus macaques, chimpanzees and other monkey and ape species; farm animals, such as cattle, sheep, pigs, goats and horses; domestic mammals, such as dogs and cats; laboratory animals, including rabbits, mice, rats and guinea pigs; birds, including domestic, wild, and game birds, such as chickens, turkeys, ducks, and geese. The term does not denote a particular age or gender. Thus, adult, young, and newborn individuals are intended to be covered as well as male and female subjects. In certain embodiments, a sample is obtained from a human subject ( e.g ., a stool or biopsy sample). In some embodiments, the subject is a non-human subject such as a primate, rodent, canine, feline, equine, ovine, porcine, and the like. In some embodiments, the sample may be obtained from a healthy, normal subject (i.e., obtained from a subject not suspected of, or having, a gastrointestinal-related or liver-related condition) and in other instances may be obtained from a subject diagnosed with, or suspected of having, a gastrointestinal-related or liver- related condition.

[0110] The term“host” as used herein refers to the source of the sample assessed for a gastrointestinal-related or liver-related condition. In one embodiment, the host is a human subject and the sample is a stool sample. In another embodiment, the host is a human subject and the sample is a blood, serum, plasma, or biopsy sample. In one aspect, the host’s sample can be assessed for the abundance of human rRNA, bacterial rRNA and/or fungal rRNA present in the host’s sample. In another embodiment, the host can be a non-human animal, such as a primate or companion animal (e.g., cat, dog, etc.). In this instance, the host’s sample can be assessed for host rRNA, bacterial rRNA and/or fungal rRNA in the host’s sample. Ribosomal RNA can be extracted from a host sample, for example, using an RNeasy Plus Universal Mini Kit (Qiagen, Cat No. 73404). Other protocols for rRNA isolation are well-known in the art and can be used. It is not a requirement that the host’s rRNA be physically separated from other forms (populations) of rRNA in order to perform the methods disclosed herein, although in some embodiments the host rRNA may be separated from other non-host rRNA present in the sample or reaction mixture.

[0111] The terms“analyzing,”“analyze,” and“analyzed” as used herein include chemical, biological, physical, and/or statistical manipulation of a sample. In some embodiments, the terms include drawing a conclusion or providing relative confidence whether the sample contains a specific characteristic or feature. In some embodiments, the sample is a sample from a subject, and the sample is analyzed using molecular biology techniques to determine the amount or quantity of human rRNA, bacterial rRNA and/or fungal rRNA present in the sample. In certain embodiments, the sample is a sample from a subject, and the sample is analyzed to determine a ratio of human rRNA to bacterial rRNA, a ratio of bacterial rRNA to human rRNA, a ratio of human rRNA to fungal rRNA, or a ratio of fungal rRNA to human rRNA in the sample. Quantification of host rRNA, or other populations of rRNA in the sample can be performed by any method known in the art. Common methods used for the quantification of rRNA include PCR-based methods, such as reverse-transcriptase quantitative PCR (RT-qPCR) (Freeman et al, BioTechniques, 26: 1, 112-122 (1999) and Pitkanen et al, Environ. Sci. Technol, 47(23): 13611-20 (2013)). In some aspects, biosensors may be employed that recognize sequence-specific duplexes, including DNA/RNA duplexes (see, e.g., Johnson and Mutharasan, Environ. Sci. Technol., 47(21): 12333-41 (2013)). Alternatively, antibodies may be employed that recognize sequence-specific duplexes, including DNA/RNA duplexes (see, e.g., Boguslawski et al, J. Immunol Methods, l;89(l): 123-30 (1986) and Yehle et al., Mol. Cell Probes, 1(2): 177-93 (1987)).

[0112] The term“classifying” includes“to associate” or“to categorize” a sample with a disease state. In certain instances,“classifying” is based on statistical evidence, empirical evidence, or both. In certain embodiments, the methods use a so-called reference set of samples (i.e., reference value) having a known disease state or represent normal, healthy control samples (i.e., samples obtained from subjects not suspected of, or having, a gastrointestinal-related or liver-related condition). Once established, the reference data set serves as a basis, model, or template against which the features of an unknown sample are compared, in order to characterize the unknown sample and potentially its disease state. In certain instances, classifying the sample is akin to diagnosing the disease state of the sample. In certain other instances, classifying the sample is akin to differentiating the disease state of the sample from another disease state (e.g., active versus inactive).

[0113] The term“oligonucleotide” or“probe” refers to a single-stranded oligomer or polymer of RNA, DNA, RNA/DNA hybrid, and/or a mimetic thereof. In certain instances, oligonucleotides are composed of naturally-occurring (i.e., unmodified) nucleobases, sugars, and intemucleoside (backbone) linkages. In certain other instances, oligonucleotides comprise modified nucleobases, sugars, and/or intemucleoside linkages. In a particular embodiment, oligonucleotides that form a duplex with a target rRNA are single-stranded DNA oligonucleotides.

[0114] The term“duplex” or“duplexed” as used herein includes the formation of a double- stranded nucleic acid structure such as the hybridization of two distinct nucleic acid sequences to each other. In a preferred embodiment, the term includes, but is not limited to, ribosomal RNA (e.g., human 18S rRNA present in a sample) hybridizing (e.g., under stringent conditions) to a synthetic oligonucleotide having complementarity or a high degree of sequence identity over its length to the ribosomal RNA in the sample (e.g., a pan fungal 18S rRNA probe, pan bacterial 16S rRNA probe, or human 18S rRNA probe).

[0115] As used herein, the term“mismatch” or“mismatch region” refers to a portion of an oligonucleotide that does not have 100% complementarity to its complementary nucleic acid sequence. An oligonucleotide may have at least one, two, three, four, five, six, or more mismatch regions. The mismatch regions may be contiguous or may be separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more nucleotides. The mismatch motifs or regions may comprise a single nucleotide or may comprise two, three, four, five, or more nucleotides. In a preferred embodiment, oligonucleotides of the present invention that hybridize to target rRNA molecules do not contain a mismatch.

[0116] The term“pan bacterial probe” refers to an oligonucleotide having a nucleic acid sequence that specifically hybridizes to 16S rRNA of bacterial phyla (e.g., Clostridium, E. coli, Streptococcus, Bacillus, Yersinia, Pseudomonas, and the like). In some embodiments, the pan bacterial probe can be used to identify bacterial 16S rRNA present in the sample of a subject. In another embodiment, a pan bacterial probe can be used to distinguish human 18S rRNA and/or fungal 18S rRNA from bacterial 16S rRNA. In some embodiments, a pan bacterial probe comprises between 15 and 50 nucleotides in length, more preferably, 15-40, 15-30, or 15-25 nucleotides in length. In a preferred embodiment, a pan bacterial probe includes a detectable label at a location within the probe. In some embodiments, the pan bacterial probe includes an acridinium ester (AE) or a derivative thereof along the backbone of the oligonucleotide. In certain embodiments, the AE or its derivative is incorporated at a single internal position along the backbone of the oligonucleotide (e.g., adjacent to flanking nucleotides) as opposed to incorporation at the 5’ or 3’ terminus of the oligonucleotide. In some aspects, the pan bacterial probe includes a single AE or a derivative thereof at about a central region (i.e., within about 5 to about 10 nucleotides of the mid-point of the oligonucleotide sequence) such that hybridization of the pan bacterial probe to a complementary nucleic acid sequence (e.g., in a sample) results in incorporation of the AE or derivative thereof into the minor groove of a resulting duplex nucleic acid structure. In a preferred embodiment, a chemiluminescent signal is emitted from the AE or its derivative after incorporation into a duplex nucleic acid structure (e.g., hybridization of the pan bacterial probe to a complementary bacterial 16S rRNA sequence present in the sample). In a preferred embodiment, the pan bacterial probe is designed such that it can hybridize to a plurality of bacterial species present in a single sample ( i.e ., pan bacterial). In some embodiments, the pan bacterial probe comprises a nucleic acid sequence that is highly conserved (i.e., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or identical homology) to a region (e.g., at least 10 nucleotides, and preferably between 15 and 50 nucleotides) of the 16S rRNA gene. In some embodiments, the bacterial probe is capable of hybridizing to one, two, three, or more bacterial phyla present in a sample. In some embodiments, the bacterial probe is capable of hybridizing to one, two, three, or more bacterial species present in a sample. In certain embodiments, the pan bacterial probe comprises or consists of SEQ ID NO: 1.

[0117] The term “human probe” refers to an oligonucleotide having a nucleic acid sequence that is specifically designed to hybridize to 18S rRNA of humans. In some embodiments, the human probe can be used to identify human 18S rRNA present in the sample of a subject. In another embodiment, a human probe can be used to distinguish bacterial 16S rRNA and/or fungal 18S rRNA from human 18S rRNA. In some embodiments, a human probe comprises between 15 and 50 nucleotides in length, more preferably, 15-40, 15-30, or 15-25 nucleotides in length. In a preferred embodiment, a human probe comprises an acridinium ester (AE) or a derivative thereof along the backbone of the oligonucleotide. In certain embodiments, the AE or its derivative is incorporated internally along the backbone of the oligonucleotide as opposed to incorporation at the 5’ or 3’ terminus of the oligonucleotide. In some aspects, the human probe includes a single AE or derivative thereof at a central or mid-point location along the length of the oligonucleotide backbone such that hybridization of the human probe to a complementary human 18S rRNA sequence in a sample results in incorporation of the AE into the minor groove of the duplexed nucleic acid sequence. In a preferred embodiment, a chemiluminescent signal is emitted from the AE or its derivative after incorporation into a duplex nucleic acid sequence (e.g., hybridization of the human probe to a complementary human 18S rRNA sequence in a sample). The human probe is designed such that it can hybridize to a plurality of human 18S rRNA nucleic acid sequences obtained from distinct sources (e.g., multiple human stool samples provided by closely (i.e., parent, child, siblings, grandparents) or genetically unrelated persons (i.e., persons from different ethnic backgrounds, etc.). In some embodiments, the human 18S probe comprises a nucleic acid sequence that is highly conserved (i.e., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or identical homology) to a region (e.g., at least 10 nucleotides, and preferably between 15 and 50 nucleotides) of the 18S rRNA gene. In some embodiments, the human probe is capable of hybridizing to one, two, three, or more distinct sources of human 18S rRNA. In certain embodiments, the human probe comprises or consists of SEQ ID NO: 2.

[0118] The term“pan fungal probe” refers to an oligonucleotide having a nucleic acid sequence that is specifically designed to hybridize to 18S rRNA of fungal phyla ( e.g ., Ascomycota, Basidiomycota, Chytridomycota, Zygomycota, and the like). In some embodiments, the pan fungal probe can be used to identify 18S fungal rRNA present in the sample of a subject. In another embodiment, a pan fungal probe can be used to distinguish human 18S rRNA and/or bacterial 16S rRNA from fungal 18S rRNA. In some embodiments, a pan fungal probe comprises between 15 and 50 nucleotides in length, more preferably, 15- 40, 15-30, or 15-25 nucleotides in length. In a preferred embodiment, a pan fungal probe comprises an acridinium ester (AE) or a derivative thereof along the backbone of the oligonucleotide. In one embodiment, the AE or its derivative is incorporated internally along the backbone of the oligonucleotide as opposed to incorporation at the 5’ or 3’ terminus of the oligonucleotide. In some aspects, the pan fungal probe includes a single AE or derivative thereof at a central or mid-point location along the length of the oligonucleotide backbone such that hybridization of the pan fungal probe to a complementary fungal 18S rRNA sequence results in incorporation of the AE into the minor groove of the duplexed nucleic acid sequence. In a preferred embodiment, a chemiluminescent signal is emitted from the AE or its derivative after incorporation of the AE into a duplex structure (e.g., hybridization of the pan fungal probe to a complementary fungal 18S rRNA sequence in a sample). In some embodiments, the pan fungal probe is designed such that it can hybridize to a plurality of fungal species (i.e., pan fungal) present in a single sample. In some embodiments, the pan fungal probe comprises a nucleic acid sequence that is highly conserved (i.e., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or identical homology) to a region (e.g., at least 10 nucleotides, and preferably between 15 and 50 nucleotides) of the 18S rRNA gene. In some embodiments, the fungal probe is capable of hybridizing to one, two, three, or more fungal phyla. In some embodiments, the fungal probe is capable of hybridizing to one, two, three, or more fungal species. In some embodiments, the fungal probe comprises or consists of SEQ ID NO: 3.

[0119] Incorporation of AE protects the probes disclosed herein (i.e., human, pan bacterial, and pan fungal probes) from degradation when exposed to basic conditions, e.g., sodium hydroxide. In contrast, unbound probes or probes that form a mismatch duplex nucleic acid structure are washed away or degraded ( e.g ., hydrolyzed) in solutions with higher pH (e.g., above 7.5). As such, a chemiluminescent signal is only derived from probes that are sufficiently hybridized to the corresponding rRNA.

[0120] The term“hybridization protection assay” or“HP A” as used herein includes, but is not limited to: (1) hybridization of a probe to a complementary nucleic acid sequence present in a sample; (2) differential hydrolysis of the sample to remove unbound or mismatched probe; and (3) detection of a detectable label bound to the probe (see, e.g., Arnold et al. , Clin. Chem., 35:1588-1594 (1989); Dhingra et al., Blood , 77:2, 238-242 (1991); and U.S. Pat. Nos. 5,283,174 and 5,639,604). In some embodiments, a detectable label includes, but is not limited to, radioisotopes, chemiluminescent molecules, enzymes, and haptens. In a preferred embodiment, the detectable label is an acridinium ester (AE) or derivative thereof.

[0121] The term“acridinium ester” or“AE” includes, but is not limited to, a derivative of acridine possessing a quaternary nitrogen center and derivatized at the 9 position to yield a labile phenyl ester moiety, specifically, 4-(2-succinimidyloxy carbonyl ethyl) phenyl- 10- methylacridiainium 9-carboxylate fluorosulfonate:

[0122] Additionally, in some embodiments, acridinium ester moieties of the following general type can be used:

wherein Ri is alkyl, alkenyl, aryl, substituted alkyl, substituted alkenyl, substituted aryl, alkoxy, aryloxy, or is absent when X=halogen;

R.2 is H, alkyl, alkenyl aryl substituted alkyl substituted alkenyl, substituted aryl, alkoxy, aryloxy, if X is N.

R.3 is H, amino, hydroxy, thiol, halogen, nitro, amino, amido, acetyl, alkyl, alkenyl, aryl, substituted acetyl substituted, alkyl, substituted alkenyl, substituted aryl, alkoxy, aryloxy;

R₄ is alkyl, alkenyl, aryl, substituted alkyl, substituted alkenyl, substituted aryl;

X is O, N, S, halogen, substituted phosphorus, substituted sulfur, substituted boron, or substituted arsenic;

Y is O, S, or NH; and

Ri and/or R₂ and/or R₃ and/or R₄ has a reactive site which allows chemical conjugation.

[0123] Methods of synthesizing acridinium esters are well known in the art and include, but are not limited to, methods set forth in US Pat. No. 5,948,899.

[0124] Acridinium esters may be attached at a number of different sites on probes as described in International PCT Publication No. WO 89/002476 and US Patent No. 5,283,174. In some embodiments, attachment of AE to the probe includes the ability to label nucleotide bases, the phosphate backbone, the 3'-terminus, the 5'-terminus as well as the non-nucleotide monomeric units of mixed nucleotide/non-nucleotide oligonucleotides.

[0125] Incorporation of an AE into a nucleic acid duplex structure protects the AE from differential hydrolysis, whereas mismatched or unbound AE-probes are rapidly hydrolyzed under basic conditions. FIG. 1 provides a schematic of an exemplary embodiment of an AE- probe based HPA (see, e.g., Nelson, Critical Reviews in Clinical Laboratory Sciences , 35:5, 369-414 (1998)). [0126] Descriptions of nucleic acid hybridization as a procedure for detecting particular nucleic acid sequences are given by Kohne in U.S. Pat. No. 4,851,330, and by Hogan et al. in U.S. Pat. Nos. 5,541,308 5,593,841, and 5,681,698 (each of which is incorporated herein by reference in its entirety). These references also describe methods for determining the presence of RNA-containing organisms in a sample. The procedures typically require probes that are sufficiently complementary to the rRNA of an organism of interest, such as Staphylococcus (e.g., 16S rRNA). According to the HPA method, nucleic acids from a sample to be tested and an appropriate probe are first mixed and incubated under specified hybridization conditions. Conventionally, the probe will be labeled with a detectable label. The resulting hybridization reaction is then assayed to detect and/or quantify the amount of labeled probe (measured as Relative Light Units (RLUs)) that has formed a duplex nucleic acid structure, thereby detecting the presence of the target rRNA in the test sample.

[0127] In a preferred embodiment, HPA probes of the present invention are single-stranded DNA probes. In certain instances, the HPA probes are single-stranded DNA probes having an acridinium ester detectable label. In certain embodiments, the acridinium ester is a methyl- or fluoro-acridinium ester, or a combination thereof. In some embodiments, the HPA is a homogeneous assay, i.e., requiring no physical separation steps (e.g., isolation and/or purification of hybridized nucleic acids) after administration of the HPA probe to the sample. In certain embodiments, the HPA probe can have a length of up to 60 nucleotides.

[0128] As used herein, the term“detectable label” includes a molecule attached to, or synthesized as part of, a nucleic acid probe that under appropriate conditions produces a detectable signal. In one embodiment, the detectable label can include a chemiluminescent label. In some embodiments, the detectable label can include a radiolabel or isotope. In another embodiment, the detectable label can include a binding moiety such as biotin and/or a hapten such as digoxygenin. In another embodiment, the detectable label can include a fluorescent dye such as, but not limited to, minor groove binders (MGB), such as, 6- carboxyfluorescein (FAM™), tetachlorofluorescein (TET™), tetramethylrhodamine (TAMRA™), 6-earboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein (JOE™), hexachlorofluorescein (HEX™), and carboxy-X-rhodamine (ROX™); Alexa Fluor^® dyes, such as, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 594, Alexa Fluor 647, and Alexa Fluor 750; and Li-Cor IR^® dyes, such as, IRDye 700 and IRDye 800. The detectable signal can include fluorescence, radioactivity, colorimetry, gravimetry, x-ray diffraction, absorption, magnetism, enzymatic activity, binding affinity, mass spectrometry, and the like, and facilitates detection of the nucleic acid probe in the sample. In preferred embodiments, the detectable label is covalently linked to an HPA probe. In certain embodiments, the detectable label can be uniquely detectable ( e.g ., a label requiring a specific wavelength of light (e.g., 488 nm or 610 nm) to produce a detectable signal, allowing the HPA probe to be detected as a result. In another embodiment, the methods disclosed herein can include a plurality of different (unique) HPA probes in a single reaction mixture or sample. For example, a reaction mixture can include two HPA probes (i.e., multiplex), each probe being specific for a target rRNA under assessment (e.g., a human 18S rRNA probe, a bacterial 16S rRNA probe, or a 18S rRNA fungal probe), wherein each HPA probe contains a different detectable label (e.g., a first HPA probe containing an AE label and a second HPA probe containing a radiolabel). In another embodiment, the detectable label can be detected due to differing physical properties. One example is the use of chemiluminescent AEs with differing rates of photo-emission. These have been referred to as the“Dual Kinetic Assay,” which differentiates between probe signals that are“flashers” or“glowers” (see, e.g., Nelson et ah, Biochem., 35:8429-8438, (1996)). The differing rates of photo-emission allow the proportions of bound probe containing a“flasher” or“glower” to be readily determined, for example, through the use of a luminometer. In certain embodiments, the simultaneuous detection of different target rRNA can be accomplished using the Dual Kinetic Assay, where two or more different probes are used in a single reaction mixture or sample, e.g., a first probe specific for human rRNA and a second probe specific for bacterial rRNA in the same reaction mixture. Alternatively, the wavelength of light emitted by two different chemiluminescent AEs (e.g., a methyl-AE and a fluoro-AE) can be used to resolve the proportions of each AE present in a single sample or reaction mixture.

[0129] It is contemplated by the present invention that the methods disclosed herein can encompass other detectable labels such as adamantyl-stabilized l,2-dioxetanes, that can be used to label HPA probes. In this embodiment, an HPA probe can be labeled with adamantyl-stabilized l,2-dioxetanes and the dioxetane hydrolyzed with enzymes such as alkaline phosphatase or beta-D-galactosidase to produce a chemiluminescent signal (see, e.g., Nelson and Kacian, Clin. Chim. Acta., 17;194(1):73-90, 1990).

[0130] Furthermore, it is also contemplated that the HPA probe can include a chemiluminescent label and a quencher molecule such that in a non-hybridized state the chemiluminescent label is in sufficiently close proximity to the quencher molecule that the chemiluminescent signal is attenuated. In contrast, hybridization of the HPA probe to a target rRNA changes the conformation of the HPA probe such that the quencher molecule is removed from the proximity of the chemiluminescent label, resulting in no significant attenuation of the chemiluminescent signal (see, e.g., U.S. Pat. No. 7,169,554).

[0131] As used herein, the terms“amplifies,”“amplifying,” and“amplification” include, but are not limited to, the replication of a nucleic acid sequence, typically in a template- dependent manner such that the resulting nucleic acid sequence is a representation of the original nucleic acid sequence. Nucleic acid amplification techniques are well known in the art, including but not limited to, PCR, reverse transcription-PCR (RT-PCR), quantitative PCR, nested-PCR, isothermal amplification, transcription-mediated amplification (TMA), Nucleic Acid Sequence-Based Amplification (NASBA), Strand Displacement Amplification (SDA), Ligase Chain Reaction (LCR), and the like. In some embodiments, rRNA present in a sample is amplified using RT-PCR, isothermal amplification or transcription-mediated amplification (TMA), prior to hybridization of an HPA probe to the amplified rRNA molecules. This is a particularly beneficial method for samples that contain low levels of target rRNA (e.g., minute amounts of fungal 18S rRNA) and high levels of non-target rRNA (e.g., bacterial 16S rRNA and/or human 18S rRNA). In some embodiments, target rRNA molecules in the sample are not amplified prior to exposure to the HPA probe. In certain instances, the sample is a stool lysate and is directly applied to components of the HPA, thereby allowing for direct detection of human, bacterial and/or fungal rRNA in the stool lysate. In some embodiments, amplification of rRNA molecules in the sample may include the use of a thermostable polymerase. In certain embodiments, amplified rRNA is isolated from the sample such that purified or extracted rRNA is obtained (i.e., a non-homogeneous assay). In some embodiments, rRNA is extracted from a sample in order to reduce contamination or possible cross-reactivity with other nucleic acids (RNA and/or DNA) in the sample. In some embodiments, isolated rRNA (e.g., human 18S rRNA, bacterial 16S rRNA, fungal 18S rRNA, or a combination thereof) is used in the HPA.

[0132] The term“abundance” as used herein refers to a quantitative measurement of a characteristic or feature. In a preferred embodiment, abundance refers to the quantity or amount of a biomarker present in a sample. In one embodiment, abundance refers to the amount or quantity of a target rRNA molecule in a sample. In one embodiment, abundance includes the number of rRNA copies/mg of stool for human 18S rRNA in a stool sample. In another embodiment, abundance can include the number of rRNA copies/mg of stool for fungal 18S rRNA in a stool sample. In yet another embodiment, abundance can include the number of rRNA copies/mg of stool for bacterial 16S rRNA in a stool sample. A particular preferred technique for measuring human rRNA, bacterial rRNA and/or fungal rRNA is HP A, using for example nucleic acid probes having specificity for each of the rRNA populations in the sample. In a preferred embodiment, HPA can be performed on a sample (. e.g ., a stool or serum sample) using any of the probes set forth herein, including but not limited to, SEQ ID NOs: l-3. In some embodiments, abundance can be directly determined using a fecal lysate sample in the absence of an RNA purification reagent or procedure. In another embodiment, abundance can be determined using a fecal lysate sample in a homogeneous HPA. In some embodiments, abundance of one or more populations of target rRNA can be determined concurrently in a single sample. In another embodiment, abundance of different rRNA populations in a sample can be determined sequentially. In yet another embodiment, abundance of different rRNA populations in a sample can be determined independently.

[0133] The term“reference value” refers to a specific value, dataset, ratio or numerical range that corresponds to: (1) a first rRNA population (e.g., human 18S rRNA, bacterial 16S rRNA or fungal 18S rRNA) in a first sample compared to a second rRNA population in the same sample; (2) a first rRNA population in a first sample compared to a first rRNA population in a second sample; (3) a first biomarker in a first sample compared to a second biomarker in the first sample; (4) a first biomarker in a first sample compared to the first biomarker in a second sample; (5) an internal conrol; (6) a human 18S rRNA:bacterial 16S rRNA ratio (or vice versa); (7) a human 18S rRNATungal 18S rRNA ratio (or vice versa); or (8) the abundance of human 18S rRNA, bacterial 16S rRNA, and/or fungal 18S rRNA. In some embodiments, the reference value comprises an internal control added or spiked-in to any of the methods disclosed herein. One benefit of using an internal control is the ability to normalize or calibrate a sample based on the presence of the internal control in the sample. In some embodiments, the internal control is a known quantity of a nucleic acid sequence added to the sample. In one embodiment, the internal control is a naturally occurring or synthetic nucleic acid sequence (e.g., locked nucleic acid (LNA). In some embodiments, the internal control is a synthesized oligonucleotide. In another embodiment, the synthesized nucleic acid sequence can comprise one or more non-natural nucleotides or nucleosides (e.g., an AE-labeled or isotopically labeled RNA or DNA base or nucleoside). In some embodiments, the nucleic acid sequence is chemically treated (e.g., to increase thermal stability of the nucleic acid sequence) or can include a modification to introduce steric hindrance ( e.g ., a LNA or 2’-0-methyl (2’-OMe)). In another embodiment, the nucleic acid sequence of the internal control can be encapsulated via natural (e.g., a virus or bacteria encoding the nucleic acid sequence) or synthetic means (e.g., coating a protein or phospholipid to the nucleic acid sequence). In some embodiments, the internal control can comprise a serial dilution of a known quantity of the nucleic acid sequence such that the internal control can be detected, and preferably measured, at the serial dilution concentrations across one or more samples to plot a standard curve. In one embodiment, the internal control can normalize a detectable signal observed in any of the methods disclosed herein (e.g., chemiluminescent detection in a HPA or fluorescent detection in a RT-qPCR assay). In some embodiments, the internal control can normalize a detectable signal between samples or within a single sample. In some embodiments, the internal control can be detected and/or quantitated by any methods known in the art, including but not limited to, HPA, PCR, RT- PCR, RT-qPCR, FISH, chromatography, and electrophoresis. Normalization may be beneficial when variations arise from the amount of sample used, when variations arise from a cell lysis step, when variations arise from different nucleic acid isolation techniques, or when other steps prior to the detecting step are subject to fluctuation or variation within or between samples (e.g., pipetting or operator variation).

[0134] In some embodiments, the reference value can be used to compare against other samples or comprises an internal data point that can be used to compare later or earlier assessments. For example, a reference value can be compared against: (1) one or more unknown test samples or samples of interest; (2) samples obtained from subjects diagnosed with a gastrointestinal-related or liver-related condition containing known levels of rRNA species in the sample (e.g., human 18S rRNA, bacterial 16S rRNA, and/or fungal 18S rRNA); (3) a dataset obtained from a prior testing period (e.g., before, during, or after clinical treatment of a subject with a gastrointestinal -related or liver-related condition). In some embodiments, a reference value is obtained from historical data obtained from a cohort of subjects that do not have or are not suspected of having a gastrointestinal-related or liver- related condition. In some embodiments, a reference value is obtained from historical data obtained from a cohort of subjects diagnosed with a gastrointestinal-related or liver-related condition such as, but not limited to, IBS, IBD, NASH, liver cancer, liver cirrhosis, autoimmune hepatitis, NAFLD, graft versus host disease, and celiac disease. In some embodiments, the reference value can be a single cut-off value, such as a median or mean. In some embodiments, the reference value can include a specific value equally applicable to all samples of interest ( e.g ., for example, a baseline reading). In a preferred embodiment, the reference value is a predetermined value or numerical range. In some embodiments, the quantity of human, bacterial and/or fungal rRNA in a sample is compared to a reference value, and in some instances, a reference value may exist for each population of rRNA (e.g., a first reference value for human 18S rRNA, a second reference value for fungal 18S rRNA, and a third reference value for bacterial 16S rRNA). The comparison may be performed by any methods known in the art, for example statistical methods, in particular a standard curve can be used to determine optimal cut-offs or thresholds for sensitivity and specificity.

[0135] The term“dataset” as used herein refers to a collection of data. In one embodiment, a dataset is the output, tabulation, observation, notation, or recordation of one or more data points from one or more reactions. In a preferred embodiment, a dataset includes the recordation of a chemiluminescent reaction (e.g., measured as RLUs) for a plurality of samples associated with, or suspected of being associated with, a gastrointestinal-related or liver-related condition. For example, a dataset can include the abundance of human 18S rRNA from a plurality of normal healthy samples (i.e., samples not suspected of, or having, a gastrointestinal-related or liver-related condition). In another embodiment, a dataset can include the abundance of human 18S rRNA in a plurality of samples from subjects diagnosed with Crohn’s Disease. In another embodiment, a dataset can include the abundance of human 18S rRNA in a plurality of samples from subjects diagnosed with NASH. In yet another embodiment, a dataset can include the abundance of human 18S rRNA in a plurality of samples from subjects diagnosed with celiac disease. In another embodiment, a dataset can include the abundance of fungal 18S rRNA in a plurality of samples from subjects diagnosed with IBS. In another embodiment, a dataset can include the abundance of fungal 18S rRNA in a plurality of stool samples from healthy subjects (i.e., subjects not suspected of, or having, a gastrointestinal-related or liver-related condition). In another embodiment, a dataset can include the abundance of bacterial 16S rRNA in a plurality of samples from subjects diagnosed with Ulcerative Colitis. In another embodiment, a dataset can include the abundance of bacterial 16S rRNA in a plurality of samples from healthy subjects (i.e., subjects not suspected of, or having, a gastrointestinal -related or liver-related condition). In some embodiments, the dataset can include a specific value such as the median or mean of a plurality of data points, reactions, or samples. In some embodiments, the dataset can include statistical analysis of a plurality of data points, reactions, or samples. [0136] The term“ratio” as used herein refers to the relationship between two numbers. In a preferred embodiment, a ratio is a measure comparing the dataset of one biomarker with the dataset of second biomarker. For example, a ratio can include comparing the median or mean dataset for a first biomarker ( e.g ., human 18S rRNA) from a first subject population (e.g., one or more healthy subjects) with the median or mean dataset for the same biomarker from a second subject population (e.g., one or more subjects diagnosed with Crohn’s Disease). In another embodiment, a ratio can include comparing the abundance of one biomarker with the abundance of second biomarker from the same sample. In one embodiment, a ratio can include comparing a quantitative measurement of one biomarker (e.g., abundance of 18S human rRNA) in a sample with the quantitative measurement of a second biomarker (e.g., abundance of bacterial 16S rRNA or fungal 18S rRNA) from the same sample. A ratio does not require that both biomarkers be assessed at the same time (concurrently) or sequentially. A ratio does not require that the biomarkers be assessed in the same reaction mixture. However, in a preferred embodiment, multiple biomarkers can be assessed from the same sample (e.g., one stool sample or nucleic acid extract prepared therefrom). In one aspect, the first biomarker is the denominator and the second biomarker is the numerator in the ratio. It will be apparent that the allocation of one biomarker as the numerator and a second biomarker as the denominator can be reversed. The resulting numerical value is a ratio of the two biomarkers in the sample.

[0137] In one embodiment, a ratio may be calculated by applying the abundance of human 18S rRNA in a sample (e.g., the numerator) against the abundance of bacterial 16S rRNA from the same sample (e.g., the denominator), or vice versa. In another embodiment, a ratio may be calculated by applying the abundance of human 18S rRNA in a sample (e.g., the numerator) against the abundance of fungal 18S rRNA from the same sample (e.g., the denominator), or vice versa. In yet another embodiment, a ratio may be calculated by applying the abundance of human 18S rRNA in a dataset of normal, healthy samples (e.g., the numerator) against the abundance of human 18S rRNA from the dataset of samples obtained from subjects diagnosed with Crohn’s Disease (e.g., the denominator), or vice versa.

[0138] The term“statistical analysis” or“statistical method,” unless otherwise clear from the context, can include a learning statistical classifier system. In some embodiments, statistical analysis includes the Mann-Whitney U Test. In some instances, the learning statistical classifier system is selected from the group consisting of a random forest, classification and regression tree, boosted tree, neural network, support vector machine, general chi-squared automatic interaction detector model, interactive tree, multiadaptive regression spline, machine learning classifier, and combinations thereof. In certain instances, the statistical analysis comprises a single learning statistical classifier system. In other embodiments, the statistical analysis comprises a combination of at least two learning statistical classifier systems. In some instances, the at least two learning statistical classifier systems are applied in tandem. Non-limiting examples of statistical analysis suitable for use in the invention are described in International PCT Publication No. WO 12/054532, the disclosure of which is hereby incorporated by reference in its entirety for all purposes.

[0139] As used herein, the terms “nucleic acid,” “nucleotide sequence,” and “polynucleotide” are interchangeable and refer to a polymeric form of nucleotides. The nucleotides may be deoxyribonucleotides (DNA), ribonucleotides (RNA), analogs thereof, or combinations thereof, and may be of any length. Polynucleotides may perform any function and may have any secondary and tertiary structures. The terms encompass known analogs of natural nucleotides and nucleotides that are modified in the base, sugar and/or phosphate moieties. Analogs of a particular nucleotide have the same base-pairing specificity ( e.g ., an analog of A base pairs with T). A polynucleotide may comprise one modified nucleotide or multiple modified nucleotides. Examples of modified nucleotides include fluorinated nucleotides, methylated nucleotides, and nucleotide analogs. Nucleotide structure may be modified before or after a polymer is assembled. Following polymerization, polynucleotides may be additionally modified via, for example, conjugation with a labeling component or target binding component. A nucleotide sequence may incorporate non-nucleotide components. The terms also encompass nucleic acids comprising modified backbone residues or linkages, that are synthetic, naturally occurring, and non-naturally occurring, and have similar binding properties as a reference polynucleotide (e.g., DNA or RNA). Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs), Locked Nucleic Acid (LNA) and morpholino structures.

[0140] As used herein,“sequence identity” or“sequence similarity” generally refers to the percent identity of nucleotide bases or amino acids comparing a first polynucleotide or polypeptide to a second polynucleotide or polypeptide using algorithms having various weighting parameters. Sequence identity between two polynucleotides or two polypeptides can be determined using sequence alignment by various methods and computer programs (e.g., BLAST, CS-BLAST, FASTA, HMMER, L-ALIGN, and the like) available through the worldwide web at sites including but not limited to GENBANK (http://www.ncbi.nlm.nih.gov/genbank) and EMBL-EBI (https://www.ebi.ac.uk)_· Sequence identity between two polynucleotides or two polypeptide sequences is generally calculated using standard default parameters of the various methods or computer programs. A high degree of sequence identity, as used herein, between two polynucleotides or two polypeptides is typically between about 90% identity and 100% identity, for example, about 90% identity or higher, preferably about 95% identity or higher, more preferably about 98% identity or higher. A moderate degree of sequence identity, as used herein, between two polynucleotides or two polypeptides is typically between about 80% identity to about 85% identity, for example, about 80% identity or higher, preferably about 85% identity. A low degree of sequence identity, as used herein, between two polynucleotides or two polypeptides is typically between about 50% identity and 75% identity, for example, about 50% identity, preferably about 60% identity, more preferably about 75% identity. For example, a human stool sample can have a high degree of sequence identity, over its length to a human 18S rRNA probe (e.g., SEQ ID NO: l) and a low a high degree of sequence identity, over its length to a fungal 18S rRNA probe (e.g., SEQ ID NO:3).

[0141] As used herein,“hybridization,”“hybridize,” or“hybridizing” is the process of combining two complementary single-stranded DNA or RNA molecules so as to form a single double-stranded molecule (DNA/DNA, DNA/RNA, RNA/RNA) through hydrogen base pairing. Hybridization stringency is typically determined by the hybridization temperature and the salt concentration of the hybridization buffer (e.g., high temperature and low salt provide high stringency hybridization conditions). Examples of salt concentration ranges and temperature ranges for different hybridization conditions are as follows: high stringency, approximately 0.01M to approximately 0.05M salt, hybridization temperature 5°C to l0°C below Tm (melting temperature); moderate stringency, approximately 0.16M to approximately 0.33M salt, hybridization temperature 20°C to 29°C below T_m; and low stringency, approximately 0.33M to approximately 0.82M salt, hybridization temperature 40°C to 48°C below Tm of duplex nucleic acids is calculated by standard methods well- known in the art (see, e.g., Maniatis et al., Molecular Cloning: A Laboratory Manual , Cold Spring Harbor Laboratory Press: New York (1982); Casey et al. , Nucleic Acids Research 4: 1539-1552 (1977); Bodkin et al., Journal of Virological Methods l0(l):45-52 (1985); Wallace et al. , Nucleic Acids Research 9(4):879-894 (1981)). Algorithm prediction tools to estimate Tm are also widely available. High stringency conditions for hybridization typically refer to conditions under which a nucleic acid having complementarity to a target sequence predominantly hybridizes with the target sequence, and substantially does not hybridize to non-target or off-target sequences. In the context of the present invention, hybridization conditions are typically of moderate stringency, preferably high stringency.

[0142] The term“about” in relation to a reference numerical value can include a range of values plus or minus 10% from that value. For example, the amount“about 10” includes amounts from 9 to 11, including the reference numbers of 9, 10, and 11. The term“about” in relation to a reference numerical value can also include a range of values plus or minus 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% from that value.

III. DESCRIPTION OF THE EMBODIMENTS

A. Preparation Of Biological Samples

[0143] It is contemplated that a number of methods could be employed to obtain biological samples of interest. Such methods include the collection of a fluid sample ( e.g ., urine, plasma, serum or whole blood) (US Patent No. 7,207,951) or the collection of a solid sample (e.g., biopsy, tumor or stool sample). In some embodiments, a preferred biological sample is a stool sample. The stool sample can include a fresh crude stool sample, a fecal swab, or a portion thereof, placed, for example, in a container for further processing. The stool sample can be placed in a container and shipped in the presence of transport media including reagents that stabilize nucleic acid or microbial communities in the stool sample (e.g., FecalSwab™ Cary Blair Collection and Transport systems, Thermo Fisher Scientific, Waltham, MA). Additionally, the transport media can contain a DNA or RNA oligonucleotide that is used as an internal control when assessing the contents of the stool sample.

B. Isolation of Nucleic Acids

[0144] Nucleic acids can be isolated using techniques well known to those of skill in the art, though in particularly preferred embodiments, methods for isolating RNA molecules can be employed (e.g., US Patent No. 8,357,672). Chromatography is a process often used to separate or isolate nucleic acids from protein or from other nucleic acids. Such methods can involve electrophoresis with a gel matrix, filter columns, alcohol precipitation, and/or other chromatography. [0145] When rRNA from cells is to be used, methods can involve lysing the cells with detergent ( e.g ., sodium tri-isopropylnaphthalene sulfonate or N-lauroyl sarcosine), chaotropic agent (e.g., guanidinium isothiocyanate or urea), and/or sonication (e.g., Bioruptor^® Standard, Diagenode SA, Belgium, Cat No. UCD-200), prior to implementing methods for isolating particular populations of RNA.

[0146] Methods may involve the use of organic solvents and/or alcohol to isolate RNA, particularly rRNA used in methods and compositions disclosed herein. Some embodiments are described in U.S. Patent Publication No. 2005/0059024 and US Patent No. 8,404,439.

[0147] In certain aspects, the disclosure provides methods for efficiently isolating RNA molecules from cells comprising adding a lysis buffer to the cells to form a reaction mixture, and mechanical disruption followed by heating the reaction mixture to lyse the cells. In some embodiments, the heated reaction mixture can be centrifuged for a brief time (e.g., less than 10 minutes) to separate the cell debris from the RNA of interest, forming a clarified stool lysate. In some aspects, the clarified stool lysate can be used in subsequent nucleic acid assays without further purification (i.e., a homogeneous assay). It is also contemplated that the clarified stool lysate can be further purified by using various commercially available purification reagent kits such as, but not limited to, RNeasy Universal Mini Kit, (Qiagen, Germany), and GeneJET RNA Purification Kit (Thermo Fisher Scientific, Waltham, MA).

[0148] In one aspect, RNA concentration and/or downstream purification can be performed on stool lysate samples. For example, to concentrate RNA present in a stool lysate sample, an aliquot of the stool lysate sample can be added to a target capture solution containing poly- T coated magnetic microparticles to form a reaction mixture. The reaction mixture can be heated to about 70°C for 30 minutes and then cooled to room temperature. A magnetic wash station may be used to wash the reaction mixture and resuspend the sample in resuspension buffer to remove specimen residuals. In some instances, downstream purification of the stool lysate may be performed using a rRNA purification reagent. For example, RNeasy PowerClean^® Pro CleanUp Kit can be used to remove biological inhibitors from the stool lysate sample (Qiagen, Germany, Catalog No. 13997-50).

C. Nucleic Acid Assays

[0149] It is contemplated that a number of assays can be employed to analyze rRNA populations in biological samples. Such assays include, but are not limited to, hybridization protection assay (HP A) (Gen-Probe Inc.), array hybridization, solution hybridization, nucleic amplification, polymerase chain reaction, quantitative PCR, RT-PCR, in situ hybridization, Northern hybridization, digital PCR, ddPCR (digital droplet PCR), nCounter (nanoString), BEAMing (Beads, Emulsions, Amplifications, and Magnetics) (Inostics), ARMS (Amplification Refractory Mutation Systems), RNA-Seq, TAm-Seg (Tagged- Amplicon deep sequencing) PAP (Pyrophosphorolysis-activation polymerization, branched DNA (bDNA) assay (Chiron), rolling circle amplification (RCA), single molecule hybridization detection (ETS Genomics), invader assay (ThirdWave Technologies), and/or Oligo Ligation Assay (OLA), hybridization, and array analysis. LT.S. Patent Nos. 7,888,010 and 8,173,611; ETS Patent Publication Nos. 2009/0131348 and 2009/0092974 are incorporated by reference in their entirety. In a preferred embodiment, detection of various populations of rRNA molecules in a biological sample ( e.g stool sample) can be achieved using HPA.

D. Hybridization Protection Assay

[0150] The hybridization protection assay (HPA) was originally developed by Gen-Probe Inc., and successfully identified small genetic differences in closely related organisms in vitro (Arnold et al, Clin Chem., 35:8, 1588-1594 (1989)). The HPA typically requires incorporation of an acridinium ester along the backbone of a single-stranded DNA probe. The HPA probe hybridizes to its target nucleic acid by locating a complementary nucleic acid sequence in the reaction mixture. However, mismatches between the HPA probe and the target nucleic acid sequence leave the HPA probe susceptible to hydrolysis under basic conditions. As such, unbound and mismatched duplex nucleic acid structures formed in a reaction mixture will be degraded by an increase in pH, leaving only complementary duplex nucleic acid structures remaining. The complementary duplex nucleic acid structures can be detected via various means including, but not limited to, the use of a luminometer.

[0151] Acridinium esters (AE) and derivatives thereof are useful labels that have been used extensively in immunoassays and nucleic acid assays. ET.S. Pat. Nos. 4,745,181; 4,918,192; 5,110,932; 5,241,070; 5,283,174; 5,538,901; 5,639,604; 5,663,074 and 5,656,426 disclose a variety of stable acridinium esters with different functional groups for conjugation to a variety of biologically active molecules. Considerable effort has also been directed towards the design of acridinium esters whose emission wavelength can be altered by either incorporating unique structural features in the acridinium ester or by employing the principle of energy transfer {see, U.S. Patent Nos. 5,395,752; 5,702,887; 5,879,894; 6,165,800; and 6,355,803). [0152] Various acridinium compounds are suitable chemiluminescent substrates for the formation of stable duplex nucleic acid structures with target nucleic acids under appropriate HPA conditions. For example, suitable acridinium compounds of the invention include, but are not limited to, acridinium esters (AEs), such as methyl-AEs and fluoro-AEs. Other suitable acridinium compounds include carboxy-substituted acridinium salts, which have been used as labels in immunoassays (Weeks et al. , Clin. Chem., 29, 1474-79 (1983); ET.S. Pat. No. 4,946,958; and McCapra et al. , ETC Patent No. GB 1,461,877) and 5-methyl- phenanthridinium-6-carboxylic acid aryl esters, which are isomeric with the acridinium aryl esters, and have been used as labels in immunoassays (ETS Patent No. 4,687,747). Acridinium compounds such as acridinium sulfonylamides and isomers set forth in ETS Patent Nos. 5,468,646 and 7,875,467 are also suitable for use in the methods disclosed herein. In some embodiments, the HPA is a homogeneous HPA, which is defined as an analytical assay where measurement of the molecule(s) of interest is performed without any physical separation procedure ( e.g ., in the absence of an RNA purification step).

[0153] It is also contemplated that other forms of chemiluminescent substrates can be used in the methods disclosed herein, including Fluorescence Resonance Energy Transfer (FRET) assays. FRET is a well-known phenomenon that has been widely used to study proximity effects in biomolecules. In FRET, an electronically excited fluorescent donor molecule transfers its electronic energy to a second, acceptor molecule through dipole-dipole coupling. This energy transfer causes fluorescence quenching of the donor. If the acceptor is fluorescent, its fluorescence is then observed. The efficiency of energy transfer is inversely proportional to the distance separating the donor and acceptor moieties and also depends on the fluorescent quantum yield of the donor and the extinction coefficient of the acceptor at the wavelength of maximal emission of the donor. Because of the distance dependence, FRET is normally not observed at distances >10 nm. Homogeneous immunoassays based on chemiluminescent energy transfer have been described using isoluminol as the chemiluminescent donor and fluorescein as the acceptor (Patel et al., Clin. Chem., 29:9, 1604-1608 (1983)).

IV. METHODS

[0154] The present invention provides nucleic acid based assays for the detection, quantification, and optionally, classification of a biological sample from a subject with respect to a gastrointestinal-related or liver-related condition. [0155] In one embodiment, the present invention provides an in vitro method for analyzing a sample from a subject having or suspected of having a gastrointestinal -related or liver- related condition to determine the relative abundance of the subject’s rRNA in the sample, the method comprising (a) determining the abundance of the subject’s rRNA in the sample; and (b) comparing the abundance of the subject’s rRNA in the sample to a reference value, thereby determining the relative abundance of the subject's rRNA in the sample. In a preferred embodiment, the subject’s rRNA is human 18S rRNA. In one aspect, the reference value is the median or mean value of a dataset for a cohort of normal, healthy stool samples (i.e., samples obtained from subjects not having or suspected of having a gastrointestinal- related or liver-related condition). In some embodiments, the reference value is a baseline or control value associated with samples obtained from a pool of normal, healthy subjects. In some embodiments, the reference value is a pre-determined cut-off value which is lower than the abundance of the subject’s rRNA in the sample. In some embodiments, the relative abundance of the subject’s rRNA in the sample is substantially higher (i.e., at least 10%, 20%, 30%, 40%, 50%, or more) than the reference value and is useful in predicting or diagnosing the subject who provided the sample as being afflicted with a gastrointestinal- related or liver-related condition.

[0156] In some embodiments, the method further comprises determining the abundance of bacterial rRNA in the sample, and comparing the abundance of the bacterial rRNA in the sample to the reference value, thereby determining the relative abundance of the bacterial rRNA in the sample. In a preferred embodiment, the bacterial rRNA is bacterial 16S rRNA. In one aspect, the reference value is the median or mean value of a dataset for a cohort of normal, healthy stool samples (i.e., samples obtained from subjects not having or suspected of having a gastrointestinal-related or liver-related condition). In some embodiments, the reference value is a baseline or control value associated with samples obtained from a pool of healthy subjects. In some embodiments, the reference value is a pre-determined cut-off value which is lower than the abundance of bacterial rRNA in the sample. In some embodiments, the reference value is greater than the abundance of bacterial rRNA in the sample. In another aspect, the reference value is the median or mean value of a dataset for a cohort of gastrointestinal-related or liver-related condition samples, such as CD or UC samples. In some embodiments, the reference value is a baseline or control value associated with samples obtained from subjects diagnosed with a gastrointestinal -related or liver-related condition. In some embodiments, the reference value is lower than the abundance of bacterial rRNA in a gastrointestinal-related or liver-related condition sample. In some embodiments, the reference value is greater than the abundance of bacterial rRNA in a gastrointestinal-related or liver-related condition sample. In some embodiments, the relative abundance of the bacterial rRNA in the sample is substantially higher (i.e., at least 10%, 20%, 30%, 40%, 50%, or more) than the reference value and is therefore useful in predicting or diagnosing the subject who provided the sample as being afflicted with a gastrointestinal -related or liver- related condition. In some embodiments, the relative abundance of the bacterial rRNA in the sample is substantially lower (i.e., at least 10%, 20%, 30%, 40%, 50%, or more) than the reference value and is therefore useful in predicting or diagnosing the subject who provided the sample as being afflicted with a gastrointestinal-related or liver-related condition.

[0157] In some embodiments, the method further comprises determining the abundance of fungal rRNA in the sample, and comparing the abundance of the fungal rRNA in the stool sample to the reference value, thereby determining the relative abundance of the fungal rRNA in the sample. In a preferred embodiment, the fungal rRNA is fungal 18S rRNA. In one aspect, the reference value is the median or mean value of a dataset for a cohort of normal, healthy samples (i.e., samples obtained from subjects not having or suspected of having a gastrointestinal-related or liver-related condition). In some embodiments, the reference value is a baseline or control value associated with a pool of samples obtained from healthy subjects (i.e., samples obtained from subjects not having or suspected of having a gastrointestinal-related or liver-related condition). In some embodiments, the reference value is a pre-determined cut-off that is lower than the abundance of fungal rRNA in the sample. In some embodiments, the reference value is greater than the abundance of fungal rRNA in the sample. In another aspect, the reference value is the median or mean value of a dataset for a cohort of gastrointestinal-related or liver-related condition samples, such as CD or UC samples. In some embodiments, the reference value is a baseline or control value associated with samples obtained from subjects diagnosed with a gastrointestinal-related or liver-related condition. In some embodiments, the reference value is lower than the abundance of fungal rRNA in a gastrointestinal-related or liver-related condition sample. In some embodiments, the reference value is greater than the abundance of fungal rRNA in a gastrointestinal-related or liver-related condition sample. In some embodiments, the relative abundance of the fungal rRNA in the sample is substantially higher (i.e., at least 10%, 20%, 30%, 40%, 50%, or more) than the reference value and is therefore useful in predicting or diagnosing the subject who provided the sample as being afflicted with a gastrointestinal-related or liver-related condition. In some embodiments, the relative abundance of the fungal rRNA in the sample is substantially lower (i.e., at least 10%, 20%, 30%, 40%, 50%, or more) than the reference value and is therefore useful in predicting or diagnosing the subject who provided the sample as being afflicted with a gastrointestinal-related or liver-related condition. In some embodiments, the sample is a stool sample.

[0158] In one embodiment, the method further comprises determining the amount of calprotectin in the sample. In some embodiments, an amount of calprotectin over 100 mg/kg may be useful in combination with the methods disclosed herein to predict or diagnose a subject with a gastrointestinal -related or liver-related condition. In another embodiment, an amount of calprotectin over 110 mg/kg may be useful in combination with the methods disclosed herein to predict or diagnose a subject with a gastrointestinal-related or liver-related condition. In yet another embodiment, an amount of calprotectin over 120 mg/kg may be useful in combination with the methods disclosed herein to predict or diagnose a subject with a gastrointestinal-related or liver-related condition. In one embodiment, an amount of calprotectin between 30-50 mg/kg may be useful in combination with the methods disclosed herein to predict or diagnose a subject with a gastrointestinal-related or liver-related condition. In another embodiment, an amount of calprotectin of less than 50 mg/kg may be useful in combination with the methods disclosed herein to predict or diagnose a subject with a gastrointestinal-related or liver-related condition.

[0159] In one embodiment, the sample is a stool sample, wherein the stool sample comprises a stool (fecal) lysate or ribonucleic acids isolated from the stool sample. In one embodiment, the stool lysate is prepared by dissolving a portion of crude fecal matter in a cell lysis buffer and heating the sample between 60°C and l00°C for 3 to 10 minutes, whereupon the sample is centrifuged at high speed ( e.g 20,000 g) to remove cell debris and specimen residuals from the supernatant. In one embodiment, the supernatant can be directly applied to the methods disclosed herein without further purification or concentration. In some embodiments, the determining step is therefore performed directly on the stool lysate. In another embodiment, the method does not include amplifying any portion of the rRNA present in the subject’s stool sample prior to the determining step. However, in some embodiments, the method includes a pre-amplification step wherein the stool sample or ribonucleic acids isolated from the stool sample are amplified prior to the determining step. In some embodiments, the amplification can include RT-PCR, PCR, TMA, or other methods of amplification known in the art. In some embodiments, one or more methods of amplification may be used to amplify the ribonucleic acids present in the sample prior to the determining step.

[0160] In one aspect, the abundance of the subject’s rRNA in the sample is determined by hybridization protection assay (HP A). In a preferred embodiment, the HPA probes of the present invention are single-stranded DNA probes. In certain instances, the HPA probes are single-stranded DNA probes having an acridinium ester detectable label. In certain embodiments, the acridinium ester is a methyl- or fluoro-acridinium ester, or a combination thereof. In some embodiments, the HPA is a homogeneous assay requiring no physical separation steps after administration of the HPA probes to the sample. In certain embodiments, the HPA probe can have a length of up to 60 nucleotides.

[0161] In one aspect, a detectable label is covalently linked to the HPA probe. In certain embodiments, the detectable label can be uniquely detectable ( e.g ., a label requiring a specific wavelength of light (e.g., 430 nm or 610 nm) to produce a detectable signal, allowing the HPA probe to be detected as a result. In another embodiment, the methods disclosed herein can include a plurality of different (unique) HPA probes in a single reaction mixture or sample. For example, a reaction mixture can include two HPA probes, each probe being specific for a target rRNA under assessment (e.g., a human 18S rRNA probe, a bacterial 16S rRNA probe or a 18S rRNA fungal probe), wherein each HPA probes contains a different detectable label (e.g., a first HPA probe containing an AE label and a second HPA probe containing a radiolabel).

[0162] In another embodiment, the detectable label of the HPA probe can be detected due to differing physical properties. One example is the use of chemiluminescent AEs with differing rates of photo-emission. These have been referred to as the“Dual Kinetic Assay,” which differentiates between probe signals that are“flashers” or“glowers” (Nelson et al. , Biochem., 35:8429-8438, (1996)). The differing rates of photo-emission allow the proportions of bound probe containing a“flasher” or“glower” to be readily determined, for example, through the use of a luminometer. In certain embodiments, the simultaneuous detection of different target rRNA can be accomplished using the Dual Kinetic Assay, where two or more different probes are used in a single reaction mixture or sample, e.g., a first probe specific for human rRNA and a second probe specific for bacterial rRNA in the same reaction mixture. Alternatively, the wavelength of light emitted by two different chemiluminescent AE’s ( e.g ., a methyl-AE and a fluoro-AE) can be used to resolve the proportions of each AE present in a single sample or reaction mixture.

[0163] It is contemplated that the methods disclosed herein can encompass other detectable labels on the HPA probe such as adamantyl-stabilized l,2-dioxetanes. In this embodiment, a HP A probe can be labeled with adamantyl-stabilized l,2-dioxetanes and the dioxetane hydrolyzed with enzymes such as alkaline phosphatase or beta-D-galactosidase to produce a chemiluminescent signal (Nelson and Kacian, Clin. Chim. Acta., 17;194(1):73-90, 1990).

[0164] Furthermore, it is also contemplated that the HPA probe can include a chemiluminescent label and a quencher molecule such that in a non-hybridized state the chemiluminescent label is in sufficiently close proximity to the quencher molecule that the chemiluminescent signal is attenuated. In contrast, hybridization of the HPA probe to a target rRNA changes the conformation of the HPA probe such that the quencher molecule is removed from the proximity of the chemiluminescent label, resulting in no significant attenuation of the chemiluminescent signal (see, e.g., ET.S. Pat. No. 7,169,554).

[0165] In one embodiment, abundance is the number of rRNA copies/mg of stool of human 18S rRNA in a stool sample. In another embodiment, abundance is the number of rRNA copies/mg of stool of fungal 18S rRNA in a stool sample. In yet another embodiment, abundance is the number of rRNA copies/mg of stool of bacterial 16S rRNA in a stool sample. A particularly preferred technique for measuring human rRNA, bacterial rRNA and/or fungal rRNA is HPA, using, for example, nucleic acid probes having specificity for each of the rRNA populations in the stool sample. In a preferred embodiment, the HPA can be performed on a subject’s sample using any of the probes set forth herein, including but not limited to, SEQ ID NOs: l-3. In some embodiments, the abundance can be directly determined using a fecal lysate sample in the absence of an RNA purification procedure. In another embodiment, the abundance can be determined using a fecal lysate sample in a homogeneous HPA. In some embodiments, the abundance of one or more populations of target rRNAs can be determined concurrently in a single stool sample. In another embodiment, the abundance of different rRNA populations in a subject’s stool sample can be determined sequentially. In another embodiment, the abundance of different rRNA populations in a sample can be determined independently. In one aspect, the abundance of human 18S rRNA can be determined using a HPA probe identified herein as SEQ ID NO: l. In yet another aspect, the abundance of bacterial 16S rRNA can be determined using a HPA probe identified herein as SEQ ID NO:2. In yet another embodiment, the abundance of fungal 18S rRNA can be determined using a HPA probe identified herein as SEQ ID NO:3.

[0166] In one aspect, the method further comprises classifying the subject as having a gastrointestinal-related or liver-related condition based on the relative abundance of the subject’s rRNA in the sample. In another embodiment, the method further comprises classifying the subject as having an active or inactive inflammatory bowel disease based on the relative abundance of the subject’s rRNA in the sample. In some embodiments, the gastrointestinal-related or liver-related condition is UC, CD, NASH, liver cancer, liver cirrhosis, autoimmune hepatitis, NAFLD, GvHD, or TAMA.

[0167] In another aspect, the method further comprises classifying the subject as having a gastrointestinal-related or liver-related condition based on the relative abundance of the bacterial rRNA in the sample. In one embodiment, the method further comprises classifying the subject as having an active or inactive inflammatory bowel disease based on the relative abundance of the bacterial rRNA in the sample. In some embodiments, the gastrointestinal- related or liver-related condition is UC, CD, NASH, NAFLD, liver cancer, liver cirrhosis, autoimmune hepatitis, GvHD, or TAMA.

[0168] In one aspect, the method further comprises classifying the subject as having a gastrointestinal-related or liver-related condition based on the relative abundance of the fungal rRNA in the sample. In one embodiment, the method further comprises classifying the subject as having an active or inactive inflammatory bowel disease based on the relative abundance of the fungal rRNA in the sample. In some embodiments, the gastrointestinal- related or liver-related condition is UC, CD, NASH, liver cancer, liver cirrhosis, autoimmune hepatitis, NAFLD, GvHD, or TAMA.

[0169] In one embodiment, the method further comprises determining a ratio of human 18S rRNA to bacterial 16S rRNA in the subject’s sample. In one embodiment, the ratio can be obtained by dividing the abundance of human 18S rRNA in the subject’s sample with the abundance of bacterial 16S rRNA in the subject’s sample. In another embodiment, the method further comprises determining a ratio of bacterial 16S rRNA to human 18S rRNA in the subject’s sample. In one embodiment, the ratio of human 18S rRNA:bacterial 16S rRNA or bacterial 16S rRNA:human 18S rRNA in the subject’s sample can be used to diagnose or classify the subject as having a gastrointestinal -related or liver-related condition. In another embodiment, the ratio of human 18S rRNA:bacterial 16S rRNA or bacterial 16S rRNA:human 18S rRNA in the subject’s sample can be used to classify the subject as having an active inflammatory bowel disease. In some embodiments, the sample is a stool sample, serum sample, blood sample, tissue sample or gastrointestinal biopsy.

[0170] In another embodiment, the method further comprises determining a ratio of human 18S rRNA to fungal 18S rRNA in the subject’s sample. In one embodiment, the ratio can be obtained by dividing the abundance of human 18S rRNA in the subject’s sample with the abundance of fungal 18S rRNA in the subject’s sample. In another embodiment, the method further comprises determining a ratio of fungal 18S rRNA to human 18S rRNA in the subject’s sample. In one embodiment, the ratio of human 18S rRNA:fungal 18S rRNA or fungal 18S rRNA:human 18S rRNA in the subject’s sample can be used to diagnose or classify the subject as having a gastrointestinal -related or liver-related condition. In another embodiment, the ratio of human 18S rRNA:fungal 18S rRNA or fungal 18S rRNA:human 18S rRNA in the subject’s sample can be used to classify the subject as having an active inflammatory bowel disease.

[0171] In one embodiment, the subject is a human. In another embodiment, the subject is a non-human animal such as, but not limited to, a feline, canine, equine, porcine, ovine, bovine, and the like. In one embodiment, the subject has been diagnosed with a gastrointestinal- related or liver-related condition. In another embodiment, the subject has been diagnosed with Crohn’s Disease or Ulcerative Colitis. In yet another embodiment, the subject has been diagnosed with IBS. In one embodiment, the subject has been diagnosed with active Crohn’s Disease or active Ulcerative Colitis. In another embodiment, the subject has been diagnosed with non-alcoholic steatohepatitis, liver cancer, liver cirrhosis, autoimmune cirrhosis or non alcoholic fatty liver disease. In one aspect, the subject may be experiencing one or more symptoms associated with an active gastrointestinal-related or liver-related condition, such as diarrhea, abdominal pain and/or constipation. In another embodiment, the subject may be in an inactive state of a gastrointestinal-related or liver-related condition. In some embodiments, the subject can include a subject having, or reporting, a“flare-up” associated with Crohn’s Disease or Ulcerative Colitis.

[0172] In one embodiment, the method can be applied to a sample from a subject diagnosed with a gastrointestinal-related or liver-related condition who is not experiencing symptoms associated with the gastrointestinal-related or liver-related condition. In some embodiments, the method can be used to determine that the subject is in an inactive state of the gastrointestinal-related or liver-related condition. In one embodiment, determining whether the subject is in an inactive state of the gastrointestinal -related or liver-related condition is predicated on the abundance of the subject’s rRNA, bacterial rRNA or fungal rRNA in the sample. In a preferred embodiment, determining whether the subject is in an inactive state of the gastrointestinal-related or liver-related condition is predicated on the abundance of the subject’s 18S rRNA, bacterial 16S rRNA or fungal 18S rRNA in the sample. In another embodiment, determining whether the subject is in an active state of a gastrointestinal-related or liver-related condition is predicated on the relative abundance of the subject’s rRNA, bacterial rRNA or fungal rRNA in the sample. In a preferred embodiment, determining whether the subject is in an active state of the gastrointestinal- related or liver-related condition is predicated on the abundance of the subject’s 18S rRNA, bacterial 16S rRNA or fungal 18S rRNA in the sample.

[0173] In some embodiments, the method is applied to a sample from a subject who is undergoing clinical treatment for a gastrointestinal-related or liver-related condition (such as, but not limited to, Humira^® (adalimumab), Remicade™ (infliximab), and prednisone treatment) to determine efficacy of the clinical treatment on the gastrointestinal-related or liver-related condition. In one embodiment, determining whether the clinical treatment is efficacious to the subject is predicated on the abundance of the subject’s rRNA, bacterial rRNA and/or fungal rRNA in the sample. In another embodiment, determining whether the clinical treatment is efficacious to the subject is predicated on the relative abundance of the subject’s 18S rRNA, bacterial 16S rRNA and/or fungal 18S rRNA in the sample.

[0174] In one aspect, the present invention provides a method for detecting human and microbiome ribosomal RNA changes associated with a gastrointestinal-related or liver- related condition, the method comprising: (a) performing a first assay to determine the abundance of a human subject’s 18S rRNA in a sample from the subject to generate a first dataset, wherein the first dataset is optionally compared to a reference value; (b) performing a second assay to determine the abundance of bacterial 16S rRNA in the sample from the subject to generate a second dataset, wherein the second dataset is optionally compared to the reference value; and (c) optionally, performing a third assay to determine the abundance of fungal 18S rRNA in the sample from the subject to generate a third dataset, wherein the third dataset is optionally compared to the reference value. [0175] In one embodiment, the first and second datasets are applied to calculate a human 18S rRNA:bacterial 16S rRNA ratio. In one embodiment, the ratio is calculated by dividing the abundance of human 18S rRNA in the subject’s sample from the first assay with the abundance of bacterial 16S rRNA in the subject’s sample from the second assay. In another embodiment, the ratio is calculated by dividing the abundance of bacterial 16S rRNA in the subject’s sample from the second assay with the abundance of human 18S rRNA in the subject’s sample from the first assay. In one embodiment, the ratio of human 18S rRNA:bacterial 16S rRNA or bacterial 16S rRNA:human 18S rRNA in the subject’s sample can be used to diagnose or classify the subject as having a gastrointestinal-related or liver- related condition. In another embodiment, the ratio of human 18S rRNA:bacterial 16S rRNA or bacterial 16S rRNA:human 18S rRNA in the subject’s sample can be used to classify the subject as having an active inflammatory bowel disease. In one embodiment, the ratio of human 18S rRNA:bacterial 16S rRNA or bacterial 16S rRNA:human 18S rRNA in the subject’s sample is indicative of the subject having or developing a gastrointestinal-related or liver-related condition. In another embodiment, the ratio of human 18S rRNA:bacterial 16S rRNA or bacterial 16S rRNA:human 18S rRNA in the subject’s sample is indicative of the subject having or developing inflammatory bowel disease.

[0176] In another embodiment, the first and third datasets are applied to calculate a human 18S rRNA:fungal 18S rRNA ratio. In another embodiment, the ratio is calculated by dividing the abundance of human 18S rRNA in the subject’s sample from the first assay with the abundance of fungal 18S rRNA in the subject’s sample from the third assay. In another embodiment, the ratio is calculated by dividing the abundance of fungal 18S rRNA in the subject’s sample from the third assay with the abundance of human 18S rRNA in the subject’s sample from the first assay. In one embodiment, the ratio of human 18S rRNA:fungal 18S rRNA or fungal 18S rRNA:human 18S rRNA in the subject’s sample can be used to diagnose or classify the subject as having a gastrointestinal -related or liver-related condition. In another embodiment, the ratio of human 18S rRNA:fungal 18S rRNA or fungal 18S rRNA:human 18S rRNA in the subject’s sample can be used to classify the subject as having an active inflammatory bowel disease. In one embodiment, the ratio of human 18S rRNA:fungal 18S rRNA or fungal 18S rRNA:human 18S rRNA in the subject’s sample is indicative of the subject having or developing a gastrointestinal -related or liver-related condition. In another embodiment, the ratio of human 18S rRNA:fungal 18S rRNA or fungal 18S rRNA:human 18S rRNA in the subject’s sample is indicative of the subject having or developing inflammatory bowel disease.

[0177] In one embodiment, the gastrointestinal-related or liver-related condition is selected from the group consisting of Crohn’s Disease, Ulcerative Colitis, non-alcoholic steatohepatitis, non-alcoholic fatty liver disease, liver cancer, liver cirrhosis, automimmune hepatitis, Celiac disease and Graft versus Host disease. In one aspect, the subject may be experiencing one or more symptoms associated with an active gastrointestinal-related or liver-related condition, such as diarrhea, abdominal pain and/or constipation. In another embodiment, the subject may be in an inactive state of a gastrointestinal-related or liver- related condition. In some embodiments, the subject can include a subject having, or reporting, a“flare-up” associated with Crohn’s Disease or Ulcerative Colitis.

[0178] In one embodiment, the method distinguishes between active or inactive inflammatory bowel disease based on the relative abundance of the human 18S rRNA in the sample. In another embodiment, the method distinguishes between active or inactive inflammatory bowel disease based on the relative abundance of the bacterial 16S rRNA in the sample. In yet another embodiment, the method distinguishes between active or inactive inflammatory bowel disease based on the relative abundance of the fungal 18S rRNA in the sample.

[0179] In one embodiment, the method identifies active inflammatory bowel disease by detecting an increase in the abundance of the human 18S rRNA in the subject’s stool sample as compared to the abundance of human 18S rRNA in a normal, healthy stool sample tested under the same conditions. In another embodiment, the normal healthy stool sample can comprise a mean or median value from a historical cohort of normal, healthy stool samples.

[0180] In one embodiment, the method distinguishes between active and inactive inflammatory bowel disease based on the human 18S rRNA:bacterial 16S rRNA ratio. In another embodiment, the method distinguishes between active and inactive inflammatory bowel disease and normal healthy stool samples (i.e., samples obtained from subjects not having or suspected of having a gastrointestinal-related or liver-related condition) based on the human 18S rRNA:bacterial 16S rRNA ratio. In yet another embodiment, the method distinguishes between active and inactive inflammatory bowel disease based on the human 18S rRNA:fungal 18S rRNA ratio. [0181] In one embodiment, the first, second and/or third assay of the method is an HPA. In some embodiments, each of the first, second and third assays is an HPA. In yet another embodiment, the first and second assays are an HPA and the third assay is a qPCR assay. In one embodiment, prior to performing the third assay, a portion of the fungal 18S rRNA is amplified. In some embodiments, the methods disclosed herein do not include an amplification procedure of one or more rRNA populations present in the sample prior to performing the first, second or third assays. In another embodiment, the method comprises amplifying at least one rRNA population present in the sample prior to performing one of the first, second or third assays. In some embodiments, the method comprises amplifying fungal 18S rRNA and/or bacterial 16S rRNA present in the sample prior to performing any one of the first, second or third assays. In yet another embodiment, the method includes amplifying at least a portion of 16S or 18S rRNA present in the sample prior to performing one or more of the first, second or third assays. In some embodiments, amplification of fungal 18S rRNA can include a PCR ( e.g reverse transcription PCR) or TMA process.

[0182] In one aspect, the first assay comprises: (a) contacting the subject’s sample with a first AE-labeled DNA probe; (b) hybridizing the first AE-labeled DNA probe to a target rRNA present in the subject’s sample; (c) separating the hybridized first AE-labeled DNA probe from unhybridized or mismatched first AE-labeled DNA probe; and (d) detecting a chemiluminescent signal from the hybridized first AE-labeled DNA probe under chemiluminescent conditions. In one embodiment, the target rRNA in the first assay is human 18S rRNA. In one embodiment, the AE-labeled DNA probe of the first assay is a human 18S rRNA probe. In a preferred embodiment, the AE-labeled DNA probe of the first assay is SEQ ID NO:l.

[0183] In another aspect, the second assay comprises (a) contacting the subject’s sample with a second AE-labeled DNA probe; (b) hybridizing the second AE-labeled DNA probe to a target rRNA present in the subject’s sample; (c) separating the hybridized second AE- labeled DNA probe from unhybridized or mismatched second AE-labeled DNA probe; and (d) detecting a chemiluminescent signal from the hybridized second AE-labeled DNA probe under chemiluminescent conditions. In one embodiment, the target rRNA in the second assay is bacterial 16S rRNA. In one embodiment, the AE-labeled DNA probe of the second assay is a pan bacterial 16S rRNA probe. In a preferred embodiment, the AE-labeled DNA probe of the second assay is SEQ ID NO:2. [0184] In certain aspects, the third assay comprises (a) contacting the subject’s sample with a third AE-labeled DNA probe; (b) hybridizing the third AE-labeled DNA probe to a target rRNA present in the subject’s sample; (c) separating the hybridized third AE-labeled DNA probe from unhybridized or mismatched third AE-labeled DNA probe; and (d) detecting a chemiluminescent signal from the hybridized third AE-labeled DNA probe under chemiluminescent conditions. In some embodiments, the target rRNA in the third assay is fungal 18S rRNA. In one embodiment, the AE-labeled DNA probe of the third assay is a pan fungal 18S rRNA probe. In a preferred embodiment, the AE-labeled DNA probe of the third assay is SEQ ID NO:3.

[0185] In some embodiments, the first, second and third assays are performed concurrently on the subject’s sample ( e.g ., within a single reaction mixture). In another embodiment, the first, second and third assays are performed sequentially on the subject’s sample (e.g., within one, two, or three reaction mixtures). In another embodiment, the first, second and third assays are performed independently on the subject’s sample ( i.e ., in three separate reaction mixtures). In some embodiments, the first, second and third assays comprise AE-labeled DNA probes having distinct physical properties such that the chemiluminescent signal from each AE-labeled DNA probe is detectable independent of one or more different AE-labeled DNA probes present in the sample. In another embodiment, the first, second and third assays comprise AE-labeled DNA probes having different AE compounds (e.g., methyl-AE, acridinium sulfonylamide, fluoro-AE) such that the chemiluminescent signal from each AE- labeled DNA probe is detectable independent of one or more different AE-labeled compounds present in the sample.

[0186] In one aspect, separating the hybridized AE-labeled DNA probe from unhybridized or mismatched AE-labeled DNA probe is performed by degrading the unhybridized or mismatched AE-labeled DNA probes. Degradation may include various chemical treatments known in the art; however it is generally preferred that the sample be subjected to basic conditions. In one embodiment, separating the hybridized AE-labeled DNA probe from unhybridized or mismatched AE-labeled DNA probe requires hydrolysis of the unhybridized or mismatched AE-labeled DNA probes. In one aspect, hydrolysis of the unhybridized or mismatched AE-labeled DNA probe is performed by increasing the pH of the sample to above pH 7.5. In one embodiment, separating the hybridized AE-labeled DNA probe from unhybridized or mismatched AE-labeled DNA probe comprises hydrolyzing the unhybridized or mismatched AE-labeled DNA probes by forming basic conditions in the first, second, and third assays prior to determining the abundance of each target rRNA in the sample. In some embodiments, the separating does not require any physical separation steps ( e.g isolating or purifying the hybridized AE-labeled DNA probe from the unhybridized or mismatched AE- labeled probe). In some embodiments, the separating comprises hydrolysis of the unhybridized or mismatched AE-labeled DNA probes such that the unhybridized or mismatched AE-labeled probes are no longer chemiluminescent.

[0187] In one aspect, the method detects or predicts a gastrointestinal-related or liver- related condition in a human subject. In one embodiment, the method detects or predicts a gastrointestinal-related or liver-related condition based on the abundance of human 18S rRNA or the abundance of bacterial 16S rRNA in the subject’s sample. In another embodiment, the method detects or predicts inflammatory bowel disease based on the abundance of human 18S rRNA or the abundance of bacterial 16S rRNA in the subject’s sample.

[0188] In one aspect, the invention provides a method for testing the efficacy of a clinical treatment in a human subject having a gastrointestinal -related or liver-related condition, the method comprising: (a) measuring the abundance of human 18S rRNA, bacterial 16S rRNA and optionally, fungal 18S rRNA in a pre-clinical treatment sample and a post-clinical treatment sample from the human subject, (b) calculating a human 18S rRNA:bacterial 16S rRNA ratio and/or a human 18S rRNA:fungal 18S rRNA ratio based on the abundance of human 18S rRNA, bacterial 16S rRNA and fungal 18S rRNA present in the pre-clinical treatment and post-clinical treatment samples; and (c) determining if the clinical treatment provides efficacy for the gastrointestinal-related or liver-related condition in the human subject based on the human 18S rRNA:bacterial 16S rRNA ratio and/or human 18S rRNA:fungal 18S rRNA ratio.

[0189] In some embodiments, the clinical treatment provides efficacy for the gastrointestinal -related or liver-related condition in the human subject if the post-clinical treatment human 18S rRNA:bacterial 16S rRNA ratio is lower than the pre-clinical treatment human 18S rRNA:bacterial 16S rRNA ratio. In one embodiment, the post-clinical treatment human 18S rRNA:bacterial 16S rRNA ratio is at least 10%, 20%, 30%, 40%, or 50%, lower than the pre-clinical treatment human 18S rRNA:bacterial 16S rRNA ratio.

[0190] Numerous clinical treatments for gastrointestinal or liver disorders are known in the prior art and are contemplated by the instant invention. For example, the clinical treatment can comprise a monoclonal antibody. Alternatively, the clinical treatment can comprise a small molecule (i.e., a compound with a molecular weight of less than about 900 Daltons). In one aspect, the clinical treatment is an antibiotic, aminosalicylate, corticosteroid, immunosuppressant or monoclonal antibody-based therapy. In one embodiment, the gastrointestinal-related or liver-related condition is selected from the group consisting of Crohn’s Disease, Ulcerative Colitis, non-alcoholic steatohepatitis, liver cancer, liver cirrhosis, autoimmune hepatitis, non-alcoholic fatty liver disease, Celiac disease and graft versus host disorder. In one aspect, clinical efficacy can be evaluated even though the human subject may still be experiencing one or more symptoms associated with the gastrointestinal-related or liver-related condition, such as diarrhea, abdominal pain and/or constipation. In some embodiments, the pre-clinical treatment and post-clinical treatment samples are collected when the subject is experiencing one or more symptoms associated with the gastrointestinal- related or liver-related condition. In another embodiment, the pre-clinical treatment sample can be collected from the human subject when the subject is experiencing one or more symptoms associated with the gastrointestinal-related or liver-related condition and the post- clinical treatment sample is collected after administration of a treatment for the gastrointestinal-related or liver-related condition. In some embodiments, the post-clinical treatment sample may be collected from the human subject when the human subject is not experiencing one or more symptoms associated with the gastrointestinal-related or liver- related condition.

[0191] In one aspect, the invention provides a method for predicting or identifying a gastrointestinal -related or liver-related condition in a human subject, the method comprising: (a) performing a first hybridization protection assay to detect human 18S rRNA in a sample from the human subject or ribonucleic acids isolated from the human subject’s sample; (b) calculating the abundance of human 18S rRNA in the subject’s sample based on the assay of step (a); (c) performing a second hybridization protection assay to detect bacterial 16S rRNA in the subject’s sample or ribonucleic acids isolated from the subject’s sample; (d) calculating the abundance of bacterial 16S rRNA in the subject’s sample based on the assay of step (c); (e) determining a ratio of human 18S rRNA:bacterial 16S rRNA based on the values obtained in steps (b) and (d); and (f) identifying the subject as having a gastrointestinal-related or liver-related condition or predicting the subject to develop a gastrointestinal -related or liver- related condition based on the ratio of step (e). [0192] In some embodiments, the method further comprises performing a third hybridization protection assay to detect fungal 18S rRNA in the subject’s sample or ribonucleic acids isolated from the subject’s sample; calculating the abundance of fungal 18S rRNA in the subject’s sample; determining a ratio of human 18S rRNA:fungal 18S rRNA; and identifying the subject as having a gastrointestinal-related or liver-related condition or predicting the subject to develop a gastrointestinal-related or liver-related condition based on the ratio of human 18S rRNA:fungal 18S rRNA. In one embodiment, the sample is a stool sample.

[0193] In one embodiment, the abundance of human 18S rRNA in the first HPA is the number of rRNA copies/mg of stool in the stool sample. In another embodiment, the abundance of bacterial 16S rRNA in the second HPA is the number of rRNA copies/mg of stool in the stool sample. In yet another embodiment, the abundance of fungal 18S rRNA in the third HPA is the number of rRNA copies/mg of stool in the stool sample. A particularly preferred technique for measuring human 18S rRNA, bacterial 16S rRNA and/or fungal 18S rRNA comprises the use of single-stranded DNA probes having specificity for each of the rRNA populations in the stool sample. In a preferred embodiment, the HPA can be performed on a subject’s sample using any of the probes set forth herein, including but not limited to, SEQ ID NOs: l-3. In some embodiments, the abundance can be directly determined using a fecal lysate sample in the absence of a RNA purification procedure. In another embodiment, the abundance can be determined using a fecal lysate sample in a homogeneous HPA. In some embodiments, the abundance of one or more populations of target rRNAs can be determined concurrently in a single stool sample. In another embodiment, the abundance of different rRNA populations in a subject’s stool sample can be determined sequentially. In another embodiment, the abundance of different rRNA populations in the sample can be determined independently.

[0194] In a preferred embodiment, the first, second and third hybridization protection assays comprise AE-labeled DNA probes. In one embodiment, the abundance of human 18S rRNA in the first HPA can be determined using a HPA probe identified herein as SEQ ID NO: l. In another embodiment, the abundance of bacterial 16S rRNA in the second HPA can be determined using a HPA probe identified herein as SEQ ID NO:2. In yet another embodiment, the abundance of fungal 18S rRNA in the third HPA can be determined using a HPA probe identified herein as SEQ ID NO:3. It will be apparent to one of skill in the art that other nucleic acid sequences can be designed as probes for the purpose of detecting rRNA targets in a biological sample. Specifically, it is contemplated that additional probes can be designed for use in the methods disclosed herein based on a comparison or alignment of nucleic acid sequence homology of any intended probe against the 16S rRNA sequence of one or more bacterial species or genus. For example, an AE-labelled oligonucleotide sequence having complementarity over a length of about 15 to about 50 nucleotides with a bacterial 16S rRNA sequence from 2, 3, 4, 5, 6, 7, 8, 9, 10, or more bacterial species is preferred. Additionally, it is contemplated that additional probes can be designed for use in the methods disclosed herein based on a comparison or alignment of nucleic acid sequence homology of any intended probe against the 18S rRNA sequence of one or more fungal species or genus. For example, an AE-labelled oligonucleotide sequence having complementarity over a length of about 15 to about 50 nucleotides with a fungal 18S rRNA sequence from 2, 3, 4, 5, 6, 7, 8, 9, 10, or more fungal species is preferred. In another embodiment, additional probes can be designed having complementarity to another region within the human 18S rRNA sequence. For example, an AE-labelled oligonucleotide sequence having complementarity over a length of about 15 to about 50 nucleotides with human 18S rRNA sequence is preferred.

[0195] In one embodiment, the gastrointestinal-related or liver-related condition is identified or predicted when the human 18S rRNA:bacterial 16S rRNA ratio in the subject’s sample is equal to, or greater than, one, two, three, four, five or more, standard deviations above a human 18S rRNA:bacterial 16S rRNA mean ratio obtained from a population of normal, healthy stool samples (/.<?., samples obtained from subjects not having or suspected of having a gastrointestinal-related or liver-related condition) under the same conditions.

[0196] In another embodiment, the gastrointestinal-related or liver-related condition is identified or predicted when the humanl8S rRNA:bacterial 16S rRNA ratio in the subject’s sample is about two-fold, three-fold, four-fold, or five-fold greater than a human 18S rRNA:bacterial 16S rRNA mean ratio obtained from a population of normal, healthy stool samples (/.<?., samples obtained from subjects not having or suspected of having a gastrointestinal-related or liver-related condition) under the same conditions.

V. KITS

[0197] In one aspect, the present invention provides a kit for analyzing a sample from a subject to determine the abundance of the subject’s 18S rRNA, bacterial 16S rRNA, and optionally, 18S fungal rRNA in the sample. In one embodiment, the kit comprises: (1) a first oligonucleotide having a nucleic acid sequence that is complementary to a region of human 18S rRNA; (2) a second oligonucleotide having a nucleic acid sequence that is complementary to a region of bacterial 16S rRNA; and optionally, (3) a third oligonucleotide having a nucleic acid sequence that is complementary to a region of fungal 18S rRNA. In a preferred embodiment, the kit comprises: (1) an oligonucleotide comprising SEQ ID NO: l; (2) an oligonucleotide comprising SEQ ID NO:2; and optionally, (3) an oligonucleotide comprising SEQ ID NO:3.

[0198] In one embodiment, the kit is useful to analyze a sample from a subject having or suspected of having a gastrointestinal-related or liver-related condition. In another embodiment, the kit is useful to analyze a sample from a subject having or suspected of having an inflammatory bowel disease such as CD or UC. In yet another embodiment, the kit is useful to analyze a sample from a subject having or suspected of having IBS, celiac disease or GvHD. In another embodiment, the kit is useful to analyze a sample from a subject having or suspected of having liver cancer, liver cirrhosis, or autoimmune hepatitis. In a preferred embodiment, the kit is useful to analyze a sample from a human subject. In another embodiment, the kit is useful to analyze a sample from a non-human animal subject. In one embodiment, the kit is useful to analyze a stool sample from a human subject. In yet another embodiment, the kit is useful to analyze a blood sample, serum sample or intestinal biopsy sample from a subject.

[0199] In one aspect, each of the oligonucleotides in the kit are up to 60 nucleotides in length. In some embodiments, each of the oligonucleotides of the kit is between 15 and 50 nucleotides in length, more preferably, 15-40, 15-30, or 15-25 nucleotides in length. In another embodiment, at least one of the oligonucleotides in the kit is a single-stranded DNA oligonucleotide. In a preferred embodiment, each of the oligonucleotides in the kit is a single-stranded DNA oligonucleotide.

[0200] In one embodiment, one or more of the oligonucleotides in the kit contains an acridinium ester (AE) or a derivative thereof along the backbone of the oligonucleotide. In certain embodiments, the AE or its derivative is incorporated internally along the backbone of the oligonucleotide as opposed to incorporation at the 5’ or 3’ terminus of the oligonucleotide. In some aspects, hybridization of the oligonucleotide to a complementary region of human 18S rRNA sequence, bacterial 16S rRNA sequence or fungal 18S rRNA sequence in the sample results in incorporation of the AE or its derivative into a minor groove of the resulting duplexed nucleic acid structure.

[0201] In another aspect, one or more of the oligonucleotides in the kit can contain a detectable label such that hybridization of the one or more oligonucleotides to a complementary nucleic acid sequence in the sample results in emission of a detectable signal from the detectable label. In some embodiments, the detectable label comprises a chemiluminescent label. In some embodiments, the detectable label includes a radiolabel or isotope. In another embodiment, the detectable label includes a binding moiety such as biotin and/or a hapten such as digoxygenin. In another embodiment, the detectable label can include a fluorescent dye such as, but not limited to, minor grrove binders (MGB), such as, 6- carboxyfluorescein (FAM™), tetachlorofluorescein (TET™), tetramethylrhodamine (TAMRA™), 6-carboxy-4‘, 5'-di chloro-2', 7'-di m ethoxyfl uorescein (JOE™), hexachlorofluorescein (HEX™), and carboxy-X-rhodamine (ROX™); Alexa Fluor® dyes, such as, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 594, Alexa Fluor 647, and Alexa Fluor 750; and Li-Cor IR® dyes, such as, IRDye 700 and IRDye 800. In yet another embodiment, the detectable signal can include fluorescence, radioactivity, colorimetry, gravimetry, x-ray diffraction, absorption, magnetism, enzymatic activity, binding affinity, mass spectrometry, and the like, and facilitates detection of the oligonucleotide containing the detectable label in the sample. In preferred embodiments, the detectable label is covalently linked to the one or more oligonucleotides in the kit. In certain embodiments, the detectable label can be uniquely detectable ( e.g ., a label requiring a specific wavelength of light (e.g., 488 nm or 610 nm) to produce a unique detectable signal, allowing the oligonucleotide(s) to be detected as a result.

[0202] In another embodiment, the kit can include oligonucleotides having different detectable labels (e.g., a first oligonucleotide containing an AE label and a second oligonucleotide containing a radiolabel). In another embodiment, detectable labels of the oligonucleotides can be detected due to differing physical properties. One example is the use of chemiluminescent AEs with differing rates of photo-emission (e.g., the“Dual Kinetic Assay” which differentiates between detectable signals that are“flashers” or“glowers” (Nelson et al., Biochem., 35:8429-8438, (1996)). The differing rates of photo-emission allow the proportions of bound oligonucleotide containing a“flasher” or“glower” to be readily determined, for example, through the use of a luminometer. In a preferred embodiment, the kit comprises a set of oligonucleotides each containing a detectable label for detection of two or more target rRNAs in a sample (e.g., in the same sample or a single reaction mixture), wherein the oligonucleotides are suitable for HPA. In some embodiments, the sample utilized with the kit is a stool sample.

[0203] Furthermore, it is contemplated that an oligonucleotide of the kit can include a chemiluminescent label and a quencher molecule such that in a non-hybridized state the chemiluminescent label is in sufficiently close proximity to the quencher molecule that the chemiluminescent signal is attenuated. In contrast, hybridization of the oligonucleotide to a target rRNA in the sample changes the conformation of the oligonucleotide such that the quencher molecule is removed from the proximity of the chemiluminescent label, resulting in no significant attenuation of the chemiluminescent signal (e.g., U.S. Pat. No. 7,169,554).

[0204] In some embodiments, the kit further comprises an oligonucleotide having a nucleic acid sequence that is complementary to a region of a gene encoding calprotectin. In one embodiment, the oligonucleotide having a nucleic acid sequence that is complementary to a region of a gene encoding calprotectin is up to 60 nucleotides in length. In some embodiments, the calprotectin oligonucleotide of the kit is between 15 and 50 nucleotides in length, more preferably, 15-40, 15-30, or 15-25 nucleotides in length. In another embodiment, the oligonucleotide having a nucleic acid sequence that is complementary to a region of calprotectin is a single-stranded DNA oligonucleotide. In one embodiment, the kit comprises reagents to amplify a gene encoding calprotectin. In another embodiment, the kit comprises reagents to amplify non-rRNA targets in the sample prior to detection of at least one rRNA population (e.g., bacterial 16S rRNA; human 18S rRNA or fungal 18S rRNA) in the sample. In yet another embodiment, the kit comprises reagents to amplify non-RNA targets in the sample prior to the amplification and detection of at least one rRNA population in the sample.

[0205] In one aspect, the present invention provides a kit for analyzing a sample from a subject to determine the relative abundance of the subject’s 18S rRNA, bacterial 16S rRNA, and optionally, 18S fungal rRNA in the sample. In one embodiment, the kit comprises: (1) a first oligonucleotide having a nucleic acid sequence that is complementary to a region of human 18S rRNA; (2) a second oligonucleotide having a nucleic acid sequence that is complementary to a region of bacterial 16S rRNA; and optionally, (3) a third oligonucleotide having a nucleic acid sequence that is complementary to a region of fungal 18S rRNA. In one embodiment, the kit comprises: (1) an oligonucleotide comprising SEQ ID NO:l; (2) an oligonucleotide comprising SEQ ID NO:2; and optionally, (3) an oligonucleotide comprising SEQ ID NO:3.

[0206] In one embodiment, the kit is useful to analyze a sample from a subject having or suspected of having a gastrointestinal-related or liver-related condition. In another embodiment, the kit is useful to analyze a sample from a subject having or suspected of having an inflammatory bowel disease such as CD or UC. In yet another embodiment, the kit is useful to analyze a sample from a subject having or suspected of having IBS, NASH, NAFL, liver cancer, liver cirrhosis, autoimmune hepatitis, celiac disease or GvHD.

[0207] In one embodiment, the kit further comprises one or more purification reagents ( e.g ., RNeasy Universal Mini Prep Kit, Qiagen, Germany) or a cell lysis buffer (e.g., ASL

Stool Lysis Buffer, Qiagen, Germany) to treat the sample, prior to incubating the sample with the one or more oligonucleotides of the kit. In some embodiments, the kit further comprises instructional materials containing directions (i.e., protocols) for the practice of the methods described herein (e.g., instructions for using the kit to determine the abundance of a target rRNA in a sample). While the instructional materials typically comprise written or printed materials, they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this invention. Such media include, but are not limited to, electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD-ROM), and the like. Such media may include addresses to internet sites that provide such instructional materials.

VI. EXAMPLES

[0208] Aspects of the present invention are illustrated in the following Examples. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, concentrations, percent changes, and the like) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, temperature is in degrees Celsius and pressure is at or near atmospheric. It should be understood that these Examples are given by way of illustration only and are not intended to limit the scope of what the inventors regard as various aspects of the present invention. EXAMPLE 1: Direct Detection Of Human 18S rRNA And Bacterial 16S rRNA In Human Stool Samples Using Hybridization Protection Assay

Specimen Collection:

[0209] Crude raw stool samples were collected from subjects and shipped at ambient, cooled (2-8^°C), or frozen temperatures to Applicants facility (-20^°C) for processing. Fecal swabs swirled in transport media containing lithium lauryl sulfate (LLS) and lithium succinate (or comparable detergent) were used to obtain some stool samples.

Sample Preparation:

[0210] In this example, three main steps (sample preparation, HPA probe hybridization and detection) were performed to directly detect various forms of rRNA in human stool samples.

[0211] Here, a cohort of 100 stool samples (30 normal stool samples, 35 CD stool samples (18 active/l7 inactive disease) and 35 UC stools samples (18 active/l7 inactive disease) were tested. Characterization of a stool sample as CD or UC was based on endoscopic scoring. Fecal lysates (1.5 ml) for each of the 100 stool samples were prepared using Qiagen’s ASL Stool Lysis Buffer (Catalog No: 19082), performed essentially according to the manufacturer’s instructions. Briefly, ASL lysis buffer was added to about 200 mg of stool sample and vortexed with mechanical disruption for 5 minutes followed by heating at about 70^°C for 5 minutes to lyse cells and solubilize rRNA. The sample was centrifuged at 20,000 g for about 5 minutes to sediment debris or allowed to settle at room temperature for one hour to clarify the stool lysate.

[0212] The clarified stool lysates were aliquoted into 350 mΐ portions and stored at -80^°C until required. Clarified stool lysates were then directy applied to the HPA (below).

Hybridization Protection Assay:

[0213] The HPA requires specific probes for each target nucleic acid under investigation. Here, specific DNA probes are used to detect human 18S rRNA (SEQ ID NO: l), bacterial 16S rRNA (SEQ ID NO:2) and fungal 18S rRNA (SEQ ID NO:3). Each DNA probe was labeled with an AE at an internal location along the backbone of the oligonucleotide. The DNA probes generate a chemiluminescent signal from the AE moiety when the DNA probes bind to a complementary nucleic acid sequence in the stool sample. [0214] The probe sequences were prepared in probe storage buffer (PSB) containing 10 mM lithium succinate (pH 5.0) and 0.1% lithium lauryl sulfate. The AE-probe in PSB was diluted to ~ 0.1 pmol/pL, which is -10⁷ RLU/pl. A hybridization buffer (2X Hyb) containing 0.2 M lithium succinate, pH 5.0, 4 mM EDTA, 4 mM EGTA, and 17% (w/v) lithium lauryl sulfate was also prepared. An aliquot of 2x Hyb was spiked with AE-probe in PSB to form a reaction mixture. The reaction mixture was vortexed and incubated at 60°C for 30 minutes during which time the probe hybridizes to its target nucleic acid sequence (i.e., human 18S rRNA, bacterial 16S rRNA or fungal 18S rRNA). Next, 300 mΐ of differential hydrolysis buffer (DH buffer) containing 0.15 M sodium tetraborate (pH 7.6), 5% Triton X-100 was added to the reaction mixture to hydrolyze unbound probe and incubated for a further 8-15 minutes at 60°C. After which, the reaction mixtures were placed on ice.

Detector Solutions:

[0215] Each sample remained on ice until a luminometer and detector solutions were prepared. RLETs were measured for reagent blank samples and for each stool sample using an automated reagent-injection luminometer (Hologic Leader 50i, Marlborough, MA) and two detector solutions. The first detector solution contained 5 mM HNCb and 0.1% H2O2; and the second detector solution contained 1M NaOH. Here, 200 mΐ of hydrogen peroxide is followed after a 1 second delay by injection of 200 mΐ of NaOH solution. The resulting chemiluminescence is integrated for 2 to 5 seconds.

Results:

[0216] The results from this experiment are presented in FIGS. 2A-2C, where standard curves for all 100 stool samples (segregated into five sets of 20 samples) are provided for detection of bacterial 16S rRNA, human 18S rRNA, and fungal 18S rRNA. All 100 stool samples for bacterial 16S rRNA were above a threshold RLET cut-off value (set at 2 standard deviations over the RLET value of a control probe), while 14 of the human 18S rRNA samples, and 8 of the fungal 18S rRNA samples were eliminated for further processing based on RLETs below the threshold cut-off value.

[0217] Next, the samples were assessed based on disease state. FIGS. 3A-3C show detection of bacterial 16S rRNA (measured as copies rRNA/mg of stool) for the 100 human stool samples from healthy (i.e., samples obtained from subjects not having or suspected of having a gastrointestinal -related or liver-related condition), CD and ETC subjects. In order to calculate copies rRNA/mg of stool, the standard curves (FIGS. 2A-2C) were converted to template copies of rRNA and then it was determined how much fecal material was added to the HPA reaction based on the fecal lysate or nucleic acid extract used. FIG. 3 A illustrates a statistically significant difference between normal (i.e., samples obtained from subjects not having or suspected of having a gastrointestinal-related or liver-related condition) and diseased stool samples (UC and CD). There is a statistically significant difference between the normal set of samples and the two diseased sample sets (CD, p=0.0044 and UC, p= <0.0001). The p-values were calculated using Mann-Whitney U test. FIG. 3B illustrates a statistically significant difference between normal and CD stool samples based on disease severity (active CD). When the CD data was parsed into active versus inactive disease based on endoscopy, there was a clear statistically significant difference between CD active (CDa) and normal sample sets (p=0.004l). Removing the outlier in the CD inactive set still produced a statistically significant p-value of 0.0083. FIG. 3C illustrates a statistically significant difference between normal and UC stool samples based on disease severity (active or inactive UC). When the UC data was parsed into active versus inactive disease based on endoscopy, there was a clear statistically significant difference between UC (active/inactive) and normal sample sets (p=0.0005). In comparison to both the normal and CD sample sets, it was noted there was a substantial drop in bacterial counts in UC patients regardless of the severity of UC disease.

[0218] FIGS. 4A-4C show detection of human 18S rRNA (measured as rRNA copies/mg of stool sample) for the 100 human stool samples, from healthy (i.e., samples obtained from subjects not having or suspected of having a gastrointestinal-related or liver-related condition), CD and UC subjects. FIG. 4 A illustrates no statistical difference between normal (i.e., samples obtained from subjects not having or suspected of having a gastrointestinal- related or liver-related condition) and diseased stool sample sets (UC and CD) (CD, p=0.2248 and UC, p= <0.7044). The p-values were calculated using Mann-Whitney U test. FIG. 4B illustrates a statistically significant difference between normal (i.e., samples obtained from subjects not having or suspected of having a gastrointestinal-related or liver-related condition) and CD stool samples based on disease severity (active CD). When the CD data was parsed into active versus inactive disease based on endoscopy, there was a clear statistically significant difference between CD active (CDa) and normal sample sets (p=0.0063). FIG. 4C illustrates a statistically significant difference between normal (i.e., samples obtained from subjects not having or suspected of having a gastrointestinal-related or liver-related condition) and UC stool samples based on disease severity (active or inactive UC). When the UC data was parsed into active versus inactive disease based on endoscopy, there was a clear statistically significant difference between UC (active) and normal sample sets (p=0.0092). The observation of higher levels of human rRNA present in the stool samples of an active CD subject is consistent with findings in the field related to increased epithelial cell shedding in CD and UC subjects (Duckworth and Watson, Methods Mol. Biol. 763: 105-14 (2011)). While not being bound by the following, it is believed that active CD and UC subjects are more likely to be affected by sites of inflammation along the GI tract and neutrophils recruited to the GI tract in response to the inflammation can cause lysis of inflamed epithelial cells (for example, through the release of various proteases), resulting in release of rRNA from those cells, thereby increasing the copies of rRNA in active UC and CD subject’s samples as compared to a healthy, normal samples ii.e., samples obtained from subjects not having or suspected of having a gastrointestinal-related or liver-related condition).

Example 2: Ratio Of Human 18S rRNA To Bacterial 16S rRNA As An Indicator Of Active CD Or UC Disease

[0219] The abundance data from Example 1 for the 100 human stool samples was manipulated to produce a ratio of human 18S rRNA to bacterial 16S rRNA. FIG. 5A shows a corresponding plot of that data for the 100 human stool samples. FIG. 5 A illustrates a statistical difference between normal ii.e., samples obtained from subjects not having or suspected of having a gastrointestinal-related or liver-related condition) and diseased stool sample sets (UC and CD) (CD, r=0.0161 and UC, p= <0.0066). The p-values were calculated using the Mann-Whitney U test. When the CD data was parsed into active versus inactive disease based on endoscopy, there was a strong statistically significant difference between CD active (CDa) and normal sample sets (p=0.0009) (FIG. 5B). FIG. 5C illustrates a statistically significant difference between normal ii.e., samples obtained from subjects not having or suspected of having a gastrointestinal-related or liver-related condition) and UC stool samples based on disease severity (active or inactive UC). When the UC data was parsed into active versus inactive disease based on endoscopy, there was a clear statistically significant difference between UC (active) and normal sample sets (p=0.0054). The observation of higher levels of human rRNA present in the stool samples of an active CD subject is consistent with findings in the field related to increased epithelial cell shedding in CD and UC subjects (Duckworth and Watson, Methods Mol. Biol., 763: 105-14 (2011)). While not being bound by the following, it is believed that active CD and UC subjects are more likely to be affected by sites of inflammation along the GI tract and neutrophils recruited to the GI tract in response to the inflammation can cause lysis of inflamed epithelial cells (for example, through the release of various proteases), resulting in release of rRNA from the cells, thereby increasing the copies of rRNA in active UC and CD subject’s stool samples as compared to a healthy, normal stool samples.

Example 3: Indirect Detection Of Human 18S rRNA And Bacterial 16S rRNA In Human Stool Samples Using Hybridization Protection Assay

[0220] A subset of the 100 fecal lysate samples from Example 1 was compared with fecal lysate samples that underwent additional nucleic acid extraction procedures. This comparison was performed to ensure that direct detection of various forms of rRNA in the stool samples by way of clarified stool lysates (as set forth in Example 1) were comparable with extracted and purified stool samples. Initially, all stool samples were prepared according to Example 1. Briefly, ASL lysis buffer was added to about 200 mg of stool sample and vortexed for 5 minutes and then heated at 70^°C for 5 minutes to lyse cells and solubilize rRNA. The sample was then centrifuged at 20,000 g for 5 minutes to sediment debris or allowed to settle at room temperature for one hour to clarify the stool lysate.

[0221] Clarified stool lysates were then either (1) purified using a nucleic acid extraction procedure (RNeasy Plus Universal Mini Kit (Qiagen, Cat No. 73404)) essentially according to the manufacturer’s instructions or (2) applied directly to the HPA. RLUs for each of the purified rRNA fecal samples and clarified fecal lysate samples were recorded as set forth in Example 1.

[0222] FIG. 6A shows the results of fecal lysate samples purified using the Qiagen RNA kit. The purified fecal samples were then applied to the HPA using the DNA probe set forth as SEQ ID NO:2. When the data was parsed into normal versus disease state sample sets (CD or UC), there was a statistically significant difference between diseased and normal sample sets (CD p=0.0366; UC p=0.030l2) (FIG. 6A).

[0223] FIG. 6B shows the results of clarified fecal lysate samples in the absence of a nucleic acid purification step (/.<?., a homogeneous assay/direct detection), where the fecal sample was dissolved in a portion of lysis buffer, vortexed with mechanical disruption, heated at 70^°C to disrupt cells, followed by a short, high-speed centrifugation to clear the fecal lysate solution. Here, bacterial 16S rRNA was detected using the DNA probe set forth as SEQ ID NO:2 and applied to the HPA (essentially as performed in Example 1). When the clarified fecal lysate sample bacterial 16S rRNA data was parsed into normal versus disease state sample sets (CD or UC), there was a statistically significant difference between diseased and normal sample sets (CD p=0.023 l; UC p=0.00055) (FIG. 6B). The p-values for the clarified fecal lysate samples are slightly improved over the RNA extracted/purified stool sample set, thus confirming that direct detection of 16S and 18S rRNA by a homogeneous assay can be achieved on human stool samples independent of a nucleic acid extraction procedure. Thus, direct detection of one or more rRNA populations in a sample can be performed according to the methods disclosed herein without negatively impacting quantitation of the one or more rRNA populations in the sample. Direct lysate testing of samples results in the production of equivalent levels of rRNA detection (for one or more rRNA populations) as compared to the level of rRNA detection of corresponding rRNA populations generated by methods including an amplification or purification procedure.

Example 4: Detection Of Various Forms Of rRNA In Human Stool Samples Using RT- qPCR

[0224] Though RT-qPCR is seen as a gold standard method, this multistep workflow may introduce bias due to loss of transcripts or mis-amplification of transcripts. Additionally, the assay requires a nucleic acid extraction step, a reverse transcriptase step, and a PCR amplification step, each of which may introduce bias. The nucleic acid extraction step may result in the loss of transcripts through incomplete homogenization, multiple wash steps and characteristics of the nucleic acid binding step (silica-membrane or magnetic beads). Reverse transcriptases are known to have issues with efficiency; and this may result in an inefficient conversion of RNA to cDNA template. Finally, preparing samples for qPCR requires dilution of the RNA template. Small fluctuations in the input material due to either dilution or intrinsic pipetting errors may result in large fluctuations in output signal after amplification. The issue of bias can also be compounded by stool -based or blood-based PCR inhibitors which are found at variable levels from specimen to specimen.

[0225] In order to evaluate the sensitivity of the direct or indirect detection of rRNA as provided in Examples 1-3, we compared the above HPA method against a comparative RT- qPCR assay. Accordingly, orthogonal RT-qPCR assays were run on the same 100 human stool samples (see, Example 1).

[0226] Nucleic acids were extracted from the stool (-200 mg) using Mo Bio Laboratories PowerMag^® Microbiome RNA/DNA isolation kit (Mo Bio Laboratories Inc., Carlsbad, CA, Catalog No. 27600-4-KF). The nucleic acid extracts were diluted and equal volume amounts were assessed on the Quantstudio Flex using the TaqMan^® RNA-to-CT™ l-Step Kit (Thermo Fisher Scientific, Waltham, CT, Catalog No. 4392938). Bacterial 16S rRNA was detected using a pan bacterial 16S qPCR Assay (Life Technologies, CA, Catalog ID: Ba04230899_sl), performed essentially accordingly to the manufacturer’s instructions. Human 18S rRNA was detected using a Human 18S rRNA qPCR Assay (Life Technologies, Inc., Carlsbad, CA, Catalog ID: A29349) performed essentially accordingly to the manufacturer’s instructions. Finally, fungal 18S rRNA was detected using a pan fungal 18S rRNA Assay (FungiQuant: A broad-coverage fungal quantitative real-time PCR assay, Liu el al. , BMC Microbiology , 12:255 (2012)). All samples regardless of target nucleic acid were run against a standard curve to quantify copy number (as described in Example 1).

Results:

[0227] FIG. 7A shows detection of bacterial 16S rRNA (measured as RT-PCR copies/mg of stool) for the 100 human stool samples, from healthy (i.e., samples obtained from subjects not having or suspected of having a gastrointestinal-related or liver-related condition), CD and UC subjects. FIG. 7A illustrates a statistical difference between normal and diseased stool sample sets (UC and CD) (CD, p=0.00l4 and UC, p= 0.0012). The p-values were calculated using the Mann-Whitney U test. FIG. 7B illustrates a statistically significant difference between normal (i.e., samples obtained from subjects not having or suspected of having a gastrointestinal-related or liver-related condition) and CD stool samples based on disease severity (active CD). When the CD data was parsed into active versus inactive disease based on endoscopy, there was a statistically significant difference between CD active (CDa) and normal sample sets (p=0.000l). FIG. 7C illustrates a statistically significant difference between normal (i.e., samples obtained from subjects not having or suspected of having a gastrointestinal-related or liver-related condition) and UC stool samples based on disease severity (active UC). When the UC data was parsed into active versus inactive disease based on endoscopy, there was a clear statistically significant difference between UC (active) and normal sample sets (p=0.003).

[0228] FIG. 8A shows detection of human 18S rRNA (measured as log copies/mg of stool) for the 100 human stool samples, from healthy (i.e., samples obtained from subjects not having or suspected of having a gastrointestinal-related or liver-related condition), CD and UC subjects. FIG. 8 A illustrates a statistical difference between normal and diseased stool sample sets (UC and CD) (CD, p=0.0004 and UC, p= 0.0169). The p-values were calculated using the Mann-Whitney U test. There was a statistically significant increase in 18S rRNA counts in both disease samples sets as compared to normal, healthy stool samples ( i.e ., samples obtained from subjects not having or suspected of having a gastrointestinal-related or liver-related condition) (consistent with the findings of Example 1). FIG. 8B illustrates a statistically significant difference between normal and CD stool samples based on disease severity (active or inactive CD). When the CD data was parsed into active versus inactive disease based on endoscopy, there was a statistically significant difference between CD active (CDa) and normal sample sets (p=<0.000l). FIG. 8C illustrates a statistically significant difference between normal and UC stool samples based on disease severity (active UC). When the UC data was parsed into active versus inactive disease based on endoscopy, there was a clear statistically significant difference between UC (active) and normal sample sets (p=<0.000l).

[0229] FIG. 9A shows detection of fungal 18S rRNA (measured as RT-PCR copies/mg of stool) for the 100 human stool samples, from healthy (i.e., samples obtained from subjects not having or suspected of having a gastrointestinal-related or liver-related condition), CD and UC subjects. FIG. 9A illustrates a statistical difference between normal and diseases stool sample sets (UC and CD) (CD, p=0.0006 and UC, p= 0.0018). The p-values were calculated using the Mann-Whitney U test. FIG. 9B illustrates a statistically significant difference between normal and CD stool samples based on disease severity (active CD). When the CD data was parsed into active versus inactive disease based on endoscopy, there was a statistically significant difference between CD active (CDa) and normal sample sets (p=0.0043). FIG. 9C illustrates a statistically significant difference between normal and UC stool samples based on disease severity (active UC). When the UC data was parsed into active versus inactive disease based on endoscopy, there was a clear statistically significant difference between UC (active) and normal sample sets (p=0.000l).

[0230] These results confirm that the data obtained using direct or indirect HPA to detect various forms of rRNA in stool samples is similar to data obtained using the industry gold standard, RT-qPCR. The novel methods for rRNA detection and quantification disclosed herein do not require nucleic acid extraction or purification steps, nor do they require additional manipulations, such as RT-PCR, which runs the risk of introducing bias. Example 5: Ratio Of Human 18S rRNA To Bacterial 16S rRNA As An Indicator Of Active CD Or UC Disease Based On RT-qPCR

[0231] The data from Example 4 was manipulated to produce a ratio of human 18S rRNA to bacterial 16S rRNA. FIG. 10A shows a plot of the ratio of human 18S rRNA to bacterial 16S rRNA (ratio 18S/16S) for the 100 human stool samples from Example 1. FIG. 10A illustrates a statistical difference between normal and disease stool sample sets based on the ratio of human rRNA to bacterial rRNA (ETC and CD) (CD, p=0.0003 and UC, p= 0.0009). The p-values were calculated using the Mann-Whitney U test. When the human 18S rRNA to bacterial 16S rRNA ratio data was parsed into active versus inactive disease based on endoscopy, there was a strong statistically significant difference between CD active (CDa) and normal sample sets (p=<0.000l) (FIG. 10B). When the human 18S rRNA to bacterial 16S rRNA ratio data was parsed into active versus inactive disease based on endoscopy, there was a strong statistically significant difference between UC active (UCa) and normal sample sets (p=<0.000l) (FIG. 10C).

[0232] Accordingly, the methods disclosed herein provide reliable and accurate methods to detect, quantify, and discriminate between different types of rRNA (rRNA populations) present in a biological sample without the need for a purification step or subsequent downstream processing prior to HPA analysis.

[0233] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, one of skill in the art will appreciate that many modifications and variations of this invention can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. The specific embodiments described herein are offered by way of example only and are not meant to be limiting in any way. It is intended that the specification and examples be considered as exemplary only, with the true scope and spirit of the invention being indicated by the following claims.

[0234] All publications, patents, patent applications or other documents cited herein are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, or other document was individually indicated to be incorporated by reference for all purposes. INFORMAL SEQUENCE LISTING

SEQ ID NO: l (human 18S rRNA probe):

5’ -CGGAACCCAAAGACTTT{X}GGTTTCCCGG-3’

{X} is a RXL phosphoramidite conjugation between nucleotides 17 and 18

SEQ ID NO:2 (bacterial 16S rRNA probe):

5’-GCTCGTTGCGGGACTT{X}AACCCAACAT-3’

{X} is a RXL phosphoramidite conjugation between nucleotides 16 and 17 SEQ ID NO : 3 (fungal 18 S rRNA probe) :

5’-CCCGACCGTCCCT {X} ATTAATCATTACGATGG-3’

{X} is a RXL phosphoramidite conjugation between nucleotides 13 and 14

Claims

WHAT IS CLAIMED IS:

1. An in vitro method for analyzing a sample from a subject to determine the relative abundance of the subject’s rRNA in the sample, the method comprising:

(a) determining the abundance of the subject’s 18S rRNA in the sample; and

(b) comparing the abundance of the subject’s 18S rRNA in the sample to a reference value, thereby determining the relative abundance of the subject’s

18S rRNA in the sample.

2. The method of claim 1, further comprising:

(c) determining the abundance of bacterial 16S rRNA in the sample; and

(d) comparing the abundance of the bacterial 16S rRNA in the sample to the reference value, thereby determining the relative abundance of the bacterial 16S rRNA in the sample.

3. The method of claim 1 or 2, further comprising:

(e) determining the abundance of fungal 18S rRNA in the sample; and

(f) comparing the abundance of the fungal 18S rRNA in the sample to the reference value, thereby determining the relative abundance of the fungal 18S rRNA in the sample.

4. The method of any one of claims 1, 2, or 3, wherein the subject has or is suspected of having a gastrointestinal -related or liver-related condition.

5. The method of any one of claims 1 to 4, further comprising determining an amount of calprotectin in the subject’s sample.

6. The method of any one of claims 1 to 5, wherein the abundance of 16S or 18S rRNA in the subject’s sample is determined by hybridization protection assay (HP A).

7. The method of any one of claims 1 to 6, wherein the sample comprises a stool sample, wherein the stool sample comprises a stool lysate or ribonucleic acids isolated from the stool sample.

8. The method of any one of claims 1 to 7, wherein the determining step is performed directly on the sample in the absence of a purification step.

9. The method of any one of claims 1 to 8, wherein the method does not include amplifying any portion of the rRNA present in the subject’s sample prior to the determining step.

10. The method of any one of claims 1 to 9, further comprising classifying the subject as having an active or inactive inflammatory bowel disease based on the relative abundance of the subject’s 18S rRNA in the sample.

11. The method of any one of claims 2 to 9, further comprising classifying the subject as having an active or inactive inflammatory bowel disease based on the relative abundance of the bacterial 16S rRNA in the sample.

12. The method of any one of claims 3 to 9, further comprising classifying the subject as having an active or inactive inflammatory bowel disease based on the relative abundance of the fungal 18S rRNA in the sample.

13. The method of any one of claims 1 to 12, wherein said subject is a human.

14. The method of any one of claims 1 to 13, wherein the subject has been diagnosed with Crohn’s Disease (CD) or Ulcerative Colitis (UC).

15. The method of any one of claims 1 to 13, wherein the subject has inactive Crohn’s Disease (CD) or inactive Ulcerative Colitis (UC).

16. The method of any one of claims 1 to 13, wherein the subject has active Crohn’s Disease (CD) or active Ulcerative Colitis (UC).

17. The method of any one of claims 1 to 13, wherein the subject has non alcoholic steatohepatitis (NASH), liver cancer, liver cirrhosis, autoimmune hepatitis or non alcoholic fatty liver disease (NAFLD).

18. The method of any one of claims 1 to 17, wherein the reference value comprises a median or mean value for abundance of host 18S rRNA, bacterial 16S rRNA or fungal 18S rRNA from a cohort of samples obtained from a subject not having or suspected of having a gastrointestinal-related or liver-related condition.

19. The method of any one of claims 1 to 17, wherein the reference value comprises a median or mean value selected from the group consisting of abundance of human 18S rRNA from a cohort of samples obtained from a subject not having or suspected of having a gastrointestinal-related or liver-related condition, abundance of bacterial 16S rRNA from a cohort of samples obtained from a subject not having or suspected of having a gastrointestinal-related or liver-related condition, abundance of fungal 18S rRNA from a cohort of samples obtained from a subject not having or suspected of having a gastrointestinal-related or liver-related condition, abundance of human 18S rRNA from a cohort of samples from subjects diagnosed with a gastrointestinal-related or liver-related condition, abundance of bacterial 16S rRNA from a cohort of samples from subjects diagnosed with a gastrointestinal-related or liver-related condition, and abundance of fungal 18S rRNA from a cohort of samples from subjects diagnosed with a gastrointestinal -related or liver-related condition.

20. A method for detecting human and microbiome ribosomal RNA changes associated with a gastrointestinal-related or liver-related condition, the method comprising:

21. The method of claim 20, wherein the first and second datasets are applied to calculate a human 18S rRNA:bacterial 16S rRNA ratio.

22. The method of claim 20, wherein the first and third datasets are applied to calculate a human 18S rRNATungal 18S rRNA ratio.

23. The method of claim 21 or 22, wherein the human 18S rRNA:bacterial 16S rRNA ratio and/or the human 18S rRNATungal 18S rRNA ratio is indicative of the subject having or developing a gastrointestinal-related or liver-related condition.

24. The method of any one of claims 20 to 23, wherein the gastrointestinal-related or liver-related condition is selected from the group consisting of Crohn’s Disease (CD), Ulcerative Colitis (UC), non-alcoholic steatohepatitis (NASH), liver cancer, liver cirrhosis, autoimmune hepatitis, non-alcoholic fatty liver disease (NAFLD), Celiac disease and Graft versus Host Disorder (GvHD).

25. The method of claim 20, wherein the method distinguishes between an active or inactive gastrointestinal-related or liver-related condition based on the abundance of the human 18S rRNA in the sample.

26. The method of claim 20, wherein the method distinguishes between an active or inactive gastrointestinal-related or liver-related condition based on the abundance of the bacterial 16S rRNA in the sample.

27. The method of claim 20, wherein the method distinguishes between an active or inactive gastrointestinal-related or liver-related condition based on the abundance of the fungal 18S rRNA in the sample.

28. The method of claim 20, wherein the method identifies an active gastrointestinal-related or liver-related condition by detecting an increase in the abundance of human 18S rRNA in the subject’s sample as compared to the abundance of human 18S rRNA in a sample not having or suspected of having a gastrointestinal-related or liver-related condition, tested under the same conditions.

29. The method of claim 21, wherein the method distinguishes between an active or inactive gastrointestinal-related or liver-related condition based on the human 18S rRNA:bacterial 16S rRNA ratio.

30. The method claim 21, wherein the method distinguishes between an active inflammatory bowel disease and a sample not having or suspected of having a gastrointestinal-related or liver-related condition based on the human 18S rRNA:bacterial 16S rRNA ratio.

31. The method of any one of claims 20 to 30, wherein the first, second, and third assays each comprise a hybridization protection assay (HP A).

32. The method of any one of claims 20 to 30, wherein the first and second assays comprise a hybridization protection assay (HP A) and the third assay comprises a qPCR assay.

33. The method of any one of claims 20 to 32, wherein the third assay comprises amplifying at least a portion of the fungal 18S rRNA prior to performing the third assay.

34. The method of any one of claims 20 to 33, wherein the first assay comprises:

(b) hybridizing the first acridinium ester labeled-DNA probe to a target rRNA present in the subject’s sample or ribonucleic acids isolated from the subject’s sample;

(c) separating the hybridized first acridinium ester labeled-DNA probe from unhybridized or mismatched acridinium ester labeled-DNA probe; and

35. The method of any one of claims 20 to 34, wherein the second assay comprises:

(a) contacting the subject’s sample or ribonucleic acids isolated from the subject’s sample with a second acridinium ester labeled-DNA probe;

(b) hybridizing the second acridinium ester labeled-DNA probe to a target rRNA present in the subject’s sample or ribonucleic acids isolated from the subject’s sample;

(c) separating the hybridized second acridinium ester labeled-DNA probe from unhybridized or mismatched acridinium ester labeled-DNA probe; and

36. The method of any one of claims 20 to 35, wherein the third assay comprises:

(b) hybridizing the third acridinium ester labeled-DNA probe to a target rRNA present in the subject’s sample or ribonucleic acids isolated from the subject’s sample;

(c) separating the hybridized third acridinium ester labeled-DNA probe from unhybridized or mismatched acridinium ester labeled-DNA probe; and (d) detecting a chemiluminescent signal from the hybridized third acridinium ester labeled-DNA probe under chemiluminescent conditions.

37. The method of any one of claims 20 to 36, wherein the first, second and third assays are performed sequentially, simultaneously, or independently on the subject’s sample.

38. The method of claim 34, wherein the first acridinium ester labeled-DNA probe comprises a human 18S rRNA probe.

39. The method of claim 35, wherein the second acridinium ester labeled-DNA probe comprises a pan bacterial 16S rRNA probe.

40. The method of claim 36, wherein the third acridinium ester labeled-DNA probe comprises a pan fungal 18S rRNA probe.

41. The method of claim 34, wherein the first acridinium ester labeled-DNA probe comprises the sequence set forth in SEQ ID NO: 1.

42. The method of claim 35, wherein the second acridinium ester labeled-DNA probe comprises the sequence set forth in SEQ ID NO:2.

43. The method of claim 36, wherein the third acridinium ester labeled-DNA probe comprises the sequence set forth in SEQ ID NO:3.

44. The method of any one of claims 34 to 43, wherein the separating comprises hydrolyzing the unhybridized or mismatched acridinium ester-labeled DNA probe.

45. The method of claim 20, wherein the method detects or predicts inflammatory bowel disease based on the abundance of human 18S rRNA, the abundance of bacterial 16S rRNA or the abundance of fungal 18S rRNA in the subject’s sample.

46. A method for testing the efficacy of a clinical treatment in a human subject having a gastrointestinal-related or liver-related condition, the method comprising:

(b) calculating a human 18S rRNA:bacterial 16S rRNA ratio and/or a human 18S rRNATungal 18S rRNA ratio based on the abundance of humanl8S rRNA, bacterial 16S rRNA and fungal 18S rRNA present in the pre-clinical treatment and post-clinical treatment samples; and

(c) determining if the clinical treatment provides efficacy for the gastrointestinal- related or liver-related condition in the human subject based on the humanl8S rRNA:bacterial 16S rRNA ratio and/or the human 18S rRNA:fungal 18S rRNA ratio.

47. The method of claim 46, wherein the clinical treatment provides efficacy for the gastrointestinal-related or liver-related condition in the human subject if the post-clinical treatment human 18S rRNA:bacterial 16S rRNA ratio is lower than the pre-clinical treatment human 18S rRNA:bacterial 16S rRNA ratio.

48. The method of claim 46 or 47, wherein the clinical treatment is an antibiotic, aminosalicylate, corticiosteroid, immunosuppressant or monoclonal antibody-based therapy.

49. A method for predicting or identifying a gastrointestinal-related or liver- related condition in a human subject, the method comprising:

(a) performing a first hybridization protection assay to detect human 18S rRNA in a sample from the human subject or ribonucleic acids isolated from the human subject’s sample;

(f) identifying the subject as having a gastrointestinal-related or liver-related condition or predicting the subject to develop a gastrointestinal -related or liver-related condition based on the ratio of step (e).

50. The method of claim 49, further comprising:

(g) performing a third hybridization protection assay to detect fungal 18S rRNA in the subject’s sample or ribonucleic acids isolated from the subject’s sample; (h) calculating the relative abundance of fungal 18S rRNA in the subject’s sample based on the assay of step (g);

(i) determining a ratio of human 18S rRNATungal 18S rRNA based on the values obtained in steps (b) and (h); and

(j) identifying the subject as having a gastrointestinal -related or liver-related condition or predicting the subject to develop a gastrointestinal -related or liver-related condition based on the ratio of step (i).

51. The method of claim 50, wherein the first, second and third hybridization protection assays are performed sequentially, simultaneously, or independently on the subject’s sample.

52. The method of claim 50 or 51, wherein the first, second and third hybridization protection assays comprise acridinium ester labeled-DNA probes.

53. The method of any one of claims 49 to 52, wherein the first hybridization protection assay comprises an acridinium ester labeled-DNA probe capable of hybridizing to human 18S rRNA.

54. The method of any one of claims 49 to 53, wherein the second hybridization protection assay comprises an acridinium ester labeled-DNA probe capable of hybridizing to bacterial 16S rRNA.

55. The method of claim 50 to 54, wherein the third hybridization protection assay comprises an acridinium ester labeled-DNA probe capable of hybridizing to fungal 18S rRNA.

56. The method of claim 53, wherein the acridinium ester labeled-DNA probe comprises the sequence set forth in SEQ ID NO: 1.

57. The method of claim 54, wherein the acridinium ester labeled-DNA probe comprises the sequence set forth in SEQ ID NO:2.

58. The method of claim 55, wherein the acridinium ester labeled-DNA probe comprises the sequence set forth in SEQ ID NO:3.

59. The method of any one of claims 49 to 58, wherein the gastrointestinal-related or liver-related condition is identified or predicted when the human 18S rRNA:bacterial 16S rRNA ratio in the subject’s sample is equal to, or greater than, two standard deviations above a human 18S rRNA:bacterial 16S rRNA mean ratio obtained from a population of samples obtained from subjects not having or not suspected of having a gastrointestinal -related or liver-related condition, under the same conditions.

60. The method of any one of claims 49 to 58, wherein the gastrointestinal-related or liver-related condition is identified or predicted when the human 18S rRNA:bacterial 16S rRNA ratio in the subject’s sample is about 3.5-fold greater than a humanl8S rRNA:bacterial 16S rRNA mean ratio obtained from a population of samples obtained from subjects not having or suspected of having a gastrointestinal-related or liver-related condition, under the same conditions.

61. A kit compri sing :

(b) a second oligonucleotide having a nucleic acid sequence that is complementary to a region of bacterial 16S rRNA; and optionally,

(c) a third oligonucleotide having a nucleic acid sequence that is complementary to a region of fungal 18S rRNA.

62. The kit of claim 61, wherein the first, second and/or third oligonucleotide each comprise an acridinium ester.

63. The kit of claim 61 or 62, comprising:

(a) an oligonucleotide comprising SEQ ID NO: l

(b) an oligonucleotide comprising SEQ ID NO:2; and optionally,

(c) an oligonucleotide comprising SEQ ID NO:3.

64. The kit of any one of claims 61 to 63, further comprising a cell lysis buffer and/or rRNA purification reagent.

65. The method of any one of claims 1 to 6 or 10 to 60, wherein the sample is selected from the group consisting of a stool sample, a blood sample, a serum sample, a liver tissue sample, a gastrointestinal biopsy sample, and a urine sample.

66. The method of any one of claims 1 to 6 or 10 to 60, wherein the sample is a stool sample.

67. The method of any one of claims 20 to 31 or 34 to 45, wherein performing the first, second and third assay does not include amplifying any population of rRNA prior to determining the abundance of the human subject’s 18S rRNA, the abundance of the bacterial 16S rRNA or the abundance of the fungal 18S rRNA in the sample.

68. The method of any one of claims 49 to 60, wherein at least one of the first, second and third hybridization protection assays includes amplifying at least a portion of a rRNA population prior to calculating the relative abundance of the amplified rRNA in the sample.

69. The method of any one of claims 20 to 45, wherein the sample is not purified prior to the determining step.

70. The method of any one of claims 49 to 60, wherein the sample is not purified prior to performing any one of the performing steps.

71. The method of claim 39, wherein the pan bacterial 16S rRNA probe is homologous to at least two species or genus of bacteria.

72. The method of claim 40, wherein the pan fungal 18S rRNA probe is homologous to at least two species or genus of fungi.

73. The method of any one of claims 49 to 60, wherein the subject has Crohn’s Disease, Ulcerative Colitis, non-alcoholic steatohepatitis (NASH), liver cancer, liver cirrhosis, autoimmune hepatitis, or non-alcoholic fatty liver disease (NAFLD).

74. The method of any one of claims 1 to 17 or 20 to 45, wherein the reference value comprises an internal control.

75. The method of any one of claims 1 to 17 or 20 to 45, wherein the reference value comprises determining the abundance of a first rRNA population in the sample and comparing the abundance of the first rRNA population against an abundance of a second rRNA population in the sample.

76. The method of any one of claims 1 to 17 or 20 to 45, wherein the reference value comprises a ratio of human 18S rRNA to bacterial 16S rRNA, a ratio of bacterial 16S rRNA to human 18S rRNA, a ratio of fungal 18S rRNA to human 18S rRNA, or a ratio of human 18S rRNA to fungal 18S rRNA.

77. The method of claim 76, wherein the reference value is obtained from a sample having or suspected of having a gastrointestinal-related or liver-related condition.

78. The method of claim 76, wherein the reference value is obtained from a sample not having nor suspected of having a gastrointestinal-related or liver-related condition.

79. The method of any one of claims 1 to 17 or 20 to 45, wherein the reference value comprises a ratio, wherein the ratio compares a first rRNA population in a first sample with the first rRNA population in a second sample.

80. The method of claim 79, wherein the ratio is the abundance of human 18S rRNA, fungal 18S rRNA, bacterial 16S rRNA or a combination thereof.

81. The method of any one of claims 1 to 17 or 20 to 45, wherein the reference value comprises a numerical range representative of a first rRNA population in the sample.

82. The method of claim 81, wherein the numerical range is representative of human 18S rRNA, fungal 18S rRNA or bacterial 16S rRNA in the sample.