WO2022032282A1

WO2022032282A1 - Methods and reagents for microbiome analysis

Info

Publication number: WO2022032282A1
Application number: PCT/US2021/071100
Authority: WO
Inventors: John Matthew HAGGERTY; Benjamin John TULLY; Christopher Hale CORZETT; Steven Eric FINKEL; Molly B. Schmid; Sapna Shah JAIN
Original assignee: Alfred E. Mann Institute For Biomedical Engineering At The University Of Southern California
Priority date: 2020-08-06
Filing date: 2021-08-04
Publication date: 2022-02-10

Abstract

This disclosure relates to cost-effective and rapid diagnostic kits to stratify patient populations based on their microbiome profiles. This disclosure also relates to a diagnostic kit comprising synthetic primers. This disclosure also relates to synthetic nucleic acid primers. This disclosure also relates to methods of developing these synthetic primers. This disclosure further relates to products and methods that allow medical staff and physicians to differentiate patients that are more or less-susceptible to developing gastro-intestinal toxicity after chemotherapy and/or radiation-therapy.

Description

PCT PATENT APPLICATION

For

METHODS AND REAGENTS FOR MICROBIOME ANALYSIS

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of U.S. provisional patent application 63/061 ,996, entitled “Methods and Reagents for Microbiome Analysis,” filed August 6, 2020, attorney docket number AMISC.017PR. The entire content of this application is hereby expressly incorporated herein by reference in its entirety.

REFERENCE TO SEQUENCE LISTING

[0002] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled SeqListingAMISC017WO.TXT, which was created and last modified on August 4, 2021 and is 36,162 bytes in size. The information in the electronic Sequence Listing is hereby expressly incorporated by reference in in its entirety.

BACKGROUND

TECHNICAL FIELD

[0003] Embodiments of the present disclosure relates to cost-effective and rapid methods, and reagents (e.g., diagnostic kits) to stratify patient populations based on their microbiome profiles. This disclosure also relates to the synthetic nucleic acid primers. This disclosure also relates to diagnosis kits comprising such primers. This disclosure further relates to products and methods that allow medical staff and physicians to differentiate patients that are more or less-susceptible to developing gastro-intestinal toxicity after chemotherapy and/or radiation-therapy.

DESCRIPTION OF RELATED ART

[0004] The human microbiome is the collection of all the microorganisms and their genetic material that live on and within the human body. It is estimated that we have 10 times as many microbial cells as human cells, with majority of them living in our gut. Our microbiome is composed of a diverse array of microorganisms such as bacteria, archaea, fungi and viruses; with bacteria making up the majority of the population. The makeup of the microbiome can vary between healthy and diseased states of a human. [0005] Numerous research studies over the last decade have shown that the human microbiome, especially the bacterial composition and molecules secreted by them, may differentiate between healthy and disease states in conditions such as cancer, gastrointestinal illnesses such as irritable bowel disease, obesity, autoimmune conditions, diabetes, neurological diseases, and others. This dysbiotic microbiome profile has the potential to be used as a prognostic and/or diagnostic tool for various human diseases, and rapid detection and/or quantification of these target bacteria would allow doctors and patients to make personal health care-based recommendations.

[0006] Inflammation and ulceration of the mucosal membranes, such as oral or gastrointestinal mucosal membranes, is a common side effect of radio-or chemotherapy regimens and generally termed mucositis. Symptoms include pain, ulceration, bloating, nausea/vomiting, diarrhea, and constipation. Cytotoxic chemotherapy regimens cause gastrointestinal mucositis (GIM) in nearly 40% of standard dose chemotherapy patients [1 ], and 100% of high dose or radiation therapies for head/neck cancers [2], In 1 -10% of cases, these side effects are sufficiently severe that they interfere with normal nutrition [3], and can cause longer hospitalizations, increased costs, increased opioid use for pain management, and often limit or prevent continuation of an optimal chemo- or radio-therapy regimen. The incremental cost of oral mucositis in patients treated for head/neck cancer was estimated at >$17K per patient [1].

[0007] Stringer et al. demonstrated a correlation between dysbiosis of the microbiome and the onset of mucositis [4], Also, Montassier et al. disclosed changes in microbiomes of 28 patients receiving chemotherapy, some of whom acquired blood stream infections (BSI) [5]. Montassier identified a set of 13 bacterial taxa (uncultured Barnesiellaceae, uncultured Christensenellaceae, Dehalobacterium, uncultured RF39, Butytricimonas, Chritsensenella, Oscillospira, uncultured Erysipelotrichaceae, Faecalibacterium, Oxalobacter, Sutterella, Veillonella, Desulfovibrio), whose relative abundances distinguished the patients who developed BSI from those who did not. RELATED ART REFERENCES

[0008] The following publications are related art for the background of this disclosure. One-digit or two-digit numbers in the box brackets before each reference, correspond to the numbers in the box brackets used in the other parts of this disclosure. Each of these publications is incorporated herein in their entirety.

[1] Nonzee NJ, Dandade NA, Patel U, Markossian T, Agulnik M, Argiris A, Patel JD, Kern RC, Munshi HG, Calhoun EA, Bennett CL. 2008. Evaluating the supportive care costs of severe radiochemotherapy-induced mucositis and pharyngitis : results from a Northwestern University Costs of Cancer Program pilot study with head and neck and nonsmall cell lung cancer patients who received care at a county hospital, a Veterans Administration hospital, or a comprehensive cancer care center. Cancer 113:1446-1452.

[2] Peterson DE, Boers-Doets CB, Bensadoun RJ, Herrstedt J, Committee EG. 2015. Management of oral and gastrointestinal mucosal injury: ESMO Clinical Practice Guidelines for diagnosis, treatment, and follow-up. Ann Oncol 26 Suppl 5:v139-151.

[3] Peterson DE, Bensadoun RJ, Roila F, Group EGW. 2011 . Management of oral and gastrointestinal mucositis: ESMO Clinical Practice Guidelines. Ann Oncol 22 Suppl 6:vi78-84.

[4] Stringer AM, Al-Dasooqi N, Bowen JM, Tan TH, Radzuan M, Logan RM, Mayo B, Keefe DM, Gibson RJ. 2013. Biomarkers of chemotherapy-induced diarrhoea: a clinical study of intestinal microbiome alterations, inflammation and circulating matrix metalloproteinases. Support Care Cancer 21 :1843- 1852.

[5] Montassier, E., Al-Ghalith, G.A., Ward, T. et al. Pretreatment gut microbiome predicts chemotherapy-related bloodstream infection. Genome Med 8, 49 (2016).

[6] Leonardo S, Toldra A, Campas M. Biosensors Based on Isothermal DNA Amplification for Bacterial Detection in Food Safety and Environmental Monitoring. Sensors (Basel). 2021 ;21 (2):602. Published 2021 Jan 16. [7] Gupta N. DNA Extraction and Polymerase Chain Reaction. J CytoL 2019 ;36(2) : 116-117. doi : 10.4103/JOC.JOC_110_18.

SUMMARY

[0009] This disclosure relates to cost-effective and rapid methods, and reagents (e.g., diagnostic kits) to stratify patient populations based on their microbiome profiles. This disclosure also relates to the synthetic nucleic acid primers. This disclosure also relates to diagnosis kits comprising such primers. This disclosure further relates to products and methods that allow medical staff and physicians to differentiate patients that are more or less-susceptible to developing gastro-intestinal toxicity after chemotherapy and/or radiation-therapy.

[0010] This disclosure relates to a method for determination of presence/absence and/or relative/absolute abundance of a target taxon present in a microbiome sample. One such method may include obtaining the microbiome sample that potentially comprises at least one nucleic acid; having a diagnostic kit comprising at least one synthetic primer pair designed specifically for the determination of presence/absence and/or relative/absolute abundance of the target taxon; processing the microbiome sample to have the at least one nucleic acid accessible to react with the at least one synthetic primer pair, thereby forming a processed sample; mixing the processed sample with the diagnostic kit to form a microbiome reaction mixture; subjecting the microbiome reaction mixture to a reaction under which at least one section of the accessible nucleic acid is amplified, thereby forming a reaction product; analyzing the reaction product; and determining presence/absence and/or relative/absolute abundance of the target taxon in the microbiome sample. In this exemplary method, the at least one synthetic primer pair may include a synthetic forward primer and a synthetic reverse primer. In this exemplary method, the at least one synthetic primer pair may be selected from the synthetic primer pairs of FIG. 15, Primer Pair Set ID numbers 1 to 79, or a combination thereof. In some embodiments, the synthetic forward/re verse primers with primer name numbers 1 to 170 that constitute the synthetic primer pairs of FIG. 15, Primer Pair Set IDs 1 to 79 have sequences, target genes, target regions, and target locations disclosed in FIG. 14, 19. In some embodiments, the at least one synthetic primer pair may be selected from the synthetic primer pairs of FIG. 15, Primer Pair Set ID numbers 1 to 79, or a combination thereof and the synthetic forward/re verse primers with primer name numbers 1 to 170 that constitute the synthetic primer pairs of FIG. 15, Primer Pair Set IDs 1 to 79 have sequences, target genes, target regions, and target locations disclosed in FIG. 14, 19.

[0011] In this disclosure, the target taxon may be any taxon of FIG. 15, or a combination thereof.

[0012] In some embodiments, the at least one synthetic primer pair may be selected from:

Primer Pair Set ID 1 : SEQ ID NO: 1 and 2;

Primer Pair Set ID 2: SEQ ID NO: 3 and 4;

Primer Pair Set ID 3: SEQ ID NO: 5 and 6;

Primer Pair Set ID 4: SEQ ID NO: 7 and 8;

Primer Pair Set ID 5: SEQ ID NO: 9 and 10;

Primer Pair Set ID 6: SEQ ID NO: 11 and 12;

Primer Pair Set ID 7: SEQ ID NO: 13 and 14;

Primer Pair Set ID 8: SEQ ID NO: 15 and 14;

Primer Pair Set ID 9: SEQ ID NO: 16 and 17;

Primer Pair Set ID 10: SEQ ID NO: 18 and 19;

Primer Pair Set ID 11 : SEQ ID NO: 20 and 19;

Primer Pair Set ID 12: SEQ ID NO: 21 and 22;

Primer Pair Set ID 13: SEQ ID NO: 23 and 24;

Primer Pair Set ID 14: SEQ ID NO: 25 and 24;

Primer Pair Set ID 15: SEQ ID NO: 26 and 24;

Primer Pair Set ID 16: SEQ ID NO: 27 and 24;

Primer Pair Set ID 17: SEQ ID NO: 28 and 29;

Primer Pair Set ID 18: SEQ ID NO: 30 and 29;

Primer Pair Set ID 19: SEQ ID NO: 31 and 29; Primer Pair Set ID 20: SEQ ID NO 30 and 29;

Primer Pair Set ID 21 : SEQ ID NO 32 and 33;

Primer Pair Set ID 22: SEQ ID NO 34 and 35;

Primer Pair Set ID 23: SEQ ID NO 36 and 37;

Primer Pair Set ID 24: SEQ ID NO 38 and 39;

Primer Pair Set ID 25: SEQ ID NO 40 and 42;

Primer Pair Set ID 26: SEQ ID NO 41 and 42;

Primer Pair Set ID 27: SEQ ID NO 43 and 45;

Primer Pair Set ID 28: SEQ ID NO 44 and 45;

Primer Pair Set ID 29: SEQ ID NO 46 and 48;

Primer Pair Set ID 30: SEQ ID NO 47 and 48;

Primer Pair Set ID 31 : SEQ ID NO 49 and 52;

Primer Pair Set ID 32: SEQ ID NO 50 and 52;

Primer Pair Set ID 33: SEQ ID NO 51 and 52;

Primer Pair Set ID 34: SEQ ID NO 49 and 53;

Primer Pair Set ID 35: SEQ ID NO 50 and 53;

Primer Pair Set ID 36: SEQ ID NO 51 and 53;

Primer Pair Set ID 37: SEQ ID NO 54 and 55;

Primer Pair Set ID 38: SEQ ID NO 54 and 56;

Primer Pair Set ID 39: SEQ ID NO 54 and 57;

Primer Pair Set ID 40: SEQ ID NO 58 and 59;

Primer Pair Set ID 41 : SEQ ID NO 60 and 61 ;

Primer Pair Set ID 42: SEQ ID NO 62 and 63;

Primer Pair Set ID 43: SEQ ID NO 64 and 65;

Primer Pair Set ID 44: SEQ ID NO 66 and 67;

Primer Pair Set ID 45: SEQ ID NO 68 and 69;

Primer Pair Set ID 46: SEQ ID NO 70 and 71 ; Primer Pair Set ID 47: SEQ ID NO 72 and 73;

Primer Pair Set ID 48: SEQ ID NO 74 and 75;

Primer Pair Set ID 49: SEQ ID NO 76 and 78;

Primer Pair Set ID 50: SEQ ID NO 77 and 78;

Primer Pair Set ID 51 : SEQ ID NO 79 and 80;

Primer Pair Set ID 52: SEQ ID NO 81 and 82;

Primer Pair Set ID 53: SEQ ID NO 91 and 92;

Primer Pair Set ID 54: SEQ ID NO 93 and 94;

Primer Pair Set ID 55: SEQ ID NO 95 and 96;

Primer Pair Set ID 56: SEQ ID NO 97 and 98;

Primer Pair Set ID 57: SEQ ID NO 99 and 100;

Primer Pair Set ID 58: SEQ ID NO 101 and 102;

Primer Pair Set ID 59: SEQ ID NO 126 and 127;

Primer Pair Set ID 60: SEQ ID NO 128 and 129;

Primer Pair Set ID 61 : SEQ ID NO 130 and 133;

Primer Pair Set ID 62: SEQ ID NO 131 and 133;

Primer Pair Set ID 63: SEQ ID NO 132 and 133;

Primer Pair Set ID 64: SEQ ID NO 130 and 134;

Primer Pair Set ID 65: SEQ ID NO 131 and 134;

Primer Pair Set ID 66: SEQ ID NO 132 and 134;

Primer Pair Set ID 67: SEQ ID NO 145 and 146;

Primer Pair Set ID 68: SEQ ID NO 147 and 148;

Primer Pair Set ID 69: SEQ ID NO 149 and 150;

Primer Pair Set ID 70: SEQ ID NO 151 and 152;

Primer Pair Set ID 71 : SEQ ID NO 153 and 154;

Primer Pair Set ID 72: SEQ ID NO 155 and 156;

Primer Pair Set ID 73: SEQ ID NO 157 and 158; Primer Pair Set ID 74: SEQ ID NO: 159 and 160;

Primer Pair Set ID 75: SEQ ID NO: 161 and 162;

Primer Pair Set ID 76: SEQ ID NO: 163 and 164;

Primer Pair Set ID 77: SEQ ID NO: 165 and 166;

Primer Pair Set ID 78: SEQ ID NO: 167 and 168; or

Primer Pair Set ID 79: SEQ ID NO: 169 and 170.

[0013] In this disclosure, the at least one synthetic primer pair may be selected from: FIG. 15, FIG. 15, Primer Pair Set IDs 7 to 9, 31 to 39, or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Butyricimonas; and/or Primer Pair Set IDs 1 to 3, 40 to 43, or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Barnesiellaceae; and/or FIG. 15, Primer Pair Set IDs 13 to 16, 21 to 22, or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Christensenellaceae; and/or FIG. 15, Primer Pair Set IDs 25 to 30, or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Christensenella; and/or FIG. 15, Primer Pair Set IDs 44 to 46, 67 to 69, or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Veillonella; and/or FIG. 15, Primer Pair Set IDs 47 to 48, 76 to 79, or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Sutterella; and/or FIG. 15, Primer Pair Set IDs 49 to 52, 76 to 79, or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Oxalobacter; and/or FIG. 15, Primer Pair Set IDs 53 to 58, 70 to 72, or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Desulfovibrio; and/or FIG. 15, Primer Pair Set IDs 59 to 66, 73 to 75, or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Faecalibacterium.

[0014] In this disclosure, the at least one synthetic primer pair may also be selected from: FIG. 15, Primer Pair Set IDs 7, 9, 31 to 39, or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Butyricimonas; and/or FIG. 15, Primer Pair Set IDs 2, 42, 43, or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Barnesiellaceae; and/or FIG. 15, Primer Pair Set ID 13, 15, 16, 21 to 22, or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Christensenellaceae; and/or FIG. 15, Primer Pair Set IDs 26, 28, 30, or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Christensenella; and/or FIG. 15, Primer Pair Set IDs 44 to 46, or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Veillonella; and/or FIG. 15, Primer Pair Set IDs 47 for determination of presence/absence and/or relative/absolute abundance of Sutterella; and/or FIG. 15, Primer Pair Set IDs 49 to 52, or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Oxalobacter; and/or FIG. 15, Primer Pair Set IDs 58, 71 , or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Desulfovibrio; and/or FIG. 15, Primer Pair Set IDs 59 to 66, 74, 75, or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Faecalibacterium.

[0015] This disclosure also relates to a synthetic primer pair that may include a synthetic forward primer and a synthetic reverse primer, each having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology to any one of the synthetic forward primers and/or the synthetic reverse primers of FIG. 15.

[0016] This disclosure also relates to a synthetic primer pair that may include a synthetic forward primer and a synthetic reverse primer, each having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology to any one of the synthetic forward primers and/or the synthetic reverse primers of SEQ ID NO: 1-170.

[0017] This disclosure also relates to a synthetic primer pair that may include a synthetic forward primer and a synthetic reverse primer, each having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology to any one of the synthetic forward primers of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 16, 18, 20, 21, 23, 25, 26, 27, 28, 30, 31, 32, 34, 36, 38, 40, 41, 43, 44, 46, 47, 49, 50, 51, 54, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 77, 79, 81, 91, 93, 95, 97, 99, 101, 126, 128, 130, 131, 132, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, and 169 and/or the synthetic reverse primers of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 17, 19, 22, 24, 29, 33, 35, 37, 39, 42, 45, 48, 52, 53, 55, 56, 57, 59, 61 , 63, 65, 67, 69, 71 , 73, 75, 78, 80, 82, 92, 94, 96, 98, 100, 102, 127, 129, 133, 134, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, and 170.

[0018] In this disclosure, the diagnostic kit may include a chemical assay suitable for a polymerase chain reaction (PCR). The microbiome reaction mixture may be subjected to a PCR condition under which, if the target taxon is present in the microbiome sample, the at least one nucleic acid of the target taxon is amplified, and may produce a fluorescence signal. The analyzing the reaction product may include measuring amount of fluorescence signal produced.

[0019] In this disclosure, the diagnostic kit may include a chemical assay suitable for a polymerase chain reaction (PCR). The microbiome reaction mixture may be subjected to a PCR condition under which, if the target taxon is present in the microbiome sample, the at least one nucleic acid of the target taxon is amplified. The analyzing the reaction product may include a gel electrophoresis method.

[0020] This disclosure also relates to a diagnostic kit that may include a synthetic primer pair for determination of presence/absence and/or relative/absolute abundance of a target taxon present in a microbiome sample, including at least one synthetic primer pair that may be selected from the synthetic primer pairs of FIG. 15, Primer Pair Set IDs 1 to 79, or a combination thereof.

[0021] In this disclosure, the microbiome sample may be any sample that may potentially include a nucleic acid. This nucleic acid may belong to target taxa of this disclosure. For example, the microbiome sample may include a soil sample, a bodily fluid/solid sample, a tissue sample, or a combination thereof. For example, the microbiome sample may include soil, stool, saliva, skin, nasal swab, vaginal swab, blood, urine, hair, oral swab, or a combination thereof.

[0022] This disclosure also relates to a nucleic acid that may have at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology to any one of the sequences of FIG. 14, 19, p0001 to p0170, or any combination thereof.

[0023] This disclosure also relates to a nucleic acid that may have at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology to any one of the sequences of SEQ ID NO: 1-170, or any combination thereof.

[0024] This disclosure also relates to a synthetic primer pair that may include a synthetic forward primer and/or a synthetic reverse primer, each having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology to any one of the synthetic forward primers and/or the synthetic reverse primers of FIG. 14, 15, 19, p0001 to p0170.

[0025] This disclosure also relates to a synthetic primer pair that may include a synthetic forward primer and/or a synthetic reverse primer, each having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology to any one of the synthetic forward primers and/or the synthetic reverse primers of SEQ ID NO: 1-170.

[0026] This disclosure also relates to a synthetic primer pair that may include a synthetic forward primer and/or a synthetic reverse primer, each having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology to any one of the synthetic forward primers of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 16, 18, 20, 21, 23, 25, 26, 27, 28, 30, 31, 32, 34, 36, 38, 40, 41, 43, 44, 46, 47, 49, 50, 51, 54, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 77, 79, 81, 91, 93, 95, 97, 99, 101, 126, 128, 130, 131, 132, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, and 169 and/or the synthetic reverse primers of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 17, 19, 22, 24, 29, 33, 35, 37, 39, 42, 45, 48, 52, 53, 55, 56, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 78, 80, 82, 92, 94, 96, 98, 100, 102, 127, 129, 133, 134, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, and 170.

[0027] This disclosure also relates to a method of treatment of a patient. Such method may include obtaining a microbiome sample from the patient, wherein the microbiome sample potentially comprises at least one nucleic acid; having a diagnostic kit comprising at least one synthetic primer pair designed specifically for the identification of the target taxon; processing the microbiome sample to have the at least one nucleic acid accessible to react with the at least one synthetic primer pair, thereby forming a processed sample; mixing the processed sample with the diagnostic kit to form a microbiome reaction mixture; subjecting the microbiome reaction mixture to a reaction under which at least one section of the accessible nucleic acid is amplified, thereby forming a reaction product; analyzing the reaction product; determining presence/absence and/or relative/absolute abundance of the target taxon in the microbiome sample, thereby forming a taxon profile of the patient; comparing the taxon profile of the patient with the taxon profile of a healthy subject; determining whether the patient has a disease; and treating the patient if the patient has the disease. The at least one synthetic primer pair may include a synthetic forward primer and a synthetic reverse primer; and wherein the at least one synthetic primer pair may be selected from the synthetic primer pairs of FIG. 15, Primer Pair Set IDs 1 to 79, or a combination thereof.

[0028] In this disclosure, the synthetic forward primer and/or the synthetic reverse primer may be a polymerase chain reaction (PCR) primer.

[0029] In this disclosure, the patient is a human or an animal. The healthy subject may be human if the patient is human. Or the healthy subject may be an animal if the patient is an animal.

[0030] In this disclosure, the patient is more or less-susceptible to developing gastrointestinal toxicity after chemotherapy and/or radiation-therapy.

[0031] In this disclosure, the diagnostic kit may be configured to differentiate patients that are more or less-susceptible to developing gastro-intestinal toxicity after chemotherapy and/or radiation-therapy.

[0032] In this disclosure, amplifying the nucleic acid may include amplifying the nucleic acid with a PCR technique or a qPCR technique.

[0033] In this disclosure, the microbiome sample may be a microbiome sample from a patient.

[0034] Methods of this disclosure may be used to identify and categorize subjects as more or less susceptible to gastrointestinal toxicity. [0035] Methods of this disclosure may be used to identify and categorize subjects as more or less susceptible to gastrointestinal toxicity after chemotherapy and/or radiationtherapy.

[0036] In this disclosure, determining the presence/absence and/or relative/absolute abundance may include measuring a fluorescence signal during the amplifying step.

[0037] The methods of this disclosure may further include administering a therapeutic to the patient.

[0038] The methods of this disclosure may further include administering a therapeutic to the patient, and wherein the therapeutic is not chemotherapy and/or radiation.

[0039] Any combination of kits, products, and methods disclosed above or otherwise herein is within the scope of the instant disclosure.

[0040] These, as well as other components, steps, features, objects, benefits, and advantages, will now become clear from a review of the following detailed description of illustrative embodiments, the accompanying drawings, and the claims.

[0041] Exemplary embodiments of the present disclosure are provided in the following numbered alternatives:

[0042] 1 . A method for determination of presence/absence and/or relative/absolute abundance of a target taxon present in a microbiome sample, comprising: obtaining the microbiome sample that potentially comprises at least one nucleic acid; having a diagnostic kit comprising at least one synthetic primer pair designed specifically for the determination of presence/absence and/or relative/absolute abundance of the target taxon; processing the microbiome sample to have the at least one nucleic acid accessible to react with the at least one synthetic primer pair, thereby forming a processed sample; mixing the processed sample with the diagnostic kit to form a microbiome reaction mixture; subjecting the microbiome reaction mixture to a reaction under which at least one section of the accessible nucleic acid is amplified, thereby forming a reaction product; analyzing the reaction product; and determining presence/absence and/or relative/absolute abundance of the target taxon in the microbiome sample; wherein: the at least one synthetic primer pair comprises a synthetic forward primer and a synthetic reverse primer; the at least one synthetic primer pair is selected from the synthetic primer pairs of FIG. 15, Primer Pair Set ID numbers 1 to 79, or a combination thereof.

[0043] 2 .The method of Claim 1 , wherein the synthetic forward/re verse primers with primer name numbers 1 to 170 that constitute the synthetic primer pairs of FIG. 15, Primer Pair Set IDs 1 to 79 have sequences, target genes, target regions, and target locations disclosed in FIG. 14, 19.

[0044] 3. The method of Claim 1 or 2, wherein the target taxon is any taxon of FIG. 15, or a combination thereof.

[0045] 4. The method of any one of Claims 1 -3, wherein the at least one synthetic primer pair is selected from:

FIG. 15, Primer Pair Set IDs 7 to 9, 31 to 39, or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Butyricimonas; and/or

FIG. 15, Primer Pair Set IDs 1 to 3, 40 to 43, or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Barnesiellaceae; and/or

FIG. 15, Primer Pair Set IDs 13 to 16, 21 to 22, or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Christensenellaceae; and/or

FIG. 15, Primer Pair Set IDs 25 to 30, or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Christensenella; and/or FIG. 15, Primer Pair Set IDs 44 to 46, 67 to 69, or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Veillonella; and/or

FIG. 15, Primer Pair Set IDs 47 to 48, 76 to 79, or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Sutterella; and/or

FIG. 15, Primer Pair Set IDs 49 to 52, 76 to 79, or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Oxalobacter; and/or

FIG. 15, Primer Pair Set IDs 53 to 58, 70 to 72, or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Desulfovibrio; and/or

FIG. 15, Primer Pair Set IDs 59 to 66, 73 to 75, or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Faecalibacterium.

[0046] 5. The method of any one of Claims 1 -4, wherein the at least one synthetic primer pair is selected from FIG. 15, Primer Pair Set IDs 7 to 9, 31 to 39, or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Butyricimonas.

[0047] 6. The method of any one of Claims 1 -4, wherein the at least one synthetic primer pair is selected from FIG. 15, Primer Pair Set IDs 1 to 3, 40 to 43, or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Barnesiellaceae.

[0048] 7. The method of any one of Claims 1 -4, wherein the at least one synthetic primer pair is selected from FIG. 15, Primer Pair Set IDs 13 to 16, 21 to 22, or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Christensenellaceae.

[0049] 8. The method of any one of Claims 1 -4, wherein the at least one synthetic primer pair is selected from FIG. 15, Primer Pair Set IDs 25 to 30, or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Christensenella.

[0050] 9. The method of any one of Claims 1 -4, wherein the at least one synthetic primer pair is selected from FIG. 15, Primer Pair Set IDs 44 to 46, 67 to 69, or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Veillonella.

[0051] 10. The method of any one of Claims 1 -4, wherein the at least one synthetic primer pair is selected from FIG. 15, Primer Pair Set IDs 47 to 48, 76 to 79, or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Sutterella.

[0052] 11 . The method of any one of Claims 1 -4, wherein the at least one synthetic primer pair is selected from FIG. 15, Primer Pair Set IDs 49 to 52, 76 to 79, or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Oxalobacter.

[0053] 12. The method of any one of Claims 1 -4, wherein the at least one synthetic primer pair is selected from FIG. 15, Primer Pair Set IDs 53 to 58, 70 to 72, or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Desulfovibrio.

[0054] 13. The method of any one of Claims 1 -4, wherein the at least one synthetic primer pair is selected from FIG. 15, Primer Pair Set IDs 59 to 66, 73 to 75, or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Faecalibacterium.

[0055] 14. The method of any one of Claims 1 -13, wherein the at least one synthetic primer pair is selected from:

FIG. 15, Primer Pair Set IDs 7, 9, 31 to 39, or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Butyricimonas; and/or

FIG. 15, Primer Pair Set IDs 2, 42, 43, or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Barnesiellaceae; and/or FIG. 15, Primer Pair Set ID 13, 15, 16, 21 to 22, or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Christensenellaceae; and/or

FIG. 15, Primer Pair Set IDs 26, 28, 30, or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Christensenella; and/or

FIG. 15, Primer Pair Set IDs 44 to 46, or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Veillonella; and/or

FIG. 15, Primer Pair Set IDs 47 for determination of presence/absence and/or relative/absolute abundance of Sutterella; and/or

FIG. 15, Primer Pair Set IDs 49 to 52, or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Oxalobacter; and/or

FIG. 15, Primer Pair Set IDs 58, 71 , or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Desulfovibrio; and/or

FIG. 15, Primer Pair Set IDs 59 to 66, 74, 75, or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Faecalibacterium.

[0056] 15. The method of any one of Claims 1 -14, wherein the at least one synthetic primer pair is selected from FIG. 15, Primer Pair Set IDs 7, 9, 31 to 39, or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Butyricimonas.

[0057] 16. The method of any one of Claims 1 -14, wherein the at least one synthetic primer pair is selected from FIG. 15, Primer Pair Set IDs 2, 42, 43, or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Barnesiellaceae.

[0058] 17. The method of any one of Claims 1 -14, wherein the at least one synthetic primer pair is selected from FIG. 15, Primer Pair Set ID 13, 15, 16, 21 to 22, or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Christensenellaceae. [0059] 18. The method of any one of Claims 1 -14, wherein the at least one synthetic primer pair is selected from FIG. 15, Primer Pair Set IDs 26, 28, 30, or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Christensenella.

[0060] 19. The method of any one of Claims 1 -14, wherein the at least one synthetic primer pair is selected from FIG. 15, Primer Pair Set IDs 44 to 46, or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Veillonella; and/or

[0061] 20. The method of any one of Claims 1 -14, wherein the at least one synthetic primer pair is selected from FIG. 15, Primer Pair Set IDs 47 for determination of presence/absence and/or relative/absolute abundance of Sutterella.

[0062] 21 . The method of any one of Claims 1 -14, wherein the at least one synthetic primer pair is selected from FIG. 15, Primer Pair Set IDs 49 to 52, or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Oxalobacter.

[0063] 22. The method of any one of Claims 1 -14, wherein the at least one synthetic primer pair is selected from FIG. 15, Primer Pair Set IDs 58, 71 , or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Desulfovibrio.

[0064] 23. The method of any one of Claims 1 -14, wherein the at least one synthetic primer pair is selected from FIG. 15, Primer Pair Set IDs 59 to 66, 74, 75, or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Faecalibacterium.

[0065] 24. The method of any one of Claims 1 -23, wherein the synthetic primer pair comprising a synthetic forward primer and a synthetic reverse primer, each having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology to any one of the synthetic forward primers of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 16, 18, 20, 21, 23, 25, 26, 27, 28, 30, 31, 32, 34, 36, 38, 40, 41, 43, 44, 46, 47, 49, 50, 51 , 54, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 77, 79, 81 , 91 , 93, 95, 97, 99, 101, 126, 128, 130, 131, 132, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, and 169 and/or the synthetic reverse primers of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 17, 19, 22, 24, 29, 33, 35, 37, 39, 42, 45, 48, 52, 53, 55, 56, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 78, 80, 82, 92, 94, 96, 98, 100, 102, 127, 129, 133, 134, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, and 170.

[0066] 25. The method of any one of Claims 1 -24, wherein the diagnostic kit comprises a chemical assay suitable for a polymerase chain reaction (PCR); and wherein the microbiome reaction mixture is subjected to a PCR condition under which, if the target taxon is present in the microbiome sample, the at least one nucleic acid of the target taxon is amplified, and produces a fluorescence signal; and wherein analyzing the reaction product comprises measuring amount of fluorescence signal produced.

[0067] 26. The method of any one of Claims 1 -25, wherein the diagnostic kit comprises a chemical assay suitable for a polymerase chain reaction (PCR); and wherein the microbiome reaction mixture is subjected to a PCR condition under which, if the target taxon is present in the microbiome sample, the at least one nucleic acid of the target taxon is amplified; and wherein analyzing the reaction product comprises a gel electrophoresis method.

[0068] 27. A diagnostic kit comprising a synthetic primer pair for determination of presence/absence and/or relative/absolute abundance of a target taxon present in a microbiome sample, comprising at least one synthetic primer pair selected from the synthetic primer pairs of FIG. 15, Primer Pair Set IDs 1 to 79, or a combination thereof.

[0069] 28. The diagnostic kit of Claim 27, wherein the microbiome sample comprises a soil sample, a bodily fluid/solid sample, a tissue sample, or a combination thereof.

[0070] 29. The diagnostic kit of Claim 27 or 28, wherein the microbiome sample comprises soil, stool, saliva, skin, nasal swab, vaginal swab, blood, urine, hair, oral swab, or a combination thereof.

[0071] 30. A nucleic acid having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology to any one of the sequences of SEQ ID NO: 1-170, or any combination thereof.

[0072] 31 . A synthetic primer pair comprising a synthetic forward primer and/or a synthetic reverse primer, each having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology to any one of the synthetic forward primers and/or the synthetic reverse primers of SEQ ID NO: 1-170.

[0073] 32. A method of treatment of a patient, comprising: obtaining a microbiome sample from the patient, wherein the microbiome sample potentially comprises at least one nucleic acid; having a diagnostic kit comprising at least one synthetic primer pair designed specifically for the identification of the target taxon; processing the microbiome sample to have the at least one nucleic acid accessible to react with the at least one synthetic primer pair, thereby forming a processed sample; mixing the processed sample with the diagnostic kit to form a microbiome reaction mixture; subjecting the microbiome reaction mixture to a reaction under which at least one section of the accessible nucleic acid is amplified, thereby forming a reaction product; analyzing the reaction product; determining presence/absence and/or relative/absolute abundance of the target taxon in the microbiome sample, thereby forming a taxon profile of the patient; comparing the taxon profile of the patient with the taxon profile of a healthy subject; determining whether the patient has a disease; and treating the patient if the patient has the disease; wherein the at least one synthetic primer pair comprises a synthetic forward primer and a synthetic reverse primer; and wherein the at least one synthetic primer pair is selected from the synthetic primer pairs of FIG. 15, Primer Pair Set IDs 1 to 79, or a combination thereof.

[0074] 33. The method of any preceding claims or any following claims, wherein the at least one synthetic primer pair is selected from: FIG. 15, Primer Pair Set IDs 7 to 9, 31 to 39, or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Butyricimonas; and/or

FIG. 15, Primer Pair Set IDs 25 to 30, or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Christensenella; and/or

FIG. 15, Primer Pair Set IDs 44 to 46, 67 to 69, or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Veillonella; and/or

[0075] 34. The method of any preceding claims or any following claims, wherein the at least one synthetic primer pair is selected from FIG. 15, Primer Pair Set IDs 7 to 9, 31 to 39, or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Butyricimonas. [0076] 35. The method of any preceding claims or any following claims, wherein the at least one synthetic primer pair is selected from FIG. 15, Primer Pair Set IDs 1 to 3, 40 to 43, or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Barnesiellaceae.

[0077] 36. The method of any preceding claims or any following claims, wherein the at least one synthetic primer pair is selected from FIG. 15, Primer Pair Set IDs 13 to 16, 21 to 22, or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Christensenellaceae.

[0078] 37. The method of any preceding claims or any following claims, wherein the at least one synthetic primer pair is selected from FIG. 15, Primer Pair Set IDs 25 to 30, or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Christensenella.

[0079] 38. The method of any preceding claims or any following claims, wherein the at least one synthetic primer pair is selected from FIG. 15, Primer Pair Set IDs 44 to 46, 67 to 69, or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Veillonella.

[0080] 39. The method of any preceding claims or any following claims, wherein the at least one synthetic primer pair is selected from FIG. 15, Primer Pair Set IDs 47 to 48, 76 to 79, or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Sutterella.

[0081] 40. The method of any preceding claims or any following claims, wherein the at least one synthetic primer pair is selected from FIG. 15, Primer Pair Set IDs 49 to 52, 76 to 79, or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Oxalobacter.

[0082] 41 . The method of any preceding claims or any following claims, wherein the at least one synthetic primer pair is selected from FIG. 15, Primer Pair Set IDs 53 to 58, 70 to 72, or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Desulfovibrio.

[0083] 42. The method of any preceding claims or any following claims, wherein the at least one synthetic primer pair is selected from FIG. 15, Primer Pair Set IDs 59 to 66, 73 to 75, or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Faecalibacterium.

[0084] 43. The method of any preceding claims or any following claims, wherein the at least one synthetic primer pair is selected from:

FIG. 15, Primer Pair Set IDs 2, 42, 43, or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Barnesiellaceae; and/or

FIG. 15, Primer Pair Set ID 13, 15, 16, 21 to 22, or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Christensenellaceae; and/or

[0085] 44. The method of any preceding claims or any following claims, wherein the at least one synthetic primer pair is selected from FIG. 15, Primer Pair Set IDs 7, 9, 31 to 39, or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Butyricimonas. [0086] 45. The method of any preceding claims or any following claims, wherein the at least one synthetic primer pair is selected from FIG. 15, Primer Pair Set IDs 2, 42, 43, or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Barnesiellaceae.

[0087] 46. The method of any preceding claims or any following claims, wherein the at least one synthetic primer pair is selected from FIG. 15, Primer Pair Set ID 13, 15, 16, 21 to 22, or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Christensenellaceae.

[0088] 47. The method of any preceding claims or any following claims, wherein the at least one synthetic primer pair is selected from FIG. 15, Primer Pair Set IDs 26, 28, 30, or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Christensenella.

[0089] 48. The method of any preceding claims or any following claims, wherein the at least one synthetic primer pair is selected from FIG. 15, Primer Pair Set IDs 44 to 46, or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Veillonella.

[0090] 49. The method of any preceding claims or any following claims, wherein the at least one synthetic primer pair is selected from FIG. 15, Primer Pair Set IDs 47 for determination of presence/absence and/or relative/absolute abundance of Sutterella.

[0091] 50. The method of any preceding claims or any following claims, wherein the at least one synthetic primer pair is selected from FIG. 15, Primer Pair Set IDs 49 to 52, or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Oxalobacter.

[0092] 51 . The method of any preceding claims or any following claims, wherein the at least one synthetic primer pair is selected from FIG. 15, Primer Pair Set IDs 58, 71 , or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Desulfovibrio.

[0093] 52. The method of any preceding claims or any following claims, wherein the at least one synthetic primer pair is selected from FIG. 15, Primer Pair Set IDs 59 to 66, 74, 75, or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Faecalibacterium.

[0094] 53. The method of any preceding claims or any following claims, wherein the synthetic forward primer and/or the synthetic reverse primer is a polymerase chain reaction (PCR) primer.

[0095] 54. The method of any preceding claims or any following claims, wherein the patient is a human or an animal.

[0096] 55. The method of any preceding claims or any following claims, wherein the healthy subject is human, if the patient is human; or the healthy subject is an animal, if the patient is an animal.

[0097] 56. The method of any preceding claims or any following claims, wherein the patient is more or less-susceptible to developing gastro-intestinal toxicity after chemotherapy and/or radiation-therapy.

[0098] 57. The method or diagnosis kit of any preceding claims or any following claims, wherein the microbiome sample comprises a soil sample, a bodily fluid/solid sample, a tissue sample, or a combination thereof.

[0099] 58. The method of any preceding claims or any following claims, wherein the microbiome sample comprises soil, stool, saliva, skin, nasal swab, vaginal swab, blood, urine, hair, oral swab, or a combination thereof.

[00100] 59. The method or diagnosis kit of any preceding claims or any following claims, wherein the diagnostic kit is configured to differentiate patients that are more or less-susceptible to developing gastro-intestinal toxicity after chemotherapy and/or radiation-therapy.

[00101] 60. The method of any preceding claims or any following claims, wherein amplifying the nucleic acid comprises amplifying the nucleic acid with a PCR technique or a qPCR technique.

[00102] 61 . The method of any preceding claims or any following claims, wherein the microbiome sample is a microbiome sample from a patient.

[00103] 62. Use of the method of the preceding and the following claims to identify and categorize subjects as more or less susceptible to gastrointestinal toxicity. [00104] 63. Use of the method of the preceding and the following claims to identify and categorize subjects as more or less susceptible to gastrointestinal toxicity after chemotherapy and/or radiation-therapy.

[00105] 64. The method of any preceding claims or any following claims, wherein determining the presence/absence and/or relative/absolute abundance comprises measuring a fluorescence signal during the amplifying step.

[00106] 65. The method of any preceding claims or any following claims, further comprising administering a therapeutic to the patient.

[00107] 66. The method of any preceding claims or any following claims, further comprising administering a therapeutic to the patient, and wherein the therapeutic is not chemotherapy and/or radiation.

[00108] 67. Any combination of above kits, products, and methods is within the scope of the instant disclosure.

BRIEF DESC IPTION OF DRAWINGS

[00109] The drawings are of illustrative and represent non-limiting examples of embodiments provided for herein. They do not illustrate all embodiments. Other embodiments may be used in addition or instead. Details that may be apparent or unnecessary may be omitted to save space or for more effective illustration. Some embodiments may be practiced with additional components or steps and/or without all of the components or steps that are illustrated. When the same numeral appears in different drawings, it refers to the same or like components or steps.

[00110] FIG. 1. An exemplary workflow describing utility of the exemplary probes (e.g., synthetic primers) of this invention in research and clinical markets.

[00111] FIG. 2. An example of 16S primers tested against E. coli template. All AMI primers were initially tested using overnight cultures of E. coli for template DNA. Strains identified reflect the designed target taxa of the primers assessed. 16S designations reflect the intended target region within the 16S rRNA gene. Numbers below bands indicate the intended product size of amplified products. PCR reactions: 95°C, 2 min.; 95°C, 30 sec.; 55°C, 30 sec.; 72°C, 30 sec.; repeat previous 3 cycles 30 times; 72°C, 10 min.; hold at 4°C. Gel conditions: 2% agarose gel, 100V, ethidium bromide staining.

[00112] FIG. 3. An example of amplified 16S rRNA PCR Products. Universal 16S rRNA primers (27F and 1492R) were used to amplify 16S PCR products of strains obtained from DSMZ. Remaining PCR products were column purified and used as template DNA for subsequent primer assessment. PCR reactions: 95°C, 2 min.; 95°C, 30 sec.; 55°C, 30 sec.; 72°C, 45 sec.; repeat previous 3 cycles 30 times; 72°C, 10 min.; hold at 4°C. Gel conditions: 2% agarose gel, 100V, ethidium bromide staining.

[00113] FIG. 4. An example of 16S primer pairs assessed on intended target templates to confirm primers positively detect their desired targets. Primers designed to specifically amplify target taxa were assessed against strains representing their intended targets. Labels above amplified products indicates the intended target taxa, the expected product size for each primer set, and the targeted region of the 16S rRNA. Number labels below amplified products indicate the AMI strain number of the purified 16S template DNA. Subsequent sequencing of the 16S template DNA revealed AMI strains AMI-0003 and AMI-0008 were not actual members of the intended target Butyricimonas taxa. PCR reactions: 95°C, 2 min.; 95°C, 30 sec.; 55°C, 30 sec.; 72°C, 30 sec.; repeat previous 3 cycles 30 times; 72°C, 10 min.; hold at 4°C. Gel conditions: 2% agarose gel, 100V, ethidium bromide staining.

[00114] FIG. 5. An example of assessing E. coli primer specificity against Shewanella ana. Regions of 16S rRNA genes in target taxa used to design primers were also used to design primers to corresponding regions of E. coli. The specificity of these E. coli primers were initially assessed using overnight cultures of both E. coli and Shewanella ana. Top text indicates the organism and 16S region the primers were designed for (i.e. E. coli). Bottom text indicates the organism provided as template in each reaction (i.e. Shewanella or E. coli). PCR reactions: 95°C, 2 min.; 95°C, 30 sec.; 60°C, 30 sec.; 72°C, 45 sec.; repeat previous 3 cycles 30 times; 72°C, 10 min.; hold at 4°C. Gel conditions: 2% agarose gel, 100V, ethidium bromide staining.

[00115] FIG. 6. An example of assessing E. coli primer specificity against gammaproteobacteria. Regions of 16S rRNA genes in target taxa used to design primers were also used to design primers to corresponding regions of E. coli. The specificity of these E. coli primers were assessed using overnight cultures of increasingly distant relatives within the gammaproteobacteria class (Salmonella, Serratia, and Shewanella). The 16S region for which the E. coli primers were designed and the organism provided as template in each reaction are indicated. PCR reactions: 95°C, 2 min.; 95°C, 30 sec.; 63°C, 30 sec.; 72°C, 45 sec.; repeat previous 3 cycles 30 times; 72°C, 10 min.; hold at 4°C. Gel conditions: 2% agarose gel, 100V, ethidium bromide staining.

[00116] FIG. 7. An example of assessing 16S primer specificity against diverse representatives. Primer sets designed to specifically amplify products from target taxa were assessed against a panel of templates from a diverse set of representative organisms. One representative from each target taxa was included in the panel. We are currently unable to grow these bacteria in the laboratory so purified 16S DNA was used as template material. Overnight cultures were used as template material for the remaining representatives in the diverse panel. The genus and template source of each representative organism and the target taxa 16S region and primers assessed are shown. The expected product size for each primer set is shown above the amplified product. PCR reactions: 95°C, 2 min.; 95°C, 30 sec.; 55°C, 30 sec.; 72°C, 45 sec.; repeat previous 3 cycles 30 times; 72°C, 10 min.; hold at 4°C. Gel conditions: 2% agarose gel, 100V, ethidium bromide staining. Representative data illustrating differential PCR amplification of target taxa.

[00117] FIG. 8. An example of assessing 16S primers against diverse, stool-derived communities. Primer sets designed to specifically amplify products from target taxa (A, Barnesiellaceae; B, Christensenellaceae) were assessed against a dilution series of target template DNA in isolation (lower gels) or within a diverse stool-derived community (upper gels). Purified 16S DNA was serially diluted 10-fold to the concentrations indicated and used as template alone (lower gels) or spiked-in to a standard volume of a diverse stool-derived community (upper gels). Amplified products in the upper gels beyond the last product in the dilution series of template in the lower gel suggest organisms from the target taxa may be present within the stool community. PCR reactions: 95°C, 2 min.; 95°C, 30 sec.; 55°C, 30 sec.; 72°C, 45 sec.; repeat previous 3 cycles 30 times; 72°C, 10 min.; hold at 4°C. Gel conditions: 2% agarose gel, 100V, ethidium bromide staining. Representative data illustrating differential PCR amplification of target taxa.

[00118] FIG. 9. Initial primer pairs assessed against intended target organisms. All AMI primer pairs were initially tested on their intended target organisms. The taxa targeted for amplification, the designed target region, and the specific primers used in each reaction are denoted. Also indicated is the strain identity and material of the template provided in each reaction (frozen glycerol stocks diluted 1 :10 in water). PCR reactions: 95°C, 2 min.; 95°C, 30 sec.; 55°C, 30 sec.; 72°C, 30 sec.; repeat previous 3 cycles 30 times; 72°C, 10 min.; hold at 4°C. Gel conditions: 2% agarose gel, 100V, ethidium bromide staining. Representative data.

[00119] FIG. 10. Primer pairs assessed on intended target templates. Primers designed to specifically amplify products from target taxa were assessed on template from representative strains within their intended target taxa. Each primer pair was assessed against a no template (water only) control and gDNA from a representative target strain. The taxa targeted for amplification, the designed target region, and the specific primers used in each reaction are denoted. Also indicated is the template material used in each reaction (NTC, No Template Control; Vp, Veillonella parvula; Sw, Sutterella wadsorthensis; Dv, Desulfovibrio vulgaris; Fp, Faecalibacterium prausnitzii). Target organism gDNA stocks were diluted 1 OO in water and used as template material. PCR reactions: 95°C, 2 min.; 95°C, 30 sec.; 55°C, 30 sec.; 72°C, 30 sec.; repeat previous 3 cycles 30 times; 72°C, 10 min.; hold at 4°C. Gel conditions: 2% agarose gel, 50V, ethidium bromide staining. Representative data.

[00120] FIG. 11. Assessing primer pair specificity against template material from diverse representative target taxa. Primer pairs designed to specifically amplify products only from intended target taxa were assessed against a panel of templates from a diverse set of representative organisms. This figure is a compilation of cropped gel images illustrating PCR reaction products using the primer pairs shown to the right (Intended target taxa and region are bold above the specific primers used in each panel) against the template material shown above each column of lanes. The diverse template panel included a No Template Control (NTC) and material from Escherichia coli (Ec) along with one representative strain from each target taxa (Of, Oxalobacter formigenes; Sw, Sutterella wadsorthensis; Dv, Desulfovibrio vulgaris; Bi, Barnesiella intestinihominis; Bv, Butyricimonas virosa; Cm, Christensenella massiliensis; Fp, Faecalibacterium prausnitzii; Vp, Veillonella parvula). The template material in each reaction was a 1 OO dilution of either frozen glycerol stocks of viable organisms (Ec, Bi, Bv, Cm) or gDNA from representative target organisms (Of, Sw, Dv, Fp, Vp). PCR reactions: 95°C, 2 min.; 95°C, 30 sec.; 55°C, 30 sec.; 72°C, 30 sec.; repeat previous 3 cycles 30 times; 72°C, 10 min.; hold at 4°C. Gel conditions: 2% agarose gel, 100V, ethidium bromide staining. Representative data illustrating differential PCR amplification of target taxa.

[00121] FIG. 12. Assessing primer pairs against diverse stool-derived communities. Primer sets designed to specifically amplify products from target taxa were assessed against complex stool-derived communities. Each primer pair that passed previous screening criteria was tested against a No Template Control (Negative Control), template material (gDNA or viable cells) from a representative strain within their target taxa (+C, Positive Control), and two stool-derived communities. Template community V19 is a culture of intermediate complexity originally derived from an OpenBiome stool community, while OB represents a complex stool derived community sourced from OpenBiome Community in which a 1 :100 dilution of the frozen stock was used as template material. PCR reactions: 95°C, 2 min.; 95°C, 30 sec.; 55°C, 30 sec.; 72°C, 45 sec.; repeat previous 3 cycles 30 times; 72°C, 10 min.; hold at 4°C. Gel conditions: 2% agarose gel, 100V, ethidium bromide staining. Representative data.

[00122] FIG. 13. Names and sequences of primers disclosed herein.

[00123] FIG. 14. List of names of synthetic forward/reverse primers, and these primers’ target regions of 16S rRNA gene of target taxa.

[00124] FIG. 15. Synthetic forward and synthetic reverse nucleic acid primer pairs. For the nucleotide sequence of each synthetic forward and reverse primer and their properties, refer to FIG. 13-14, 16.

[00125] FIG. 16. List of identification (ID) numbers of synthetic primer sets (i.e. pairs) formed by the primers of FIG. 15, and a summary of experimental results obtained by using these primer pairs and single strain templates. [00126] FIG. 17. List of identification (ID) numbers of synthetic primer sets (i.e. pairs) formed by the primers of FIG. 15, and a summary of experimental results obtained by using these primer pairs and community templates.

[00127] FIG. 18. Internal AMI numbers and collection IDs of strains used in this disclosure.

[00128] FIG. 19. List of names of synthetic forward/reverse primers and their sequences, and these primers’ target regions, target locations, and target genes of target taxa and the target taxa’s target references.

[00129] FIG. 20. List of identification (ID) numbers of synthetic primer sets (i.e. pairs) formed by the primers of FIG. 19, and summary of experimental results obtained by using these primer pairs and single strain templates.

[00130] FIG. 21. List of identification (ID) numbers of synthetic primer sets (i.e. pairs) formed by the primers of FIG. 19, and a summary of experimental results obtained by using these primer pairs and community templates.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[00131] Illustrative and non-limiting embodiments are now described. Other embodiments may be used in addition or instead. Details that may be apparent or unnecessary may be omitted to save space or for a more effective presentation. Some embodiments may be practiced with additional components or steps and/or without all of the components or steps that are described.

[00132] The following acronyms are used in this disclosure.

[00133] ATCC: American Type Culture Collection

[00134] BES: Bioelectrochemical System

[00135] BSI: Blood Stream Infections

[00136] DNA: Deoxyribonucleic Acid

[00137] DSMZ: Deutsche Sammlung von Mikroorganismen und Zellkulturen

[00138] FMP-R: Fecal Microbiota Preparation for Research

[00139] GC: Guanine and Cytosine nitrogen bases in the DNA sequence [00140] gDNA: Genomic DNA

[00141] HAD: helicase dependent amplification

[00142] ISDPR: isothermal strand displacement polymerization

[00143] LAMP: loop mediated isothermal amplification

[00144] NCBI: National Center for Biotechnology Information

[00145] OTU: Operational Taxonomic Unit

[00146] PCR: Polymerase Chain Reaction

[00147] qPCR: quantitative polymerase chain reaction

[00148] QIIME: Quantitative Insights into Microbial Ecology

[00149] RCA: rolling circle amplification

[00150] rDNA: Ribosomal DNA

[00151] RNA: Ribonucleic Acid

[00152] rRNA: ribosomal Ribonucleic Acid

[00153] RPA: recombinase polymerase amplification

[00154] SDA: strand displacement amplification

[00155] SNP: Single Nucleotide Polymorphism

[00156] This disclosure relates to a cost-effective and rapid method, and reagents (e.g. diagnostic kits) to stratify patient populations based on their microbiome profiles. This disclosure also relates to a diagnostic kit comprising synthetic primers. This disclosure also relates to synthetic nucleic acid primers. This disclosure also relates to a method of developing these synthetic primers. This disclosure further relates to products and methods that allow medical staff and physicians to differentiate patients that are more or less-susceptible to developing gastro-intestinal toxicity after chemo-therapy and/or radiation-therapy.

[00157] This disclosure also relates to designing and developing PCR and qPCR primers for microbial biomarkers. The microbiome may be profiled using sequencing methods. Sequencing might be necessary to understand which bacteria might play an important role in health and disease. However, sequencing the entire sample may not be a suitable approach for large research studies and clinical diagnostics since it is time and resource intensive. Once the distinguishing microbial biomarker profile is identified through an initial study, targeted probes (i.e., primers) may be used to identify and quantify only the bacteria of interest. This can save time and resources, which is valuable for a clinician and a researcher. The targeted probes may be used on a variety of platforms such as sequencing, quantitative polymerase chain reaction (qPCR) arrays, or many other molecular platforms. The systems, methods, assays and kits of this disclosure may fulfill this unmet research and clinical need of developing targeted probes for bacterial identification and quantification.

[00158] This disclosure also relates to a diagnostic kit that may determine the relative amounts of bacterial biomarkers and may give clinical results in terms of Yes/No/Risk index that can be used by a clinician to make treatment decisions. This disclosure also relates to targeted primers for bacterial groups at a desired level of resolution, which may establish a platform to develop microbiome diagnostic kits for other clinical indications.

[00159] This disclosure also relates to systems, methods and assays that may be used for treatment of patients. For example, the side effects caused by chemo/radiation therapy for cancer patients, such as gastrointestinal toxicity and blood stream infections may be sufficiently severe that they may interfere with normal nutrition, and may cause longer hospitalizations, increased costs, increased opioid use for pain management, and often limit or prevent continuation of an optimal chemo- or radio-therapy regimen. Incidence of adverse events such as mucositis has been shown to increase healthcare costs >$17k per patient. Currently, there are no commercial tools to predict this risk. The systems, the methods, the essays, and the kits of this disclosure may be used, for example, to design a personalized treatment and nutrition plan for cancer patients.

[00160] The systems, the methods, the essays, and the kits of this disclosure may also be used, for example, to develop cost-effective and rapid (e.g., <24 hour turnaround time) targeted microbiome assays. These microbial biomarker assays may be used for following up validation research studies, patient stratification, prediction of treatment outcomes, non-invasive diagnosis as well as various other uses.

[00161] The microbiome may be better than any other combination of clinical markers at differentiating patients who may or may not develop BSI’s. For example, determining relative abundance of 10 taxa may predict relative susceptibility to chemo-induced BSI’s. Also, there may be external influences on a person’s gut microbiome, which may need to be considered in addition to such relative abundance of the taxa.

[00162] The target taxa of this disclosure may be Barnesiellaceae, Butyricimonas, Christensenellaceae, Chritsensenella, Faecalibacterium, Oxalobacter, Sutterella, Veillonella, Desulfovibrio, or a combination thereof. In this disclosure, the taxon may be a strain, species, genus, or a combination thereof. Presence/absence or changes in relative/absolute abundance of these taxa may be indicative of a patient’s response to a treatment.

[00163] One exemplary method of this disclosure may include obtaining the microbiome sample that potentially comprises at least one nucleic acid. In this disclosure, the nucleic acid may be a nucleic acid. For example, the nucleic acid may be a DNA, an RNA, fragments of such nucleic acids, the like, or a combination thereof.

[00164] In this disclosure, the primers used to amplify nucleic acids of the target taxa are synthetic primers. We designed these synthetic primers to target 16s rRNA or genomic DNA regions of the target taxa. These primers were synthesized by Integrated DNA Technologies (Coralville, Iowa).

[00165] This disclosure also relates to processing the microbiome sample to have the at least one nucleic acid accessible to react with the at least one synthetic primer pair. The microbiome sample may be processed by any suitable method that makes the nucleic acid accessible to the synthetic primer pair reaction. This method, for example, may be any chemical and/or physical (e.g., mechanical) method. Examples of suitable mechanical methods may include mincing, shredding, filtering, and the like. In other examples, using homogenizers, ultrasonicators, ball mills, and the like may make the nucleic acid accessible to the synthetic primer pair reaction. A combination of these mechanical methods may also be used. Examples of suitable chemical methods may include digestion of the microbiome sample by using acids, bases, and enzymes. One exemplary microbiome sample processing may include a nucleic acid extraction method well-practiced in the PCR industry. This nucleic acid extraction method, for example, may include lysing the microbiome sample. The nucleic acid may then be solubilized. The nucleic acid extraction method may also include organic extraction (phenol- chloroform method), nonorganic method (salting out and proteinase K treatment), adsorption method (silica-gel membrane), the like, or a combination thereof, as disclosed by Gupta [7],

[00166] The diagnostic kit of this disclosure may assess the relative abundances of the target taxa, using established molecular techniques, such as qPCR, hybridization or NGS methods. This diagnostic kit may be tied to an algorithm, which may allow the binary grouping of patients into more prone or less prone to GIM populations, or alternatively the algorithm may give a probability of different grades of GIM.

[00167] The diagnostic kit of this disclosure may rely upon nucleic acid amplification, which may be achieved through thermal amplification methods, such as polymerase chain reaction (PCR), or through isothermal amplification methods, such as loop mediated isothermal amplification (LAMP), rolling circle amplification (RCA), recombinase polymerase amplification (RPA), helicase dependent amplification (HAD), strand displacement amplification (SDA) or isothermal strand displacement polymerization (ISDPR). The diagnostic kit may include reagents needed to perform these reactions, and determine presence/absence, or relative/absolute abundance of the target taxa in a microbiome. For a review of such techniques, see Leonardo et al. [6]-

[00168] In this disclosure, the treatment may be any treatment of a medical condition such as disease. For example, the treatment may be a cancer treatment. The methods and reagents provided for herein may be configured to differentiate patients that are more or less-susceptible to developing gastro-intestinal toxicity after chemotherapy and/or radiation-therapy.

[00169] In this disclosure, the patient may be a human or an animal.

[00170] In this disclosure, the microbiome may be any microbiome or any sample that may potentially include a taxon or a nucleic acid. The sample may be a solid sample, a liquid sample, or a combination thereof. Examples of the samples may be a soil sample, a fecal sample, a healthy or tumor tissue sample, a bodily fluid sample, a saliva sample, a lung aspirate sample or a combination thereof. [00171] In this disclosure, the reaction product may be analyzed by a gel electrophoresis and quantitative PCR method. The reaction product may also be analyzed Next Generation Sequencing or by hybridization methods, such as hybridization to a chip.

[00172] The synthetic nucleic acid primers of this disclosure used for determination of presence/absence or relative/absolute abundance of the target taxa were synthetically synthesized.

[00173] An exemplary, workflow describing utility of the exemplary probes (e.g., synthetic nucleic acid primers) of this invention in research and clinical markets are schematically shown in FIG. 1. This workflow may start with the discovery of microbial biomarkers that distinguish healthy and disease states. These microbial biomarkers may be, for example, biomarkers of nucleic acids belonging to target taxa. Then, the synthetic nucleic acid primers may be designed, synthetically manufactured, and tested and validated against the target taxa to develop assays. These synthetic nucleic acid primers may then be used to identify and/or quantify (i.e., determine presence/absence and/or relative/absolute abundance) of the target taxa in a (microbiome) sample. The results of such determination activity may, for example, then be used to stratify patient populations, predict, or diagnose medical conditions (e.g., disease) and treatment outcomes.

[00174] In this disclosure, the target taxa were predicted from the V5 and V6 hypervariable 16S ribosomal RNA gene region annotated from the Greengenes database.

[00175] Examples of the designed and synthetically manufactured nucleic acid primers of this disclosure, their primer name used in this disclosure, their target regions and taxa, and their properties are listed in FIG. 14. Sequences for these primers are provided in FIG. 13. Results of testing of these synthetic nucleic acid primers on single strain templates are summarized in FIG. 16. Results of testing these synthetic nucleic acid primers on a variety of microbial community templates sourced from the human fecal samples are summarized in FIG. 17. The strains used in this disclosure, their AMI numbers, their strain names, and Collection-IDs are listed in FIG. 18. Synthetic forward/re verse primers, properties, these primers’ target regions, target locations, and target genes of target taxa and the target taxa’s target references are listed in FIG. 19. Sequences for these primers are provided in FIG. 13. Identification (ID) numbers of synthetic primer sets (i.e., pairs) formed by the primers of FIG. 19, and summary of experimental results obtained by using these primer pairs and single strain templates are listed FIG. 20. Identification (ID) numbers of synthetic primer sets (i.e., pairs) formed by the primers of FIG. 19, and summary of experimental results obtained by using these primer pairs and community templates are listed in FIG. 21. The synthetic forward and synthetic reverse nucleic acid primer pairs are listed in FIG. 15.

Example 1 . Testing and validation of synthetic primers.

[00176] To be an effective diagnostic, molecular probes (e.g. primers) may need to be both specific enough to amplify only designated target organisms (e.g. target taxa) within a diverse community and sensitive enough to identify those organisms at low abundance. Accordingly, our synthetic primers were rigorously tested to ensure both.

[00177] First, it was confirmed that prospective primers can amplify their intended targets by performing PCR on the organisms they were designed for. Viable cells were used as template when the target organism was cultivable, whereas purified 16S rDNA, the DNA sequence that comprises the 16S rRNA gene or genomic DNA was used when active cultures of target organisms were unavailable. Primer pairs that efficiently generated products of the proper size on their intended targets were moved on for additional evaluation.

[00178] Next, optimum thermal conditions were empirically determined for each set of primers by constructing reactions across a gradient of annealing temperatures. By optimizing the annealing temperature of PCR reactions, subsequent testing were performed at the highest possible temperature for each set of probes to ensure stringency by preventing unintended annealing and amplification.

[00179] Once optimum reaction conditions were determined for each set of primers, the synthetic primers were then assessed for specificity by testing them against a representative panel of diverse microorganisms to ensure spurious products were not amplified. Primer pairs that yielded no false positives were then subjected to more complex communities. In this disclosure, a false positive is defined as the unintended amplification of a product in a non-target organism. In this disclosure, a complex community is defined as a microbiome sample comprising many diverse microorganisms. These communities may either be mixtures of known or non-target organisms originally derived from donor stool samples.

[00180] If the complex community did not produce a product, suggesting the absence of our target organism, additional “spike-in” experiments were performed. Here, purified 16S rDNA or genomic DNA from a target strain was introduced within the context of a diverse mixed community. Successful probes selectively amplified products from their intended targets.

[00181] To assess the sensitivity of the synthetic primers, the abundance of spiked in target template was varied in the complex community. In this disclosure, the complex community is defined as a natural or artificial community that reflects the expected level of species diversity in a microbiome sampled from a human subject. Here, target DNA was introduced to PCR reactions at increasingly smaller quantities until eventually there were no copies of template DNA. Accordingly, we determined the minimum number of copies of template DNA required to obtain an amplified product.

[00182] These experiments were performed in parallel using only dilute template DNA as well as target sequence within complex communities to compare the background sensitivity of probes with idealized PCR reactions containing only target DNA as well as within the context of a complex background of non-target template. Accordingly, these findings may be used to determine the minimum abundance of target sequence required to detect the presence of a target organism.

Example 2. Assessing synthetic nucleic acid primers on intended targets.

[00183] Primer pairs suitable for diagnostics applications may need to be sufficiently specific to amplify PCR products only from their intended target taxa. The first step in assessing whether a primer might be suitable for further development is to assess primers on their intended DNA targets. Accordingly, every primer was first assessed using template DNA from a representative strain within the target taxa.

[00184] Initially, for every primer designed for a specific region of the 16S rRNA within target taxa, we also designed another primer intended to amplify that region within E. coli. These primers were used to begin assessing the feasibility of using 16S rRNA as a target for differential amplification.

[00185] Given that every primer pair had a corresponding E. coli probe, we began by assessing all primers sets against E. coli template DNA. As expected, all primers designed for E. coli 16S rRNA gene amplified products of the expected size when E. coli cultures were used as template DNA. Furthermore, as intended, no PCR products were observed with primers designed to be specific for other target taxa. Representative data are shown in FIG. 2 and summarized in FIG. 16.

[00186] To determine whether primers designed for target taxa (e.g., Barnesiellaceae, Butyricimonas, and Christensenellaceae) could amplify PCR products from their intended organisms, we obtained representatives organisms belonging to each group from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ). Bacterial strains were provided as lyophilized ampoules. Each strain was assigned a unique AMI strain identifier (FIG. 18), opened, and reconstituted. This reconstituted material was used as template material to amplify full-length 16S rDNA using universal primers (27F and 1492R). All the PCR reactions amplified products to the appropriate size (FIG. 3).

[00187] Purified full-length 16S rDNA products from each target strain were used as template to assess primers designed for each target taxa. All primers successfully amplified appropriately sized PCR products from all purified 16S product template DNA obtained from their intended target taxa (FIG. 4), with the sole exception being the forward primer targeting the Christensenellaceae 16S rRNA K3 region (designated primer p0026 on FIG. 14). Unexpectedly, two strains purchased from the DSMZ with the designation Butyricimonas did not yield PCR products using primers designed for these target taxa. However, purified 16S rDNA products were sequenced to verify strain identities and revealed strains AMI_0003 and AMI_0008 were not within the target Butyricimonas taxa.

Example 3. Assessing synthetic nucleic acid primer specificity on non-tarqet templates. [00188] Given that all primer sets amplified products on their intended targets, we began to determine how specific each primer set was by assessing each primer pair on templates from non-target strains.

[00189] Similarly, we obtained strains from our target taxa and created pure 16S rDNA, we next assessed the specificity of our target taxa primers against non-target templates. Here, a diverse panel of representative strains was used to determine whether non- target taxa templates would produce spurious products. This panel included one representative of each target taxa (Barnesiella intestinihominis, Butyricimonas virosa, and Christensenella massiliensis in the form of purified 16S rDNA) as well as additional strains representing increasingly divergent taxa (Enterococcus faecalis, Staphylococcus epidermidis, Bacillus subtilis, Pseudomonas aeruginosa, Serratia marcescens, Salmonella typhimurium, and Escherichia coll) (FIG. 7, representative data).

[00190] These assessments against non-target templates revealed varying degrees of primer specificity. Some primers performed exactly as designed and provided specific and differential amplification against all non-target templates assessed. Other primer sets occasionally produced untargeted amplification under the reaction conditions assessed to date (summarized in FIG. 16). When spurious PCR products were observed, we characterized the number, location, and context of SNPs required to obtain differential amplification of target versus non-target sequence.

[00191] To determine whether primers designed for target taxa could amplify PCR products from their intended organisms, we first obtained representatives organisms belonging to each group from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ). These strains were provided as lyophilized ampoules. Each strain was assigned a unique AMI strain identifier, opened, and reconstituted in growth medium. For strains AMI_0001 thru AMI_0011 , this reconstituted material was stored at -80°C as frozen glycerol stocks and used as template material for PCR reactions.

[00192] Initially, we designed, ordered, and assessed primers specific for three target taxa (Barnesiellaceae, Butyricimonas, Christensenellaceae) and these primers were assessed against one representative target strain in our possession (Barnesiella intestinihominis, AMI_0001 ; Butyricimonas virosa, AMI_0004; and Christensenella massiliensis, AMI_0009; respectively). See FIG. 9, summarized in FIG. 20. [00193] Frozen glycerol stocks of each representative target organisms were diluted 1 OO in water and used as template material for PCR reactions using primer pairs designed to specifically amplify product on gDNA from organisms within their target taxa. All primer pairs successfully amplified appropriately sized PCR products from all from representative organisms within their intended target taxa.

[00194] Next, we designed and ordered primers for additional target taxa (Veillonella, Sutterella, Oxalobacter, Desulfovibrio, and Faecalibacterium). To begin assessing the specificity of these primers we obtained purified gDNA for one representative organism within each taxon from the American Type Culture Collection (ATCC) and gave them AMI strain designations (FIG. 18: ATCC_0019 thru ATCC_0023). The details of the primers used in this study were listed in FIG. 19.

[00195] To determine whether primers designed for a given target taxa could amplify PCR products from their intended targets, each new primer pair was assessed against a negative (no template) control as well as purified gDNA from the representative organism within their target taxa (FIG. 10). The majority of primer pairs passed this initial screening assessment, amplifying a PCR product of the proper size on target gDNA template while producing no spurious products in negative control reactions (results summarized in FIG. 20). For every target taxon at least one primer pair successfully amplified a product of the expected size on a representative target organism and moved forward in our assessment pipeline for further evaluation against non-target templates.

Example 4. Assessing synthetic nucleic acid primers within complex communities.

[00196] Diagnostic primers need to specifically and sensitively amplify PCR products differentially from target taxa with diverse communities in complex samples. To begin assessing the specificity and sensitivity of our primers within increasingly complex samples, we selected our best primer pairs (Barnesiella: p0003, p0004; Butyricimonas: p0016, p0017; Christensenella: p0032, p0033) and assessed their performance against a diverse microbial community within a complex medium.

[00197] Primer sets were assessed against these stool-derived communities by comparing amplified products against only pure 16S target DNA at increasingly dilute concentrations, pure 16S target DNA spiked in the diverse community at various concentrations, and against the diverse community without any template DNA spiked in (FIG. 8).

[00198] The Butyricimonas and Christensenella primer sets amplified products on purified target 16S rDNA at increasingly dilute template concentrations, until template DNA was diluted to extinction (FIG. 8, lower). When the same dilutions were introduced into the background stool-derived community (Figure 8, upper), we continued to see PCR products at the same limit of detection. These results indicate the primers were as sensitive when only template DNA is present as well as when template is present within a diverse background community. Furthermore, the absence of PCR products at dilutions of target DNA past the limit of detection into a diverse community indicates the background stool-derived community does not contain any representatives from the target taxa. In some embodiments, the initial concentration of 16S rDNA for use in the amplification reactions herein may be determined by spectrophotometry (e.g.

NanoDrop), or fluorometry (e.g. Qubit Fluorometric Quantitation).

[00199] The Barnesiellaceae primers displayed similar sensitivity when assessed on dilute target DNA alone (FIG. 8, lower). However, when testing the same dilutions of primers within the diverse stool-derived community we observed PCR products at every dilution of target template, even beyond dilutions where no target DNA remained (FIG. 8, upper). These results indicate that Barnesiellaceae are present in the background diverse community. This was subsequently confirmed with sequencing to assess the composition of the complex community.

Example 5. Assessing Primer Specificity on Non-tarqet Templates

[00200] After determining which primer pairs successfully amplified products on templates from representative organisms within the intended target taxa, we next assessed the specificity of our target taxa primers against non-target templates to determine whether we observe differential amplification on target versus non-target templates.

[00201] Here, a diverse panel of representative strains was used to determine whether non-target taxa templates would produce spurious products (FIG. 11 , summarized in FIG. 20). This panel included a no template control, Escherichia coli (Ec, AMI_0024), and one representative of each target taxa (Of, Oxalobacter formigenes; Sw, Sutterella wadsorthensis; Dv, Desulfovibrio vulgaris; Bi, Barnesiella intestinihominis; Bv, Butyricimonas virosa; Cm, Christensenella massiliensis; Fp, Faecalibacterium prausnitzii; Vp, Veillonella parvula). The template material in each reaction was a 1 OO dilution of either frozen glycerol stocks of viable organisms (Ec, Bi, Bv, Cm) or gDNA from representative target organisms (Of, Sw, Dv, Fp, Vp).

[00202] These assessments against non-target templates revealed varying degrees of primer specificity. Nearly all primers performed as designed and yielded specific products of the intended size only on template material from their intended target taxa (results summarized in FIG. 20). However, others occasionally produced untargeted products on non-target templates. For example, primers p0093 and p0094 were designed to specifically target region AAH02 in Desulfovibrio, yet produced detectable products on Oxalobacter and Sutterella templates.

[00203] When spurious PCR products were observed (usually on closely related organisms as expected), we characterized the number, location, and context of SNPs required to obtain differential amplification of target versus non-target sequence. Furthermore, reaction conditions may likely be improved to increase PCR stringency and improve primer specificity against non-target sequences.

[00204] Primer pairs that passed this initial specificity assessment, producing intended PCR products exclusively on template from a representative target, were next assessed within complex communities with diverse template material.

Example 6. Assessing primers within complex communities.

[00205] Diagnostic primers will need to specifically and sensitively amplify PCR products exclusively from target taxa within diverse communities in complex samples. To begin assessing the specificity and sensitivity of our primers within increasingly complex samples, we selected primer pairs that passed our initial quality controls and assessed their performance against two diverse stool-derived microbial communities. These two complex microbial communities both originate from human stool samples obtained from OpenBiome, a non-profit stool bank that provides stool preparations to clinicians for fecal microbiota transplants and clinical scientists to support research on the human microbiome.

[00206] First, a Fecal Microbiota Preparation for Research (FMP-R) was obtained from OpenBiome. This sample comprises a mixture of fecal-derived material from numerous pre-screened, healthy human donors and intended to reflect a representative complex and healthy gut microbiome community. It is a complex sample, both in terms of the microbial community (as determined by the 16S rRNA gene community profile provided by OpenBiome) and the contents of the fecal material (only large insoluble fibers have been removed) that reflects a minimally processed stool sample. This OpenBiome community is provided as a frozen glycerol suspension and closely reflects the stool samples eventually anticipated in clinical assessments and applications.

[00207] Second, we created a stool-derived community of intermediate complexity for primer assessment. This microbial community, hereafter referred to as V19 in this disclosure, is derived from the OpenBiome FMP-R, but has subsequently been passaged through outgrowths in complex medium. These subsequent changes in environment and growth have altered the microbial composition to form the V19 microbial community. While the overall diversity of the community has decreased, the organisms present continue to reflect bacteria present in human stool. Furthermore, the differences in community (complex vs. intermediate) and sample composition (minimally processed stool vs. culture medium) provide important differences that can facilitate primer assessment.

[00208] Primer sets that previously passed quality control assessments against simple target and non-target templates were next assessed against both of these complex stool-derived communities (FIG. 12, summarized in FIG. 21). Each primer pair was tested against a negative (no template) control, a positive control (+C) including template material from a representative isolate within the intended target taxa, and both the intermediate and complex stool-derived communities.

[00209] As expected from previous results, none of the primer pairs yielded a product when no template was provided and all of the primer sets amplified a product of the intended size on strains or gDNA from a representative target organism. Against both the intermediate V19 bacterial community and the OpenBiome FMP-R, referred as OB in this disclosure, the presence or absence of PCR products was consistent with the known community compositions.

[00210] For example, all primer pairs designed to amplify regions from Faecalibacterium yielded products on OB community templates, where these organisms are abundant, yet did not amplify products on V19 community templates where this taxa was rare and expected to be below the limit of detection. Similarly, primers p0101 and p0102 targeting the AAH05 region of the Desulfovibrio taxa amplified a product on the V19 community but not on the OB community template. Again, this result was consistent with expectations based on 16S rRNA gene community profiling where Desulfovibrios is rare in the original OB community had been enriched by during the outgrowth steps, increasing in relative abundance to now exceed the limit of detection only in the V19 community template.

[00211] Together, these results are consistent with expectations for primer pairs that are specific for their intended target taxa, yielding differential amplification of PCR products on complex stool-derived template material consistent with the presence or absence of target organisms above the current limit of detection within the assessed communities.

[00212] The forward and reverse primer pairs can be found in FIG. 15.

[00213] The components, steps, features, objects, benefits, and advantages that have been discussed are merely illustrative. None of them, nor the discussions relating to them, are intended to limit the scope of protection in any way. Numerous other embodiments are also contemplated. These include embodiments that have fewer, additional, and/or different components, steps, features, objects, benefits, and/or advantages. These also include embodiments in which the components and/or steps are arranged and/or ordered differently.

[00214] Unless otherwise stated, all measurements, values, ratings, positions, magnitudes, sizes, and other specifications that are set forth in this disclosure are approximate, not exact. They are intended to have a reasonable range that is consistent with the functions to which they relate and with what is customary in the art to which they pertain. [00215] All articles, patents, patent applications, and other publications that have been cited in this disclosure are incorporated herein by reference.

[00216] In this disclosure, the indefinite article “a” and phrases “one or more” and “at least one” are synonymous and mean “at least one”.

[00217] Relational terms such as “first” and “second” and the like may be used solely to distinguish one entity or action from another, without necessarily requiring or implying any actual relationship or order between them. The terms “comprises,” “comprising,” and any other variation thereof when used in connection with a list of elements in the specification or claims are intended to indicate that the list is not exclusive and that other elements may be included. Similarly, an element preceded by an “a” or an “an” does not, without further constraints, preclude the existence of additional elements of the identical type.

[00218] In at least some of the previously described embodiments, one or more elements used in an embodiment can interchangeably be used in another embodiment unless such a replacement is not technically feasible. It will be appreciated by those skilled in the art that various other omissions, additions and modifications may be made to the methods and structures described herein without departing from the scope of the claimed subject matter. All such modifications and changes are intended to fall within the scope of the subject matter, as defined by the appended claims.

[00219] With respect to the use of substantially any plural and/or singular terms herein, those having skill in the art can translate from the plural to the singular and/or from the singular to the plural as is appropriate to the context and/or application. The various singular/plural permutations may be expressly set forth herein for sake of clarity.

[00220] It will be understood by those within the art that, in general, terms used herein, and especially in the appended claims (e.g., bodies of the appended claims) are generally intended as “open” terms (e.g., the term “including” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “having at least,” the term “includes” should be interpreted as “includes but is not limited to,” etc.). It will be further understood by those within the art that if a specific number of an introduced claim recitation is intended, such an intent will be explicitly recited in the claim, and in the absence of such recitation no such intent is present. For example, as an aid to understanding, the following appended claims may contain usage of the introductory phrases “at least one” and “one or more” to introduce claim recitations. However, the use of such phrases should not be construed to imply that the introduction of a claim recitation by the indefinite articles “a” or “an” limits any particular claim containing such introduced claim recitation to embodiments containing only one such recitation, even when the same claim includes the introductory phrases “one or more” or “at least one” and indefinite articles such as “a” or “an” (e.g., “a” and/or “an” should be interpreted to mean “at least one” or “one or more”); the same holds true for the use of definite articles used to introduce claim recitations. In addition, even if a specific number of an introduced claim recitation is explicitly recited, those skilled in the art will recognize that such recitation should be interpreted to mean at least the recited number (e.g., the bare recitation of “two recitations,” without other modifiers, means at least two recitations, or two or more recitations). Furthermore, in those instances where a convention analogous to “at least one of A, B, and C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., “a system having at least one of A, B, and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or A, B, and C together, etc.). In those instances where a convention analogous to “at least one of A, B, or C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., “ a system having at least one of A, B, or C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or A, B, and C together, etc.). It will be further understood by those within the art that virtually any disjunctive word and/or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or “B” or “A and B.”

[00221] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[00222] As will be understood by one skilled in the art, for any and all purposes, such as in terms of providing a written description, all ranges disclosed herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like include the number recited and refer to ranges which can be subsequently broken down into sub-ranges as discussed herein. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1 -3 articles refers to groups having 1 , 2, or 3 articles. Similarly, a group having 1 -5 articles refers to groups having 1 , 2, 3, 4, or 5 articles, and so forth.

[00223] While various aspects and embodiments have been disclosed herein, other aspects and embodiments will be apparent to those skilled in the art. The various aspects and embodiments disclosed herein are for purposes of illustration and are not intended to be limiting, with the true scope and spirit being indicated by the following claims.

[00224] The abstract is provided to help the reader quickly ascertain the nature of the technical disclosure. It is submitted with the understanding that it will not be used to interpret or limit the scope or meaning of the claims. In addition, various features in the foregoing detailed description are grouped together in various embodiments to streamline the disclosure. This method of disclosure should not be interpreted as requiring claimed embodiments to require more features than are expressly recited in each claim. Rather, as the following claims reflect, inventive subject matter lies in less than all features of a single disclosed embodiment. Thus, the following claims are hereby incorporated into the detailed description, with each claim standing on its own as separately claimed subject matter.

Claims

WHAT IS CLAIMED IS:

1 . A method for determination of presence/absence and/or relative/absolute abundance of a target taxon present in a microbiome sample, comprising: obtaining the microbiome sample that potentially comprises at least one nucleic acid; having a diagnostic kit comprising at least one synthetic primer pair designed specifically for the determination of presence/absence and/or relative/absolute abundance of the target taxon; processing the microbiome sample to have the at least one nucleic acid accessible to react with the at least one synthetic primer pair, thereby forming a processed sample; mixing the processed sample with the diagnostic kit to form a microbiome reaction mixture; subjecting the microbiome reaction mixture to a reaction under which at least one section of the accessible nucleic acid is amplified, thereby forming a reaction product; analyzing the reaction product; and determining presence/absence and/or relative/absolute abundance of the target taxon in the microbiome sample; wherein: the at least one synthetic primer pair comprises a synthetic forward primer and a synthetic reverse primer; and the at least one synthetic primer pair is selected from the synthetic primer pairs of FIG. 15, Primer Pair Set ID numbers 1 to 79, or a combination thereof.

2. The method of Claim 1 , wherein the synthetic forward/re verse primers with primer name numbers 1 to 170 that constitute the synthetic primer pairs of FIG. 15, Primer Pair Set IDs 1 to 79 have sequences, target genes, target regions, and target locations disclosed in FIG. 14, 19.

3. The method of Claim 1 , wherein the target taxon is any taxon of FIG. 15, or a combination thereof.

4. The method of Claim 1 , wherein the at least one synthetic primer pair is selected from:

FIG. 15, Primer Pair Set IDs 49 to 52, 76 to 79, or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Oxalobacter; and/or FIG. 15, Primer Pair Set IDs 53 to 58, 70 to 72, or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Desulfovibrio; and/or

5. The method of Claim 4, wherein the at least one synthetic primer pair is selected from FIG. 15, Primer Pair Set IDs 7 to 9, 31 to 39, or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Butyricimonas.

6. The method of Claim 4, wherein the at least one synthetic primer pair is selected from FIG. 15, Primer Pair Set IDs 1 to 3, 40 to 43, or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Barnesiellaceae.

7. The method of Claim 4, wherein the at least one synthetic primer pair is selected from FIG. 15, Primer Pair Set IDs 13 to 16, 21 to 22, or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Christensenellaceae.

8. The method of Claim 4, wherein the at least one synthetic primer pair is selected from FIG. 15, Primer Pair Set IDs 25 to 30, or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Christensenella.

9. The method of Claim 4, wherein the at least one synthetic primer pair is selected from FIG. 15, Primer Pair Set IDs 44 to 46, 67 to 69, or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Veillonella.

10. The method of Claim 4, wherein the at least one synthetic primer pair is selected from FIG. 15, Primer Pair Set IDs 47 to 48, 76 to 79, or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Sutterella.

11 . The method of Claim 4, wherein the at least one synthetic primer pair is selected from FIG. 15, Primer Pair Set IDs 49 to 52, 76 to 79, or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Oxalobacter.

12. The method of Claim 4, wherein the at least one synthetic primer pair is selected from FIG. 15, Primer Pair Set IDs 53 to 58, 70 to 72, or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Desulfovibrio.

13. The method of Claim 4, wherein the at least one synthetic primer pair is selected from FIG. 15, Primer Pair Set IDs 59 to 66, 73 to 75, or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Faecalibacterium.

14. The method of Claim 1 , wherein the at least one synthetic primer pair is selected from:

FIG. 15, Primer Pair Set IDs 44 to 46, or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Veillonella; and/or FIG. 15, Primer Pair Set IDs 47 for determination of presence/absence and/or relative/absolute abundance of Sutterella; and/or

15. The method of Claim 14, wherein the at least one synthetic primer pair is selected from FIG. 15, Primer Pair Set IDs 7, 9, 31 to 39, or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Butyricimonas.

16. The method of Claim 14, wherein the at least one synthetic primer pair is selected from FIG. 15, Primer Pair Set IDs 2, 43, or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Barnesiellaceae.

17. The method of Claim 14, wherein the at least one synthetic primer pair is selected from FIG. 15, Primer Pair Set ID 13, 15, 16, 21 to 22, or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Christensenellaceae.

18. The method of Claim 14, wherein the at least one synthetic primer pair is selected from FIG. 15, Primer Pair Set IDs 26, 28, 30, or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Christensenella.

19. The method of Claim 14, wherein the at least one synthetic primer pair is selected from FIG. 15, Primer Pair Set IDs 44 to 46, or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Veillonella.

20. The method of Claim 14, wherein the at least one synthetic primer pair is selected from FIG. 15, Primer Pair Set IDs 47 for determination of presence/absence and/or relative/absolute abundance of Sutterella.

21 . The method of Claim 14, wherein the at least one synthetic primer pair is selected from FIG. 15, Primer Pair Set IDs 49 to 52, or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Oxalobacter.

22. The method of Claim 14, wherein the at least one synthetic primer pair is selected from FIG. 15, Primer Pair Set IDs 58, 71 , or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Desulfovibrio.

23. The method of Claim 14, wherein the at least one synthetic primer pair is selected from FIG. 15, Primer Pair Set IDs 59 to 66, 74, 75, or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Faecalibacterium.

24. The method of Claim 1 , wherein the synthetic primer pair comprising a synthetic forward primer and a synthetic reverse primer, each having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology to any one of the synthetic forward primers of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 16, 18, 20, 21, 23, 25, 26, 27, 28, 30, 31, 32, 34, 36, 38, 40, 41, 43, 44, 46, 47, 49, 50, 51, 54, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 77, 79, 81, 91, 93, 95, 97, 99, 101, 126, 128, 130, 131, 132, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, and 169 and/or the synthetic reverse primers of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 17, 19, 22, 24, 29, 33, 35, 37, 39, 42, 45, 48, 52, 53, 55, 56, 57, 59, 61 , 63, 65, 67, 69, 71 , 73, 75, 78, 80, 82, 92, 94, 96, 98, 100, 102, 127, 129, 133, 134, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, and 170.

25. The method of Claim 1 , wherein the diagnostic kit comprises a chemical assay suitable for a polymerase chain reaction (PCR); and wherein the microbiome reaction mixture is subjected to a PCR condition under which, if the target taxon is present in the microbiome sample, the at least one nucleic acid of the target taxon is amplified, and produces a fluorescence signal; and wherein analyzing the reaction product comprises measuring amount of fluorescence signal produced.

26. The method of Claim 1 , wherein the diagnostic kit comprises a chemical assay suitable for a polymerase chain reaction (PCR); and wherein the microbiome reaction mixture is subjected to a PCR condition under which, if the target taxon is present in the microbiome sample, the at least one nucleic acid of the target taxon is amplified; and wherein analyzing the reaction product comprises a gel electrophoresis method.

27. A diagnostic kit comprising a synthetic primer pair for determination of presence/absence and/or relative/absolute abundance of a target taxon present in a microbiome sample, comprising at least one synthetic primer pair selected from the synthetic primer pairs of FIG. 15, Primer Pair Set IDs 1 to 79, or a combination thereof.

28. The diagnostic kit of Claim 26, wherein the microbiome sample comprises a soil sample, a bodily fluid/solid sample, a tissue sample, or a combination thereof.

29. The diagnostic kit of Claim 26, wherein the microbiome sample comprises soil, stool, saliva, skin, nasal swab, vaginal swab, blood, urine, hair, oral swab, or a combination thereof.

30. A nucleic acid having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology to any one of the sequences of SEQ ID NO: 1-170, or any combination thereof.

31 . A synthetic primer pair comprising a synthetic forward primer and/or a synthetic reverse primer, each having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology to any one of the synthetic forward primers and/or the synthetic reverse primers of SEQ ID NO: 1-170.

32. A method of treatment of a patient, comprising: obtaining a microbiome sample from the patient, wherein the microbiome sample potentially comprises at least one nucleic acid; having a diagnostic kit comprising at least one synthetic primer pair designed specifically for the identification of the target taxon; processing the microbiome sample to have the at least one nucleic acid accessible to react with the at least one synthetic primer pair, thereby forming a processed sample; mixing the processed sample with the diagnostic kit to form a microbiome reaction mixture; subjecting the microbiome reaction mixture to a reaction under which at least one section of the accessible nucleic acid is amplified, thereby forming a reaction product; analyzing the reaction product; determining presence/absence and/or relative/absolute abundance of the target taxon in the microbiome sample, thereby forming a taxon profile of the patient; comparing the taxon profile of the patient with the taxon profile of a healthy subject; determining whether the patient has a disease; and treating the patient if the patient has the disease; wherein the at least one synthetic primer pair comprises a synthetic forward primer and a synthetic reverse primer; and wherein the at least one synthetic primer pair is selected from the synthetic primer pairs of FIG. 15, Primer Pair Set IDs 1 to 79, or a combination thereof.

33. The method of any preceding claims or any following claims, wherein the at least one synthetic primer pair is selected from:

FIG. 15, Primer Pair Set IDs 7 to 9, 31 to 39, or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Butyricimonas; and/or FIG. 15, Primer Pair Set IDs 1 to 3, 40 to 43, or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Barnesiellaceae; and/or

34. The method of any preceding claims or any following claims, wherein the at least one synthetic primer pair is selected from FIG. 15, Primer Pair Set IDs 7 to 9, 31 to 39, or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Butyricimonas.

35. The method of any preceding claims or any following claims, wherein the at least one synthetic primer pair is selected from FIG. 15, Primer Pair Set IDs 1 to 3, 40 to 43, or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Barnesiellaceae.

36. The method of any preceding claims or any following claims, wherein the at least one synthetic primer pair is selected from FIG. 15, Primer Pair Set IDs 13 to 16, 21 to 22, or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Christensenellaceae.

37. The method of any preceding claims or any following claims, wherein the at least one synthetic primer pair is selected from FIG. 15, Primer Pair Set IDs 25 to 30, or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Christensenella.

38. The method of any preceding claims or any following claims, wherein the at least one synthetic primer pair is selected from FIG. 15, Primer Pair Set IDs 44 to 46, 67 to 69, or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Veillonella.

39. The method of any preceding claims or any following claims, wherein the at least one synthetic primer pair is selected from FIG. 15, Primer Pair Set IDs 47 to 48, 76 to 79, or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Sutterella.

40. The method of any preceding claims or any following claims, wherein the at least one synthetic primer pair is selected from FIG. 15, Primer Pair Set IDs 49 to 52, 76 to 79, or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Oxalobacter.

41 . The method of any preceding claims or any following claims, wherein the at least one synthetic primer pair is selected from FIG. 15, Primer Pair Set IDs 53 to 58, 70 to 72, or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Desulfovibrio.

42. The method of any preceding claims or any following claims, wherein the at least one synthetic primer pair is selected from FIG. 15, Primer Pair Set IDs 59 to 66, 73 to 75, or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Faecalibacterium.

43. The method of any preceding claims or any following claims, wherein the at least one synthetic primer pair is selected from:

FIG. 15, Primer Pair Set IDs 2, 43, or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Barnesiellaceae; and/or

- 60 - FIG. 15, Primer Pair Set IDs 59 to 66, 74, 75, or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Faecalibacterium.

44. The method of any preceding claims or any following claims, wherein the at least one synthetic primer pair is selected from FIG. 15, Primer Pair Set IDs 7, 9, 31 to 39, or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Butyricimonas.

45. The method of any preceding claims or any following claims, wherein the at least one synthetic primer pair is selected from FIG. 15, Primer Pair Set IDs 2, 43, or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Barnesiellaceae.

46. The method of any preceding claims or any following claims, wherein the at least one synthetic primer pair is selected from FIG. 15, Primer Pair Set ID 13, 15, 16, 21 to 22, or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Christensenellaceae.

47. The method of any preceding claims or any following claims, wherein the at least one synthetic primer pair is selected from FIG. 15, Primer Pair Set IDs 26, 28, 30, or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Christensenella.

48. The method of any preceding claims or any following claims, wherein the at least one synthetic primer pair is selected from FIG. 15, Primer Pair Set IDs 44 to 46, or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Veillonella.

49. The method of any preceding claims or any following claims, wherein the at least one synthetic primer pair is selected from FIG. 15, Primer Pair Set IDs 47 for determination of presence/absence and/or relative/absolute abundance of Sutterella.

50. The method of any preceding claims or any following claims, wherein the at least one synthetic primer pair is selected from FIG. 15, Primer Pair Set IDs 49 to 52, or

- 61 - a combination thereof for determination of presence/absence and/or relative/absolute abundance of Oxalobacter.

51 . The method of any preceding claims or any following claims, wherein the at least one synthetic primer pair is selected from FIG. 15, Primer Pair Set IDs 58, 71 , or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Desulfovibrio.

52. The method of any preceding claims or any following claims, wherein the at least one synthetic primer pair is selected from FIG. 15, Primer Pair Set IDs 59 to 66, 74, 75, or a combination thereof for determination of presence/absence and/or relative/absolute abundance of Faecalibacterium.

53. The method of any preceding claims or any following claims, wherein the synthetic forward primer and/or the synthetic reverse primer is a polymerase chain reaction (PCR) primer.

54. The method of any preceding claims or any following claims, wherein the patient is a human or an animal.

55. The method of any preceding claims or any following claims, wherein the healthy subject is human, if the patient is human; or the healthy subject is an animal, if the patient is an animal.

56. The method of any preceding claims or any following claims, wherein the patient is more or less-susceptible to developing gastro-intestinal toxicity after chemotherapy and/or radiation-therapy.

57. The method or diagnosis kit of any preceding claims or any following claims, wherein the microbiome sample comprises a soil sample, a bodily fluid/solid sample, a tissue sample, or a combination thereof.

58. The method of any preceding claims or any following claims, wherein the microbiome sample comprises soil, stool, saliva, skin, nasal swab, vaginal swab, blood, urine, hair, oral swab, or a combination thereof.

59. The method or diagnosis kit of any preceding claims or any following claims, wherein the diagnostic kit is configured to differentiate patients that are more or less-

- 62 - susceptible to developing gastro-intestinal toxicity after chemotherapy and/or radiationtherapy.

60. The method of any preceding claims or any following claims, wherein amplifying the nucleic acid comprises amplifying the nucleic acid with a PCR technique or a qPCR technique.

61 . The method of any preceding claims or any following claims, wherein the microbiome sample is a microbiome sample from a patient.

62. Use of the method of the preceding and the following claims to identify and categorize subjects as more or less susceptible to gastrointestinal toxicity.

63. Use of the method of the preceding and the following claims to identify and categorize subjects as more or less susceptible to gastrointestinal toxicity after chemotherapy and/or radiation-therapy.

64. The method of any preceding claims or any following claims, wherein determining the presence/absence and/or relative/absolute abundance comprises measuring a fluorescence signal during the amplifying step.

65. The method of any preceding claims or any following claims, further comprising administering a therapeutic to the patient.

66. The method of any preceding claims or any following claims, further comprising administering a therapeutic to the patient, and wherein the therapeutic is not chemotherapy and/or radiation.

67. Any combination of above kits, products, and methods is within the scope of the instant disclosure.

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