WO2007134254A2 - Surveillance et suivi de communautés virales du tractus respiratoire au moyen d'échantillons d'expectorations humaines non cultivées - Google Patents

Surveillance et suivi de communautés virales du tractus respiratoire au moyen d'échantillons d'expectorations humaines non cultivées Download PDF

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Publication number
WO2007134254A2
WO2007134254A2 PCT/US2007/068811 US2007068811W WO2007134254A2 WO 2007134254 A2 WO2007134254 A2 WO 2007134254A2 US 2007068811 W US2007068811 W US 2007068811W WO 2007134254 A2 WO2007134254 A2 WO 2007134254A2
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WIPO (PCT)
Prior art keywords
sample
nucleic acid
sputum
pellet
buffer
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PCT/US2007/068811
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WO2007134254A3 (fr
Inventor
Forest Rohwer
Mike Furlan
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San Diego State University Research Foundation
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San Diego State University Research Foundation
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay

Definitions

  • compositions e.g., kits
  • methods for surveying and characterizing the presence of viral, bacterial and/or phage nucleic acid including the complete viral, bacterial and/or phage nucleic acid (e.g., DNA and/or RNA) population found in an individual, e.g., in a human or other animal, including its complete viral, bacterial and/or phage pulmonary population, using the individual's sputum, for example, by using a human sputum sample.
  • This invention provides methods for isolating a virus nucleic acid-comprising sample from a sputum sample, the method comprising the following steps: (a) providing a sputum sample; (b) treating the sputum sample with a reducing agent; (c) adding a buffer to the treated sample of (b); (d) mechanically homogenizing (or equivalent, e.g., macerating) the sample of (c); (e) centrifuging the sample of (d) to generate a sample pellet; (f) suspending the pellet of step (e) in a buffer and preserving an aliquot with an equal amount of para- formaldehyde or equivalent; (g) centrifuging the resuspended pellet of (f) in density gradient comprising at least a 1.5 g/ml density band (or equivalent); and, (h) isolating a virus nucleic acid-comprising sample from the 1.5 g/ml density band.
  • the sputum sample is a human sputum sample.
  • the reducing agent comprises dithiothreitol (DDT), or equivalent.
  • the sputum sample can be mixed in a solution of about 5%, 6%, 7%, 8%, 9% or 10% or more dithiothreitol (DDT).
  • full strength dithiothreitol (DDT) is mixed in a magnesium-based buffer to generate the DTT solution.
  • the sputum sample has a volume of about 10 mis, or from between about 1 to 25 mis, or from about 3 to 20 mis, or from about 6 to 15 mis, or from about 8 to 12 mis.
  • the buffer comprises a magnesium-based buffer, e.g., a TRISTM buffer with magnesium salt, or equivalent.
  • the suspended pellet sample of step (f) is partially purified of (cleared of) bacterial and other cellular debris from the viral component of the suspended pellet sample before the centrifugation step.
  • the suspended pellet sample of step (f) can be partially purified of (cleared of) bacterial and other cellular debris by using one or more filters, and/or one or more chromatographs.
  • the chromatograph can comprise affinity chromatography, lectin chromatography, antibody chromatography, diethylaminoethyl cellulose chromatography, ion exchange chromatography and/or high performance liquid chromatography (HPLC).
  • HPLC high performance liquid chromatography
  • the one or more filters can be anywhere between about 0.1 to 2.0 micrometer filters, e.g., a 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 and/or 1.0 micrometer filter; any combination or order of filters can be used, in combination with or separate from use of one or more chromatographs.
  • the density gradient comprises a CsCl gradient.
  • the method further comprises isolating a bacterial or a phage nucleic acid from the nucleic acid isolated in step (h). In one aspect, the method further comprises sequencing the isolated nucleic acid. In one aspect, the method further comprises comparing the sequenced viral, phage and/or bacterial nucleic acid to databases of viral nucleic acids to identify the family or species of the isolated viral, phage and/or bacterial nucleic acid.
  • the invention provides methods for isolating a viral, phage and/or bacterial nucleic acid from a sputum sample, the method comprising the following steps: (a) providing a sputum sample; (b) treating the sputum sample with a reducing agent; (c) adding a buffer to the treated sample of (b); (d) mechanically homogenizing or macerating the sample of (c); (e) generating a virus-comprising pellet or equivalent from the sample of (d); (f) suspending the virus- comprising pellet or equivalent of step (e) in a buffer; (g) centrifuging the suspended pellet of (f) in a density gradient comprising at least a 1.5 g/ml density band; and, (h) isolating a viral, phage and/or bacterial nucleic acid sample from the 1.5 g/ml density band.
  • the invention provides methods for isolating a viral, phage and/or bacterial nucleic acid from a sputum sample, the method comprising the following steps: (a) providing a sputum sample; (b) treating the sputum sample with a reducing agent; (c) adding a buffer to the treated sample of (b); (d) mechanically homogenizing or macerating the sample of (c); (e) generating a virus-comprising, bacterial-comprising and/or phage-comprising pellet or equivalent from the sample of (d); (f) suspending the virus-comprising, bacterial-comprising and/or phage- comprising pellet or equivalent of step (e) in a buffer; and, in one aspect, (g) isolating a viral, phage and/or bacterial nucleic acid from the sample.
  • viral, phage and/or bacterial nucleic acid is isolated by a density gradient, a filter and/or a chromatograph, or any combination thereof, and the density gradient, a filter and/or a chromatograph can be used in any order.
  • the chromatograph can comprise an affinity chromatography, lectin chromatography, antibody chromatography, diethylaminoethyl cellulose chromatography, ion exchange chromatography and/or high performance liquid chromatography (HPLC).
  • HPLC high performance liquid chromatography
  • the density gradient can comprise a CsCl gradient.
  • the method comprises isolating a phage and/or bacterial nucleic acid from an isolated virus nucleic acid sample. In one aspect, the method further comprises sequencing the isolated nucleic acid. In one aspect, the method further comprises comparing the sequenced viral nucleic acid to databases of viral or phage nucleic acids to identify the family or species of the isolated virus and/or phage.
  • the method also comprises isolating phage and/or bacterial nucleic acid from the sputum sample.
  • the sputum is isolated from a human or an animal, e.g., an individual or patient with and acute or a chronic pulmonary infection, disease and/or condition, including an individual with the acute or chronic infection, disease; in one aspect, the condition is the flu, or is cystic fibrosis, or is a viral bronchial or lung infection.
  • the methods further comprise culturing the isolated virus. In one aspect of any method of the invention, the methods further comprise culturing the isolated phage. In one aspect of any method of the invention, the methods further comprise culturing the isolated bacteria.
  • the methods further comprise cloning the isolated virus nucleic acid. In one aspect of any method of the invention, the methods further comprise cloning the isolated phage nucleic acid. In one aspect of any method of the invention, the methods further comprise cloning the isolated bacterial nucleic acid.
  • the invention provides a kit comprising the appropriate solutions and instructions for practicing the invention, including a kit comprising the appropriate solutions, as set forth herein, for collecting a sample of sputum from a patient and isolating a viral sample.
  • compositions e.g., kits
  • methods for surveying and characterizing the presence of viral, bacterial and/or phage nucleic acid including the complete viral, bacterial and/or phage nucleic acid (e.g., DNA and/or RNA) population found in an individual, e.g., a human or other animal, using the individual's sputum, for example, by using a human sputum sample.
  • viral, bacterial and/or phage nucleic acid e.g., DNA and/or RNA
  • the presence of viral, bacterial and/or phage nucleic acid in the individual is determined by extraction of the nucleic acid, shotgun sequencing of the nucleic acid and subsequent comparison of the resultant sequences against existing nucleic acid (e.g., DNA) databases to identify known and, if present, novel viruses and/or phage, by their nucleic acid (DNA and/or RNA) sequences.
  • This exemplary protocol of the invention uses a combination of established techniques and novel techniques (including established techniques used in novel ways) to extract the viral, bacterial and/or phage nucleic acid (e.g., DNA and/or RNA) from the sample.
  • the sputum sample e.g., human sputum
  • DDT dithiothreitol
  • the sputum sample comprises a minimum of about 10 ml human sputum; or from about 1 to 25 mis, or from about 3 to 20 mis, or from about 6 to 15 mis, or from about 8 to 12 mis.
  • the 10% DTT solution is made by diluting full strength DTT with an appropriate volume of a buffer, e.g., a magnesium-based buffer (such as SM buffer, or TRISTM buffer with magnesium salt).
  • a buffer e.g., a magnesium-based buffer (such as SM buffer, or TRISTM buffer with magnesium salt).
  • DDT is designed to extract large cells from human sputum, exposing the viral cells to isolation.
  • SM is a typical buffer used for phage extraction, but because of the chemical base for the buffer (i.e., magnesium), this buffer can counteract the effects of the DDT.
  • a 10% solution can be effective for isolating viruses.
  • the sample is subsequently mechanically homogenized. This is necessary because although DDT is supposed to extract cells at room temperature, it was found that because of the high viscosity of human sputum samples (particularly those of patients with cystic fibrosis), it was ineffective without homogenization.
  • the sample is incubated at about 37°C, or between about 35°C to 39°C, or between about 33°C to 41°C, for about 15 minutes, or from between about 10 to 20 minutes. This allows the DDT opportunity to act on the large sputum cells.
  • the temperature and time can optimize the breakdown of the sputum while preserving the viral community.
  • a sample pellet or equivalent, is generated; for example, in one aspect, the sample is centrifuged to generate a sample pellet according to normal laboratory procedures.
  • the pellet (or equivalent) is resuspended in a buffer, e.g., a magnesium-based buffer (such as SM buffer, or TRISTM buffer with magnesium salt), and an aliquot is preserved with an equal amount of para- formaldehyde or equivalent.
  • a buffer e.g., a magnesium-based buffer (such as SM buffer, or TRISTM buffer with magnesium salt)
  • the suspension is necessary to enable the sample to be loaded into a density gradient, e.g., a CsCl gradient or equivalent, that clears bacterial and other cellular debris from the viral component.
  • the sample before loading into the density gradient, e.g., a CsCl gradient or equivalent, the sample must be filtered - or equivalent (e.g., columns having the same effect), and in one aspect, through a range of filters (or columns) that are experimentally determined based on the types of viruses found in sputum samples.
  • the density gradient e.g., a CsCl gradient or equivalent
  • viruses are subsequently isolated using standard density gradient techniques, e.g., CsCl gradient techniques, or equivalent, and the viruses extracted from the 1.5 g/ml density band.
  • Equivalents include any chromatograph protocol(s) that can remove bacterial and other cellular debris from the viral component, including affinity chromatography, such as lectin chromatography or antibody chromatography; or diethylaminoethyl cellulose chromatography, or ion exchange chromatography, or any form of high performance liquid chromatography (HPLC). These equivalents can be used in place of, or adjunct to, using standard density gradient techniques.
  • nucleic acid extraction and cleanup protocols are used, e.g., formamide/CTAB buffer DNA extraction and cleanup.
  • Techniques for the manipulation of nucleic acids such as, e.g., subcloning, labeling probes (e.g., random-primer labeling using Klenow polymerase, nick translation, amplification), sequencing, hybridization and the like are well described in the scientific and patent literature, see, e.g., Sambrook, ed., MOLECULAR CLONING: A LABORATORY MANUAL (2ND ED.), VOIS. 1-3, Cold Spring Harbor Laboratory, (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Ausubel, ed.
  • the samples are sequenced according to standard methodology (e.g., PYROSEQUENCINGTM by 454 Life Sciences, Branford, CT).
  • the recovered DNA sequences are subsequently compared with any sequence database to characterize and/or identify the organisms, including the viruses, isolated from the sputum.
  • the SEED database of the Fellowship for Interpretation of Genomes (FIG) (Burr Ridge, IL) is used to identify known and novel viral DNA sequences.
  • Amplification reactions can be used to quantify the amount of nucleic acid in a sample (such as the amount of viral nucleic acid isolated from a sputum sample), label the nucleic acid (e.g., to apply it to an array or a blot), detect the nucleic acid, or quantify the amount of a specific nucleic acid in a sample.
  • message isolated from a cell or a cDNA library are amplified.
  • the skilled artisan can select and design suitable oligonucleotide amplification primers.
  • Amplification methods are also well known in the art, and include, e.g., polymerase chain reaction, PCR (see, e.g., PCR Protocols, A Guide to Methods and Applications, ed. Innis, Academic Press, N.Y. (1990) and PCR Strategies (1995), ed. Innis, Academic Press, Inc., N.Y., ligase chain reaction (LCR) (see, e.g., Wu (1989) Genomics 4:560; Landegren (1988) Science 241:1077; Barringer (1990) Gene 89:117); transcription amplification (see, e.g., Kwoh (1989) Proc. Natl. Acad. Sci.
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • the invention provides methods and formulations for isolating viral nucleic acid, e.g., DNA or RNA, from sputum, e.g., human sputum.
  • sputum e.g., human sputum.
  • the associated utility uses existing methodology to identify and monitor the viral communities.
  • the sequencing was done using PYROSEQUENCINGTM by 454 Life Sciences, Branford, CT. This sequencing can be done with as little as five micrograms of DNA. This protocol can yield thirty million base-pairs that can be used to characterize the (isolated) viral community and search against various databases for similar sequence matches.
  • the databases include the NCBI Non-Redundant (NR) database. This particular database contains the collection of most publicly available sequences for most organisms and environments. Searches against the NR will allow us to know if the sequences are similar to known sequences or completely novel.
  • SEED which is part of the Fellowship for Interpretation of Genomes (FIG) (Burr Ridge, IL).
  • FIG uses subsystems to annotate both complete and partial genome sequences.
  • Subsystems are biochemical pathway, fragments of pathways, clusters of genes that function together, or any group of genes that any annotator considers to be related.
  • the subsystems are annotated across genomes by the annotators, providing the most reliable and consistent annotations within and between genomes.
  • the subsystems-based annotations are ongoing and at a given point in time the subsystems represent the snapshot of the best available annotation of the SEED database.
  • Example 2 Exemplary protocols [0037] The following example describes an exemplary protocol of the invention. This exemplary protocol is designed for a minimum of 10 milliliters of Human sputum sample.
  • SM buffer magnesium based buffer
  • SPUTOLYSINTM Calbiochem, San Diego, CA
  • DTT dithiothreitol
  • each of the following fractions is 1 milliliter in volume and their densities are as follows: lower faction is 1.7 grams per milliliter, middle fraction is 1.5 grams per milliliter, and the upper fraction is 1.35 grams per milliliter.
  • the densities of the layers may vary from project to project. The densities chosen can be determined through experimentation and verification with microscopy. 10. Centrifuge for two hours at twenty two thousand R. P.M. and a temperature of 4 degrees
  • Microscopy check In order to see that you are following the protocol, make slides from the parts of the protocol where 100 ul of sample was collected. To make slides follow the following protocol: a. Take three 15ml Falcon tubes and label them with the same labeling that is on the microtubes you plan to transfer fixed sample from; b. Fill each tube with 4990 ul of Sigma water; c. Transfer 10 ul of sample from the appropriate Microtube to the correctly labeled Falcon tube; d. Vortex all tubes; e. Filter trough a .02 um filter that is capable of being mounted on to a microscope slide; f.
  • CTAB DNA clean up from formide extraction

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  • Chemical & Material Sciences (AREA)
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  • General Health & Medical Sciences (AREA)
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  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
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Abstract

L'invention concerne des compositions (par exemple des trousses) et des procédés destinés à surveiller et à caractériser la présence d'acides nucléiques viraux, bactériens et/ou phagiques, incluant la population d'acides nucléiques viraux et/ou phagiques entière (par exemple l'ADN et/ou l'ARN), trouvés chez un individu, notamment un être humain ou un autre animal, y compris la population pulmonaire virale, bactérienne et/ou phagique entière, en utilisant les expectorations des individus, comme un échantillon d'expectoration humaine par exemple.
PCT/US2007/068811 2006-05-12 2007-05-11 Surveillance et suivi de communautés virales du tractus respiratoire au moyen d'échantillons d'expectorations humaines non cultivées Ceased WO2007134254A2 (fr)

Applications Claiming Priority (2)

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US79975906P 2006-05-12 2006-05-12
US60/799,759 2006-05-12

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WO2007134254A2 true WO2007134254A2 (fr) 2007-11-22
WO2007134254A3 WO2007134254A3 (fr) 2008-01-10

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PCT/US2007/068811 Ceased WO2007134254A2 (fr) 2006-05-12 2007-05-11 Surveillance et suivi de communautés virales du tractus respiratoire au moyen d'échantillons d'expectorations humaines non cultivées

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA1317860C (fr) * 1987-04-01 1993-05-18 Daniel Louis Kacian Techniques pour la preparation de specimens pour des epreuves bacteriennes
US7482116B2 (en) * 2002-06-07 2009-01-27 Dna Genotek Inc. Compositions and methods for obtaining nucleic acids from sputum

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