JP-7856427-B2 - Rapid incubation and nucleic acid enrichment for identification and analysis of microbial samples

Inventors

• ワン、クリフォード リー

Assignees

• イルミナ インコーポレイティッド

Dates

Publication Date

20260511

Application Date

20200428

Priority Date

20190429

Claims (20)

1. A method for identifying and analyzing viable and/or growing microorganisms in a sample, (a) Incubating a sample containing or suspected to contain one or more types of microorganisms in the presence of one or more types of nucleosides or nucleotide analogs, wherein the one or more types of nucleosides or nucleotide analogs are incorporated into newly synthesized microbial nucleic acids, selected from 5-ethynyl-2'-deoxyuridine (EdU) and 5-ethynyl-uridine (EU), (b) Labeling the newly synthesized microbial nucleic acid by contacting it with an azide-linker-biotin labeling reagent that selectively binds to one or more types of nucleosides or nucleotide analogs, (c) Isolating or purifying the labeled newly synthesized microbial nucleic acid using streptavidin or an avidin solid support pull-down agent, (d) Cutting the linker to obtain microbial nucleic acids isolated or purified from the pull-down agent, (e) A method comprising determining the identity of the viable and/or growing microorganisms in the sample based on sequencing or identity determination of the isolated or purified newly synthesized microbial nucleic acid.
2. The method according to claim 1, wherein the sample is obtained from a subject suspected of having a microbial infection or who is actually infected with a microbial infection.
3. The method according to claim 2, wherein the subject is suspected of having sepsis or is diagnosed with sepsis.
4. The method according to any one of claims 1 to 3, wherein the obtained sample in (a) is treated using a dehosting method before (b) to selectively remove non-microbial nucleic acids.
5. The aforementioned dehosting method, Nonmicrobial nucleic acids, The method according to claim 4, comprising: (a) selectively cleaving nonmicrobial DNA by contacting the obtained sample with a recombinant protein comprising a binding domain that selectively binds to nonmicrobial nucleic acids bound by histones or nonmicrobial nucleic acids containing methylated CpG residues, and a nuclease domain having the activity to cleave nucleic acids; or (b) removing the sample by using an affinity agent bound to a solid substrate that selectively binds to nucleic acids bound by histones or selectively binds to methylated CpG residues of nonmicrobial nucleic acids.
6. The method according to claim 1, wherein the sample is an environmental sample obtained from an environmental testing site.
7. The method according to claim 6, wherein the environmental testing site is tested for microbial contamination.
8. The method according to claim 1, wherein the sample is a sample obtained from food suspected of being contaminated with microorganisms.
9. The method according to any one of claims 1 to 8, wherein the one or more types of microorganisms are bacteria, fungi, viruses, algae, archaea, and/or protozoa.
10. The aforementioned bacteria include Actinomyces israelii, Bacillus anthracis, Bacillus cereus, Bartonella henselae, Bartonella quintana, Bordetella pertussis, Borrelia burgdorferi, Borrelia garinii, Borrelia afzelii, Borrelia recurrentis, Brucella abortus, Brucella canis, Brucella melitensis, Brucella suis, and Campylobacter jejuni. Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydophila psittaci, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani, Corynebacterium diphtheriae, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, Francisella tularensis Mycobacterium tularensis), Haemophilus influenzae, Helicobacter pylori, Legionella pneumophila, Leptospira interrogans, Leptospira santarosai, Leptospira weilii, Leptospira noguchii, Listeria monocytogenes, Mycobacterium leprae, Mycobacterium tuberculosis, Mycobacterium ulcerans, Mycoplasma pneumoniae *Neisseria pneumoniae*, *Neisseria gonorrhoeae*, *Neisseria meningitidis*, *Pseudomonas aeruginosa*, *Rickettsia rickettsia*, *Salmonella typhi*, *Salmonella typhimurium*, *Shigella sonnei*, *Staphylococcus aureus*, *Staphylococcus epidermidis*, *Staphylococcus saprophyticus*, *Streptococcus agalactiae*, *Streptococcus pneumoniae* The method according to claim 9, selected from *Pneumoniae*, *Streptococcus pyogenes*, *Treponema pallidum*, *Ureaplasma urealyticum*, *Vibrio cholerae*, *Yersinia pestis*, *Yersinia enterocolitica*, and/or *Yersinia pseudotuberculosis*.
11. The aforementioned fungi include Absidia corymbifera, Absidia ramose, Achorion gallinae, Actinomadura spp., Ajellomyces dermatididis, Aleurisma brasiliensis, Allersheria boydii, Arthroderma spp., Aspergillus flavus, Aspergillus fumigatu, Basidiobolus spp., Blastomyces spp., and Cadphora genus. spp), Candida albicans, Cercospora apii, Chrysosporium spp, Cladosporium spp, Cladothrix asteroids, Coccidioides immitis, Cryptococcus albidus, Cryptococcus gattii, Cryptococcus laurentii, Cryptococcus neoformans, Cunninghamella elegans, Dematium wernecke, Discomyces israerii israelii), Emmonsia spp., Emmonsiella capsulate, Endomyces geotrichum, Entomophthora coronate, Epidermophyton floccosum, Filobasidiella neoformans, Fonsecaea spp., Geotrichum candidum, Glenospora khartoumensis, Gymnoascus gypseus, Haplosporangium parvum, Histoplasma, Histoplasma capsulatum Capsulatum), Hormiscium dermatididis, Hormodendrum spp., Keratinomyces spp., Langeronia soudanense, Leptosphaeria senegalensis, Lichtheimia corymbifera, Lobmyces loboi, Loboa loboi, Lobomycosis, Madurella spp., Malassezia furfur, Micrococcus pelletieri, Microsporum spp., Monilia spp.), Mucor spp., Mycobacterium tuberculosis, Nannizzia spp., Neotestudina rosatii, Nocardia spp., Oidium albicans, Oospora lactis, Paracoccidioides brasiliensis, Petriellidium boydii, Phialophora spp., Piedraia hortae, Pityrosporum furfur, Pneumocystis irovetii Pneumocystis carinii (jirovecii), Pullularia gougerotii, Pyrenochaeta romeroi, Rhinosporidium seeberi, Sabouraudites (Microsporum), Sartorya fumigate, Sepedonium, Sporotrichum spp., Stachybotrys, Stachybotrys chartarum, Streptomyce spp., Tinea spp., Torula The method according to claim 9, comprising a species selected from *Trichophyton* spp, *Trichosporon* spp, and/or *Zopfia rosatii*.
12. The aforementioned viruses include Simplexvirus, Varicellovirus, Cytomegalovirus, Roseolovirus, Lympho-cryptovirus, Rhadinovirus, Mastadenovirus, α-Papillomavirus, β-Papillomavirus, X-Papillomavirus, γ-Papillomavirus, Mupapillomavirus, Nupapillomavirus, Alphapolyomavirus, Betapolyomavirus, γ-Polyomavirus, Deltapolyomavirus, and Morsipot. Molluscipoxvirus, Orthopoxvirus, Parapoxvirus, α-Torquevirus, β-Torquevirus, γ-Torquevirus, Cyclovirus, Gemycircular, Gemykibivirus, Gemyvongvirus, Erythrovirus, Dependovirus, Bocavirus, Orthohepadnavirus, Gammaretrovirus, Deltaretrovirus, Lentivirus, Simiispumavirus, Coltivirus, Rotavirus, Seadonavirus navirus), α-Coronavirus, β-Coronavirus, Torovirus, Mamastrovirus, Norovirus, Sapovirus, Flavivirus, Hepacivirus, Pegivirus, Orthohepevirus, Cardiovirus, Cosavirus, Enterovirus, Hepatovirus, Kobuvirus, Parechovirus, Rosavirus, Salivirus, Alphavirus, Rubivirus, Ebolavirus, Marburgvirus, Henipavirus, Morbillivirus ( The method according to claim 9, comprising a virus selected from Morbilivirus, respirovirus, rubulavirus, metapneumovirus, orthopneumovirus, Ledantevirus, lyssavirus, vesculovirus, mammarenavirus, orthohantavirus, orthonairovirus, orthobunyavirus, phlebovirus, alpha-influenzavirus, beta-influenzavirus, gamma-influenzavirus, quaranjavirus, thogotovirus, and/or deltavirus.
13. The method according to any one of claims 1 to 12, wherein the sample is incubated for 5 to 180 minutes in the presence of one or more types of nucleosides or nucleotide analogs.
14. The method according to claim 13, wherein the sample is incubated for 30 to 120 minutes in the presence of one or more types of nucleosides or nucleotide analogs.
15. The method according to any one of claims 1 to 14, wherein the labeled reagent binds to or with one or more types of nucleosides or nucleotide analogs via click chemistry, strain [3+2] cycloaddition, or Staudinger ligation.
16. The labeling reagent is The method according to claim 1, selected from the following.
17. The method according to any one of claims 1 to 16, wherein the linker of the labeled reagent includes a chemically cleavable linker or an enzymatically cleavable linker.
18. The method according to claim 17, wherein the severable linker is an acid-unstable linker or a disulfide linker.
19. The method according to claim 18, wherein the acid-unstable linker comprises a hydrazone or a cis-aconityl group.
20. The method according to claim 17, wherein the enzymatically cleavable linker includes a peptide-based linker or a β-glucuronide-based linker.

Description

(Cross-reference of related applications) This application claims priority under Section 119 of the United States Patent Act to U.S. Provisional Patent Application No. 62/840,322, filed on 29 April 2019, the disclosure of which is incorporated herein by reference. (Field of invention) This disclosure relates to methods, compositions, and kits for the identification and analysis of microorganisms in a sample using nucleosides or nucleotide analogs. Determining whether a patient has a microbial infection is a common clinical challenge. Sepsis is the most common cause of death among hospitalized patients, with an estimated 200,000 deaths annually in the United States. However, sepsis is an ambiguous clinical syndrome with a wide range of clinical manifestations. Diagnosis is usually based on suspected infection combined with signs of organ failure. Early diagnosis and administration of antibiotics for sepsis is crucial because progression to severe sepsis or septic shock can have serious consequences. Unfortunately, differentiating sepsis from other inflammatory conditions is often difficult in critically ill patients. Detecting bacterial infection in the blood is a critical step in diagnosing sepsis and initiating antibiotic treatment. However, blood cultures are negative in 60–70% of patients with severe sepsis, and studies have shown >80% to be negative. In addition, conventional microbiological methods are too time-consuming to influence primary therapy against pathogenic bacteria. While the development of PCR and mass spectrometry has increased the possibility of identifying bacteria in blood samples, they often rely on time-consuming pre-analytical preparations such as blood cultures to increase the pathogen load. Infection proxies include elevated levels of circulating cytokines and acute-phase proteins such as C-reactive proteins, although their concentrations also increase during physiological events such as childbirth or during pathological tissue injury such as burns. Typically, for sepsis, blood culture tests are performed to identify which type of bacteria or fungus caused the infection in the blood. Blood cultures are collected separately from other blood tests and are often taken multiple times from different veins. It can take several days to obtain the results of a blood culture. In vitro culture conditions result in only one-third to half of people with sepsis having positive blood cultures, meaning that bacteria actually grow and multiply under in vitro conditions. Currently, bacterial detection often requires culturing for two reasons: (1) to isolate bacteria for analysis, and (2) to reduce any contaminating background cells or other substances that could make analysis difficult or impossible. For example, in patients with sepsis, blood needs to be cultured to isolate the pathogen. Similarly, in food surveillance, samples need to be cultured to isolate contaminating microorganisms. Unfortunately, standard culture processes can take several days. In the case of sepsis, this lag time can lead to unnecessary antibiotic administration or misdiagnosis, potentially resulting in patient complications or death. In the food industry, this lag time delays information leading to recalls or other preventative measures. Therefore, rapid detection of active infections can enable measures that can mitigate problems and save lives. While PCR detection may not require long-term culture, unlike unbiased sequencing methods, PCR requires prior knowledge of the target organism's genome sequence. That is, researchers need to know what they are looking for, which may not be applicable to rare or undiscovered organisms. Furthermore, PCR-dependent methods only detect the presence of genetic material in a sample and cannot distinguish whether the material originates from living or dead organisms. In many cases, identifying active infections caused by living microorganisms is a critical consideration for treatment options and even for identifying contaminants in food or the environment. This disclosure provides a method for identifying and analyzing viable and/or growing microorganisms in a sample, comprising: (a) obtaining a sample having or suspected having one or more types of microorganisms; (b) incubating the sample in the presence of one or more types of nucleosides or nucleotide analogs so that the one or more types of nucleosides or nucleotide analogs are incorporated into newly synthesized microbial nucleic acids; (c) labeling the newly synthesized microbial nucleic acids by contacting them with one or more types of nucleosides or nucleotide analogs or with labeling reagents that selectively bind thereto; (d) isolating or purifying the labeled newly synthesized microbial nucleic acids; and (e) determining the identity of viable and/or growing microorganisms in the sample based on sequencing or identity determination of the isolated or purified newly synthesized microbial nucleic acids. In another embodiment,