WO2002027014A2 - Detection of fecal contamination - Google Patents
Detection of fecal contamination Download PDFInfo
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- WO2002027014A2 WO2002027014A2 PCT/US2001/030399 US0130399W WO0227014A2 WO 2002027014 A2 WO2002027014 A2 WO 2002027014A2 US 0130399 W US0130399 W US 0130399W WO 0227014 A2 WO0227014 A2 WO 0227014A2
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- 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
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
Definitions
- FIELD The present disclosures relates to nucleic acid sequences that can be used to identify the host-source of bacteria found in fecal waste.
- Fecal pollution is a persistent problem, affecting many coastal and inland waters in the United States.
- fecal contamination from many sources finds its way into our waters, jeopardizing the health of the ecosystems and everything that depends on them. The problem continues partly because current methods are unable to identify the source.
- Fecal contamination may be introduced into natural waters from a variety of sources, including ineffective sewage treatment, leaking septic systems, illegal dumping, recreational boaters, agricultural runoff, and wildlife.
- Point sources such as sewage treatment plants
- non-point sources such as runoff from urban and rural areas, are much more difficult to trace, and often constitute a significant portion of the contamination.
- pollution from agriculture and wildlife was considered "natural and uncontrollable" (Martin, /. Environ. Qual. 26:1198-203, 1997).
- Bacterial and other pathogens include organisms such as Salmonella, Shigella, E. coli 0157:H7, Cryptosporidium, and Giardia.
- Viruses such as Hepatitis, Norwalk, and other enteroviruses also are often associated with feces and are detected commonly in environmental water samples (Havelaar et al, Appl. Environ. Microbiol 59:2956-62, 1993; Paul et al, Appl. Environ. Microbiol. 63:133-8, 1997; Griffin et al, Appl. Environ. Microbiol.
- Phosphorus found in runoff from agricultural land was shown to increase as the proportion of land used in agricultural practices, such as dairy waste application, increased (McFarland et al, J. Environ. Qual. 28:836-844, 1999). This is significant because of the potential for eutrophication in phosphorus- limited freshwaters (Daniel et al, J. Environ. Qual. 27:251-7, 1998). Additionally, allochthonous material from non-point sources can induce algal blooms, some of which may be harmful (see Paerl, Limnology & Oceanography 33:823-47, 1988 for review).
- fecal pollution is frequently monitored in many coastal waters, especially those areas used for shellfisheries and recreation.
- the most commonly used measure of fecal pollution are the number of viable coliforms, fecal coliforms, or Escherichia coli in a water sample (American Public Health Association, Standard Methods for the Examination of
- Fecal coliforms are defined as gram-negative, non-sporulating, rod-shaped bacteria that ferment lactose with gas formation within 24 hours at 44.5°C (Id.). They have been used as indicator bacteria for many years and are currently the Environmental Protection Agency standard for assessing water quality.
- Ribosomal RNA is a direct gene product and is coded for by the rRNA gene.
- the DNA sequence for rRNA is used as a template to synthesize rRNA molecules.
- ribosomes are present in all cells of all life forms. About 85-90 percent of the total RNA in a typical cell is rRNA.
- a bacterium such as E. coli contains about 10 4 ribosomes per cell while a mammalian liver cell contains about 5 x 10 6 ribosomes. Since each ribosome contains one of each rRNA subunit, the bacterial cell and mammalian cell contains 10 4 and 5 x 10 6 , respectively, of each rRNA subunit.
- nucleic acid hybridization a procedure well known in the art, has been used to specifically detect extremely small or large quantities of a particular nucleic acid sequence, even in the presence of a very large excess of non-related sequences.
- Prior uses of nucleic acid hybridization are found, for example, in publications involving molecular genetics of cells and viruses, genetic expression of cells and viruses, genetic analysis of life forms, evolution and taxonomy of organisms and nucleic acid sequences, molecular mechanisms of disease processes, and diagnostic methods for specific purposes, including the detection of viruses and bacteria in cells and organisms.
- PCR Polymerase chain reaction, or PCR, produces many copies of a particular template DNA sequence in vitro.
- This process uses nucleic acid hybridization to hybridize two primers, consisting of short DNA oligonucleotide molecules, to complementary sites on either side of the DNA sequence to be copied, or amplified.
- the use of a thermally-stable DNA polymerase allows repeated cycles of template denaturation, primer annealing, and synthesis of the template sequence. Specificity of the reaction is controlled by the primer design and the reaction conditions.
- Prior uses of PCR are found, for example, in publications involving studies of genetics of cells and viruses, genetic expression within cells, evolution and systematics of organisms, and diagnostic applications in clinical, industrial, and other settings.
- the present disclosure provides probes and primers which can be used to detect fecal contamination.
- the probes and primers described herein can be used to identify the host-source of fecal bacteria.
- the term "host-source” refers to the organism in which the bacterium grows.
- the host-source of a bacterium isolated from a human is human
- the host-source of a bacterium isolated from a cow is cow.
- the probes and primers disclosed herein can be used to track any species of interest, such as environmentally important species, genetically engineered species released in the environment, and pathogens in clinical specimens.
- the disclosure provides a method of detecting fecal . contamination, and also its source, from environmental samples by using primers and probes targeting fecal Bifidobacterium and bacteria in the Bacteroides-Prevotella group of fecal anaerobic bacteria.
- the disclosure also provides methods of using the disclosed probes and primers. These methods include contacting at least one probe or primer with a sample and detecting the binding of the probe or primer to a nucleic acid sequence in the sample, wherein the presence of binding is indicative of the presence of fecal contamination in the sample. In some embodiments, these methods use amplification reactions, such as polymerase chain reaction (PCR).
- PCR polymerase chain reaction
- Samples can be obtained from a variety of sources.
- a sample is a biological sample, such as a fecal or stool sample.
- the sample is an environmental sample, such as soil, water, sediments, suspended particles, and air.
- compositions provided herein include the nucleic acid sequences shown in SEQ ID NO: 1, 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, and/or 14, variants of these sequences, and stabilized forms (such as extended forms) of these sequences.
- variant sequences are used which have 70%, 75%, 80%, 85% 90%, 92%, 95%, 98% or 99% sequence identity, which retain the ability to be used to detect fecal contamination, and in some embodiments, to identify the host-source of fecal contamination.
- the disclosure also provides extended forms of the nucleic acid sequences shown in SEQ ID NOS: 1, 3, and 5-14.
- extended forms contain additional sequences that are complementary to the target nucleic acid sequence, or contain additional residues that are non- complementary to the target sequence.
- extended probes and primers contain less than about 5, 10, 15, 20, 25, 30, 50, 75, 100, 150, or 200 additional nucleic acid residues. The extended forms of the probes and primers continue to maintain the ability to detect bacteria.
- the disclosure provides fragments of the probes and primers shown in SEQ ID NOS: 1, 3, and 5-14, as well as variants of these fragments which maintain the ability to be used to detect fecal contamination, and in some embodiments, to be used to detect and/or identify the host- source of fecal contamination.
- these fragments can be from about 5 to 19 nucleic acid residues long.
- the sequences disclosed herein, including variants, stabilized forms, extended forms, and fragments of SEQ ID NOS: 1, 3, and 5-14 have a detectable label. The detectable label does not interfere with the detection of fecal contamination.
- kits having one or more of the probes and/or primers disclosed herein, for example at least two of the probes and/or primers disclosed herein.
- kits are suitable for use in detecting fecal contamination in a sample, and in some embodiments, are suitable for identification of the host-source of bacteria present in the sample.
- the kit can be used for hybridization of the disclosed probes or primers with the sample; and/or for PCR amplification of nucleic acid sequences present in the sample using the probes and primers herein disclosed.
- the kit comprises two or more containers, each containing at least one of the probes and/or primers disclosed herein.
- a kit contains multiple, such as two or more, probes and/or primers disclosed herein which are specific for a particular environment.
- kits can designed which contain probes and/or primers specific for the host-sources expected in a particular region, such as a particular environment. Such host-sources are likely candidates as the source of fecal contamination. Instructions for use of the kit can also be included.
- the disclosed probes and primers can be used in a device that allows for DNA isolation and amplification to detect fecal contamination or its source.
- FIGS. 1 and IB show the results from LH-PCR analysis of 16S rDNA gene fragments amplified with Bac32F-FAM (SEQ ID NO: 1) and Bac303R (SEQ ID NO: 2) (FIG. 1A) and Bifl64F (SEQ ID NO: 4) and Bif601R-FAM (SEQ ID NO: 5) (FIG. IB).
- Solid lines represent community profiles from human fecal DNA
- dotted lines represent community profiles from cow fecal DNA. Samples are mixtures of DNA from 7-8 individuals. The arrows indicate cow-specific gene fragments.
- FIGS. 2 and 2B show the results from T-RFLP analysis of 16S rDNA gene fragments amplified with Bac32F-FAM (SEQ ID NO: 1) and Bac708R (SEQ ID NO: 3) and cut with Acil (FIG. 2A) or Hael ⁇ l (FIG. 2B).
- Solid lines represent community profiles from human fecal DNA
- dotted lines represent community profiles from cow fecal DNA. Arrows indicate host-specific genetic markers.
- FIGS.3A and 3B show the results from T-RFLP analysis of 16S rDNA gene fragments amplified with Bifl 64F (SEQ ID NO: 4) and Bif ⁇ O 1R-FAM (SEQ ID NO: 5) and cut with Haelll (FIG. 3A) or Taql (FIG. 3B).
- Solid lines represent community profiles from human fecal DNA; dotted lines represent community profiles from cow fecal DNA. Arrows indicate host-specific genetic markers.
- FIG. 4 shows a tree of phylogenetic relationships among partial 16S rDNA sequences (558 positions) of human (HF) and cow (CF) host-specific genetic markers identified from fecal clone libraries. The tree was inferred by neighbor-joining. Numbers above the internal branches are percentages of bootstrap replicates that support the branching order. Bootstrap values below 50% are not shown. Cytophaga fermentans was used to root the tree.
- FIGS.5A-5C show results from T-RFLP analyses of 16S rDNA gene fragments amplified from DNA extracted from Tillamook Bay water samples.
- DNA was amplified using Bac32F (SEQ ID NO: 1) and Bac708R (SEQ ID NO: 3) and digested with ctl (FIG. 5A) or Hae ⁇ ll (FIGS. 5A and 5B). Arrows indicate host-specific markers.
- FIGS. 5A and 5B show cow-specific markers 227 and 222, respectively.
- FIG. 5C shows the 119-bp human-specific marker.
- FIG. 6 shows a tree of phylogenetic relationships among partial 16S rDNA sequences (558 positions) of clones recovered from Tillamook Bay water samples (TB).
- HF and CF are host-specific genetic markers identified from human or cow fecal clone libraries, respectively.
- the tree was inferred by neighbor-joining. Numbers above the internal branches are percentages of bootstrap replicates that support the branching order. Bootstrap values below 50% are not shown. Bootstrap values for a and b dropped from 68 to 47 and 76 to 40, respectively, when TB147 was added to the analysis. Cytophaga fermentans was used to root the tree.
- FIG. 7 shows a tree of phylogenetic relationships among partial 16S rDNA sequences (558 positions) cloned from fecal DNAs.
- Cat Bl cluster, Pig B2 cluster, Dog E7 cluster, Elk A3 cluster and Pig CI cluster are Bacteroides bacterial sequences or sequence clusters that are unique to different host species: one group each from elk, dog, and cat, and two groups from pig. These host- specific bacterial sequences were used to design primers specific for each animal group.
- the tree was inferred by neighbor joining. Numbers above the branches are genetic distances, defined as the average number of sequence changes per position, and calculated by the Kimura-Nei algorithm.
- FIG. 8 shows a map of the sample testing area.
- nucleic acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood to be included by any reference to the displayed strand.
- SEQ ID NO: 1 shows the sequence of the Bac32F primer.
- SEQ ID NO: 2 shows the sequence of the Bac303R primer.
- SEQ ID NO: 3 shows the sequence of the Bac708R primer.
- SEQ ID NO: 4 shows the sequence of the Bifl 64F primer.
- SEQ ID NO: 5 shows the sequence of the Bif601R primer.
- SEQ ID NO: 6 shows the sequence of the CF128F primer.
- SEQ ID NO: 7 shows the sequence of the CF193F primer.
- SEQ ID NO 8 shows the sequence of the HF183F primer.
- SEQ ID NO 9 shows the sequence of the Elk 149F primer.
- SEQ ID NO 10 shows the sequence of the Dog 132F primer.
- SEQ ID NO 11 shows the sequence of the Cat 13 IF primer.
- SEQ ID NO 12 shows the sequence of the Pig 134F primer.
- SEQ ID NO 13 shows the sequence of the Pig 150F primer.
- SEQ ID NO 14 shows the sequence of the human HF134F primer.
- Detectable Label A molecule capable of detection.
- the label is conjugated to a nucleotide sequence.
- the label can be directly conjugated to a nucleic acid sequence (or to a particular nucleoside triphosphate thereof) or can become bound thereto by being bound to a specific binding agent that is attached to a probe or primer nucleotide sequence.
- any label that is detectable can be used.
- labels examples include, but are not limited to: isotopic or non-isotopic, catalysts such as an enzyme or a catalytic polynucleotide, promoter, dye, fluorescent molecule, chemiluminescent molecule, coenzyme, enzyme substrate, radioactive group, small organic molecule, amplifiable polynucleotide sequence, a particle such as latex or carbon, metal sol, crystallite, liposome, cell, etc., which may or may not be further labeled with a dye, catalyst, or other detectable group, and the like.
- labels include an oligonucleotide or specific polynucleotide sequence that can be added to a probe or primer to provide a template for amplification or ligation.
- a detectable label is a member of a signal-producing system which generates a detectable signal alone or together with other members of the signal-producing system.
- DNA Deoxyribonucleic acid.
- DNA is a long chain polymer which comprises the genetic material of most living organisms (some viruses have genes comprising ribonucleic acid, RNA).
- the repeating units in DNA polymers are four different nucleotides, each of which comprises one of the four bases, adenine, guanine, cytosine and thymine bound to a deoxyribose sugar to which a phosphate group is attached.
- Triplets of nucleotides, referred to as codons in DNA molecules code for amino acid in a polypeptide.
- codon is also used for the corresponding (and complementary) sequences of three nucleotides in the mRNA into which the DNA sequence is transcribed.
- DNA construct Refers to any nucleic acid molecule of cDNA, genomic DNA, synthetic
- a construct is a nucleic acid segment that may be single- or double-stranded. It is understood that such nucleotide sequences include intentionally manipulated nucleotide sequences, e.g., subjected to site-directed mutagenesis, and sequences that are degenerate as a result of the genetic code.
- Gene and Genome The terms "gene” and “genome” include dsDNA (double-stranded
- RNA DNA
- ssDNA single-stranded DNA
- RNA RNA
- a gene is a sequence of DNA or RNA that codes for a protein.
- Isolated An "isolated" biological component (such as a nucleic acid or protein or organelle) is a component that has been substantially separated or purified away from other biological components in the cell of the organism in which the component naturally occurs, i.e., other chromosomal and extra-chromosomal DNA, RNA, proteins, and organelles.
- Nucleic acids and proteins that have been “isolated” include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids and proteins prepared by recombinant expression in a host cell, as well as chemically synthesized nucleic acids.
- Nucleotide Includes, but is not limited to, a monomer that includes a base linked to a sugar, such as a pyrimidine, purine or synthetic analogs thereof, or a base linked to an amino acid, as in a peptide nucleic acid (PNA).
- a nucleotide is one monomer in a polynucleotide.
- a nucleotide sequence refers to the sequence of bases in a polynucleotide.
- Oligomers Includes both oligonucleotides and analogs. Oligonucleotide: A linear polynucleotide sequence of up to about 200 nucleotide bases in length, for example a polynucleotide (such as DNA or RNA) which is at least about 6 nucleotides, for example at least 15, 50, 100 or 200 nucleotides long.
- a polynucleotide such as DNA or RNA
- Polynucleotide A nucleic acid sequence (such as a linear sequence) of any length. Includes oligonucleotides and also gene sequences found in chromosomes.
- Probes and primers Nucleic acid probes and primers may be prepared readily based on the amino acid and nucleic acid sequences provided herein. Probes and primers detect their target by hybridization to complementary sequences.
- a "probe” comprises an isolated nucleic acid attached to a detectable label or reporter molecule. Examples of labels include, but are not limited to those listed above. Methods for labeling and guidance in the choice of labels appropriate for various purposes are discussed in, e.g., Sambrook et al.
- Primers are short nucleic acids, such as DNA oligonucleotides at least 10 nucleotides in length.
- a primer can be annealed to a complementary target DNA strand by nucleic acid hybridization to form a hybrid between the primer and the target DNA strand, and then extended along the target DNA strand by a DNA polymerase enzyme.
- Primer pairs can be used for amplification of a nucleic acid sequence, e.g., by the polymerase chain reaction (PCR), or other nucleic-acid amplification methods known in the art.
- a primer can be labeled with a detectable label and used as a probe.
- Primer and probe oligonucleotide sequences may be interchangeable.
- PCR primer pairs can be derived from a known sequence, for example, by using computer programs intended for that purpose such as Primer (Version 0.5, ⁇ 1991, Whitehead Institute for Biomedical Research, Cambridge, MA).
- Primer Version 0.5, ⁇ 1991, Whitehead Institute for Biomedical Research, Cambridge, MA.
- probes and primers may be selected that comprise, for example, 10, 20, 25, 30, 35, 40, 50 or more consecutive nucleotides.
- a purified enzyme or nucleic acid preparation is one in which the subject protein or nucleotide, respectively, is at a higher concentration than the protein or nucleotide would be in its natural environment within an organism.
- a preparation of an nucleic acid can be considered as purified if the nucleic acid content in the preparation represents at least 50%, for example at least 70%, of the total content of the preparation.
- Sample A material to be analyzed.
- a sample is a biological sample, such as a fecal sample.
- a sample is an environmental sample, such as soil, sediment water, or air.
- Environmental samples can be obtained from an industrial source, such as a farm, waste stream, or water source.
- Sequence identity The identity/similarity between two or more nucleic acid sequences, or two or more amino acid sequences, is expressed in terms of the identity or similarity between the sequences. Sequence identity can be measured in terms of percentage identity; the higher the percentage, the more identical the sequences are. Sequence similarity can be measured in terms of percentage similarity (which takes into account conservative amino acid substitutions); the higher the percentage, the more similar the sequences are.
- NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al, J. Mol. Biol. 215:403-10, 1990) is available from several sources, including the National Center for Biological Information (NCBI, National Library of Medicine, Building 38A, Room 8N805, Bethesda, MD 20894) and on the Internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. Additional information can be found at the NCBI web site.
- NCBI National Center for Biological Information
- Subject Includes any organism, for example a mammalian subject, such as a human or veterinary subject.
- a first nucleic acid is substantially similar to a second nucleic acid if, when optimally aligned (with appropriate nucleotide deletions or gap insertions) with the other nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about, for example, 50%, 75%, 80%, 85%, 90%, 92%, 95%, 98%, or 99% of the nucleotide bases.
- nucleotide sequence identity in at least about, for example, 50%, 75%, 80%, 85%, 90%, 92%, 95%, 98%, or 99% of the nucleotide bases.
- the methods disclosed herein were used to identify genetic markers from fecal bacteria from several hosts, that identify feces from these hosts.
- the primers disclosed herein were designed that are specific for each genetic marker. These primers can be used to identify the most likely sources of fecal contamination in samples.
- markers for other organisms that are likely to be a cause of pollution can be identified. For example, other sources of fecal pollution include waterfowl and deer, and other common wildlife.
- probes and primers can be designed based on the methods disclosed herein to detect fecal contamination that results from such wildlife.
- Fecal samples were collected from human and cow sources and DNAs extracted.
- the 16S rDNA fragments from these samples were amplified using primers specific for Bacteroides or Bifidobacterium.
- Length Heterogeneity Polymerase Chain Reaction (LH-PCR) and Terminal Restriction Fragment Length Polymorphism (T-RFLP) were used to identify regions of DNA that were specific to bacteria isolated from humans and cows (host-specific DNA). Two human-specific and three cow-specific markers were identified. These markers were used to isolate and clone 16S rDNA fragments. The sequencing of these fragments allowed a phylogenetic analysis to be performed. The markers clustered into three host-specific clusters.
- Samples were taken from various natural bodies of water and tested using the LH-PCR and T-RFLP techniques to determine whether the identified fecal markers could be recovered from water.
- the host-specific markers were recovered from the environmental samples and sequence analysis confirmed their identities.
- the initially identified markers then were used to clone additional rDNA fragments from polluted water. These fragments were sequenced and found to be members of the same three phylogenetic clusters. The combined sequence data (data from the initial screen and data from the additional rDNA clones) were used to develop additional PCR primers diagnostic for fecal source.
- Environmental samples were collected and analyzed using both the primers that were initially designed to identify the genetic markers and the additional primers designed from subsequent isolations. These environmental samples were collected from various sites in the state of Oregon. The samples that tested positive for rDNA derived from fecal matter were analyzed to see if they correlated with known point and non-point sources of pollution.
- Fecal samples from additional animal species were collected and DNAs were extracted as described above. Specific primers were used to amplify Bacteroides rDNAs and rDNA clone libraries were constructed. The clones were sequenced and compared by phylogenetic analysis. Gene clusters or regions of bacterial gene sequences that only occurred in a particular animal host species were identified and used to design additional primers diagnostic for fecal contamination from these animal species.
- Human fecal samples were donated by healthy adult and child volunteers from Corvallis, Oregon, including Caucasian, Asian, and Hispanic individuals. Samples were collected in sterile containers and stored at -80°C. Fresh cow fecal samples were collected from healthy Holstein dairy cows from two farms in Corvallis, Oregon, and three farms in Tillamook County, Oregon. The Corvallis cow fecal samples were collected during three different seasons from 1996 to 1998, and Tillamook County cow fecal samples during Fall 1996. Wild elk feces were collected from several locales in the Oregon Coast Range. Pig feces were collected from three farms near Corvallis, Oregon.
- Dog and cat feces were collected from pets in Corvallis, Oregon, and from the Heart of the Valley Humane Society, Corvallis, Oregon. Samples were collected with sterile utensils and placed in sterile 50-mL tubes, kept on ice for transport to the lab, and stored at -80°C.
- DNA was extracted by bead-beating using the method of Gray and Herwig (Appl. Environ. Microbiol. 62:4049-59, 1996) with the following modifications: 0.5 g of 0.1-mm diameter glass beads (acid- washed and baked) were used, polyvinylpolypyrrolidone was omitted from the lysis buffer, and crude extracts were ethanol-precipitated. The resulting pellets were dried under vacuum and resuspended in InstaGene Matrix (BioRad, Hercules, CA) or TE (10 mM Tris, 1 mM EDTA, pH 8).
- DNA extracts were purified by phenol/chloroform extractions followed by ethanol precipitation and resuspension in TE. DNAs from elk, pig, cat, and dog feces were extracted with a Qiagen DNEasy kit according to the manufacturer's directions.
- DNA from the water samples was extracted based on the method of Giovannoni et al (Appl. Environ. Microbiol. 56:2572-5, 1990) except the cesium trifluouroacetic acid (CsTFA) purification steps were omitted. Instead, samples were cleaned using one of the following methods: 1) one volume of 20 % polyethylene glycol 8000 in 2.5 M NaCl was added, samples incubated for 15 minutes at 37°C and centrifuged for 10 minutes at 12,500 rpm, and the resulting pellets washed twice with ice-cold 80% ethanol; 2) guanidine thiocyanate (Fluka, Buchs, Switzerland) purification based on the method of Pitcher et al.
- CsTFA cesium trifluouroacetic acid
- EXAMPLE 3 Analysis of Fecal DNA to Identify Host-Specific Markers Approximately 2-4 ng of fecal DNAs from individual humans, cows, elks, pigs, dogs and cats, were amplified by the PCR. In addition to analyzing individual samples, pooled PCR products from multiple individuals from each host species were also analyzed. DNAs from fourteen human samples were amplified with both Bacteroides/Prevotella and Bifidobacterium primers (Table 1). DNAs from eight Corvallis and eight Tillamook cows were amplified with Bacteroides/Prevotella primers, but only four each with Bifidobacterium primers.
- Each 50 ⁇ L PCR contained IX TAQTM polymerase buffer, 10 ⁇ M each primer, 200 ⁇ M each dNTP, 1.25 units of TAQTM polymerase, 640 ng/ ⁇ L BSA (Kreader, Appl. Environ. Microbiol. 62:1102-6, 1996), and 1.5 mM MgCl 2 .
- Bif601R (SEQ ID NO: 5) and Bac32F (SEQ ED NO: 1) were labeled with the fluorophore 6-FAM (GenSet, La Jolla, CA).
- a thermal mini-cycler (MJ Research, Watertown, MA) was used for all reactions with the followmg conditions: 35 cycles of 94°C for 30 seconds, 53°C for 1 minute, 72°C for 2 minutes, followed by a final six-minute extension at 72°C.
- the products were quantified in a 1% agarose gel by comparing the band intensity to a low molecular weight DNA mass ladder (Gibco BRL).
- Restriction enzymes were chosen based on analysis of published sequences in GenBank using Mapsort (Genetics Computer Group, Wisconsin). Enzymes that produced the greatest number of terminal restriction fragments of different length within the Bacteroides/Prevotella or Bifidobacterium 16S rDNA sequences were tested empirically. Enzymes were purchased from New England Biolabs (Beverly, MA). PCR products amplified using Bac32F (SEQ ID NO: 1) and Bac708R (SEQ ID NO: 3) were digested overnight at 37°C with either Acil or Haelll.
- PCR products amplified using Bifl64F (SEQ ID NO: 4) and Bif601R (SEQ ID NO: 5) were digested overnight with Haelll (at 37°C) or Taql (at 65°C). Each 20 ⁇ L digestion contained 20-40 ng of PCR products, 10 units of enzyme, IX enzyme buffer, and 100 ⁇ g/ml BSA (for Taql only).
- the fragments generated were separated by size on an ABI DNA sequencer with GeneScan software, allowing for the identification of DNA fragment lengths unique to either humans or cows. From these analyses, seven potential host-specific 16S rDNA genetic markers from human and cow fecal DNAs were identified by LH-PCR or T-RFLP analysis of human and cow fecal DNAs (Table 2). To be considered a host-specific genetic marker, the gene fragment had to be present in all samples from that host and be absent from all samples from the other host.
- Table 2 Potential host-specific genetic markers.
- LH-PCR analysis which detects length differences in PCR amplicons, revealed Bacteroides/Prevotella and Bifidobacterium cow-specific genetic markers (FIG. 1).
- LH-PCR analysis of 16S rDNA amplicons amplified with Bac32F (SEQ ID NO: 1) and Bac708R (SEQ ID NO: 3) from human and cow feces identified a peak at 276 bp as a potential cow-specific gene fragment, but no human-specific genetic markers were detected.
- LH-PCR analysis of 16S rDNA amplicons amplified with Bifl64F (SEQ ED NO: 4) and Bif601R (SEQ ID NO: 5) revealed a cow- specific genetic marker at 453 bp.
- Analysis of 16S rDNA amplicons from human feces and sewage amplified with Bifl64F (SEQ ED NO: 4) and Bif601R (SEQ ID NO: 5) and digested with Taql produced a human-specific peak at 313 bp (arrow, solid line), but no cow- specific peaks were detected in the amplicons from cow feces (FIG.
- Tillamook Bay is a shallow estuary on the northwest coast of Oregon (FIG. 8). It is approximately 3.2 km wide and 11.3 km long, with an estimated 3,590 hectares of surface water at high tide. Its watershed covers nearly 150,000 hectares, and is drained by five major rivers, the Miami, the Kilchis, the Tillamook, the Trask, and the Wilson Rivers.
- EXAMPLE 5 Fecal DNA Library Construction and Analysis DNAs from individual cow, human, elk, dog, cat, or pig fecal samples, or from water samples, were amplified with Bac32F (SEQ ID NO: 1) and Bac708R (SEQ ID NO: 3), and amplicons from 10 individuals from each host species were pooled. PCR products were gel-purified using the Qiaquick Gel Extraction Kit (Qiagen, Valencia, CA) and cloned using the pGEM-T Easy Cloning Kit (Promega, Madison, WI) according to the manufacture's directions.
- Bac32F (SEQ ED NO: 1) was labeled with the fluorophore 6-FAM. PCR products amplified with Bac32F (SEQ ED NO: 1) and Bac303R (SEQ ID NO: 2) were analyzed by LH-PCR. PCR products amplified with Bac32F (SEQ ID NO: 1) and Bac708R (SEQ ED NO: 3) were digested with the restriction enzymes HAEW. or ACR as described above and analyzed by T-RFLP. The clones on each microtiter plate corresponding to each genetic marker were identified by locating the intersection of a positive result in a row with a positive result in a column.
- Plasmid DNAs from overnight cultures were prepared using the Qiaprep Spin Column Purification Kit (Qiagen) according to the manufacturer's directions. DNA was quantified spectrophotometrically on a Shimadzu (Columbia, Maryland) UV/Vis spectrophotometer. Bidirectional sequences were obtained using T7 and SP6 priming sites on either side of the insert. Sequences were determined on an ABI 377 DNA Sequencer using dye- terminator chemistry.
- Sequences were subjected to analysis using BLAST v. 2.0 to obtain preliminary closest phylogenetic neighbors. The sequences were aligned manually to sequences from the Cytophaga- Flavobacter-Bacteroides group obtained from GenBank using the DNA sequence editor in GCGTM v.10 (Genetics Computer Group, Wisconsin). Sequences and alignments were verified by comparisons to the 16S rRNA secondary structure of Bacteroides fragilis and to Bacteroides signature sequences (Gherna et al, System. Appl. Microbiol. 15:513-21, 1992). Evolutionary distances were calculated using the DNADISTTM program with the Kimura 2-parameter model for nucleotide change and a transition/transversion ratio of 2.0 (Kimura, J. Mol.
- cow-specific clones None of the cow-specific clones was closely related to any characterized microorganisms. These clones formed two distinct gene clusters within the Cytophaga-Flavobacter-Bacteroides phylum (FIG. 4). Seven clones were recovered from cow feces that produced the 227 bp size fragment, when amplified with Bac32F (SEQ ID NO: 1) and Bac708R (SEQ ED NO: 3), and cut with cz'I. Partial 16S rDNA sequencing revealed five different sequences, each with the same T- RFLP profile, which formed the CF123 gene cluster. Fragment sizes estimated by T-RFLP analysis were about two bases larger than the size determined from the sequences (225 bp).
- the clones comprising the HF8 cluster were >99% similar, with the exception of HF102.
- These three clones (HF8, HF117, HF145) varied by only 1-2 nucleotides over a 700-base sequence, which falls within predicted TAQTM polymerase error rates (Saiki et al, Science 239:487-91, 1988).
- Three of the six deviant nucleotides were consistent with common TAQTM errors (Dunning et al, Nucl. Acids Res. 16:10393, 1988; and Tindall et al, Biochem. 27:6008-13, 1988), and two others were incompatible with secondary structure, indicating PCR or sequencing errors. It is possible that these three sequences are actually the same.
- HF 102 was in the same gene cluster, it had 9- 11 nucleotide differences from the other three sequences in this cluster. However, these differences were in a hypervariable region of the gene. Sequence analysis of the additional clones recovered from water samples revealed seven unique clones that corresponded to the human or cow genetic markers previously identified. All of the clones were very similar, but not identical, to clones recovered from human and cow fecal samples (FIG. 6). To confirm that the clones recovered from water samples were fecal in origin, primers were designed that were specific to two of the water clones, TB141 and TB147. These primers were used to amplify 16S rRNA genes from cow fecal DNAs.
- TB13 corresponded to the human-specific cluster, HF8, and was greater than 99% similar to other clones in this cluster.
- the TB13 sequence differed by only 1-2 bases from HF8, HF117, and HF145.
- the rermining clones corresponded to the cow- specific markers.
- TB141 had the same T-RFLP pattern as CF46, CF68, and CF151 and was 84.7% to 90.4% similar to the other CF151 clones.
- TB101, TB106, TB135 and TB146 had the same T- RFLP pattern as the other clones in the CF123 cluster and were 93.3 to 96.1% similar.
- the T-RFLP pattern of TB147 matched the patterns of the CF123 cluster, but the sequence grouped with the CF151 cluster (FIG. 6). Additionally, TB147 had the highest similarity with CF17 (88.2%), which is in the CF 123 cluster. Bootstrap values for the CF151 cluster dropped considerably when TB147 was included in the analysis.
- LH-PCR and T-RFLP proved to be highly reproducible methods, although the estimated peak size often deviated by 1-2 base pairs from the size predicted from the respective sequence. Despite these discrepancies, the methods were reproducible, with variances of ⁇ 0.3 bp for fragments up to 350 bp.
- GenBank accession numbers are as follows: AF233400, AF233401, AF233402, AF233403, AF233404, AF233405, AF233406, AF233407, AF233408, AF233409, AF233410, AF233411, AF233412, and AF233413. These sequences were used to develop initial primers to identify genetic markers.
- Species-specific sequence clusters of Bacteroides- Prevotella from elk, dog, cat, and pig were used to design host-species-specific primers for elk (CACAGCCGCTCGAAAG; SEQ ID NO: 9), dog (CCTTCCGTACACTCAGGG; SEQ ED NO: 10), Cat (ACCTGCCTTCCACTCG; SEQ ED NO: 11), and two primers for pig (TTCCCYTGTCCACGG; SEQ ID NO: 12 and ATAGCCCAGCGAAAGTTG; SEQ ED NO: 13).
- the Dog E7 cluster used to design the Dogl32F primer specific for domestic pet feces, also includes CatE5 and Cat F3 sequences.
- the Cat Bl cluster used to design the Catl31F primer specific for cat feces, also includes CatGl and Cat C8 sequences.
- the ElkA3 cluster was used to design the Elkl49F primer specific for elk feces.
- the Pig B2 cluster was used to design the Pigl34F primer, and the PigB2 cluster was used to design the Pigl50F primer, both specific for pig feces.
- Table 3 Host-species-specific primers for detection of fecal anaerobic bacteria
- PCR primers specific for each gene cluster host-specificity was confirmed by testing fecal DNAs from human and cow feces and from sewage samples. Genes corresponding to the HF8 cluster were detected in 11 out of 13 human fecal samples, all of the sewage samples, and none of the cow fecal samples (Table 4). Using the HF10 primers, a PCR product was detected in less than half of the sewage and human fecal samples. The HF10 primers allowed for the detection of products in one cow fecal sample. HF8 genes were more widely distributed among the humans, and primers for HF10 were not as specific as desired, hence, HF8 genes were tested in subsequent analyses. Genes from the CF151 and CF123 clusters were detected in all cow samples, but in none of the human or sewage samples.
- PCR sensitivity was approximately 1 x 10 "12 g DNA (10 5 gene copies) for all three plasmid DNAs. Detection of Bacteroides-Prevotella DNA was 2-4 times greater than fecal coliform detection (Table 6). Detection of CF123 genes was as sensitive as detection of fecal coliforms. Fecal coliform detection, however, was one to two times more sensitive than detection of CF151 genes. The sensitivity assay using cow fecal dilutions was repeated with feces from different cows, and similar results were obtained (Table 6).
- Table 6 Detection limits for Bacteroides-Prevotella 16S rDNA, host-specific genetic markers, and fecal coliforms.
- Results are from dilution assays using either cow feces or raw sewage.
- the seven additional unique clones isolated from natural waters corresponded to the human and cow fecal markers previously identified. Hence, host-specific genetic markers are useful for identifying non-point sources of fecal pollution in coastal waters.
- Six of the clones were close phylogenetic relatives of the clones recovered from feces.
- the seventh clone was more distantly related and its phylogenetic placement was not strongly supported by bootstrap analysis (FIG. 6). All the evidence, except the phylogeny, indicates the inclusion of this clone in the CF123 cluster.
- EXAMPLE 10 Sensitivity Analysis Serial dilutions of fresh cow feces or raw sewage were added to 1-L samples of filter- sterilized bay water. Final concentrations in the 1-L samples ranged from 2 x 10 "7 mg (wet weight)/L to 2.0 mg/L. Samples were filtered onto a 0.2 ⁇ m SuporTM filter and stored in lysis buffer at-80°C as described above. The percent solids of the fecal samples was estimated by weighing replicate samples of wet feces and drying with heat until no more weight was lost. To estimate percent solids of raw sewage, the solids were collected by centrifugation, the supernatants were decanted, and the samples were dried overnight with heat.
- DNAs extracted from the filters were amplified using Bac32F (SEQ LD NO: 1) and Bac708R (SEQ ID NO: 3) as described above, and the PCR products were visualized in a 1% agarose gel. Products were digested as described above. Samples were analyzed by T-RFLP, using 25 finoles of the most concentrated dilution (2.0 mg/L), and equivalent volumes from all other dilutions.
- FIG. 8 shows the sampling sites in Tillamook Bay and its tributaries and locations of NPDES (National Pollution Discharge Elimination System) or CAFO (Confined Animal Feeding Operation) permitted sites.
- Fecal coliforms in these samples ranged from 0 to 120 CFU/lOOmL (Table 7).
- Bacteroides/Prevotella DNA was detected in all eight samples tested, but Bifidobacterium DNA was detected in only two of these samples. Additionally, the product yield of Bifidobacterium amplicons detected in these water samples was considerably less than that obtained from the same samples using Bacteroides primers. All seven host-specific genetic markers were detected in at least one water sample (Table 7, FIG. 4).
- sequence data were used to validate the identities of the Bacteroides/Prevotella markers from water samples. Sequences from Bacteroides/Prevotella markers were recovered that belonged to the HF8, CF123, and CF151 gene clusters.
- Table 7 Fecal coliform measurements and presence/absence of Bifidobacterium and Bacteroides/Prevotella host-specific markers in water samples.
- the sensitivity of detecting host-specific DNA in water samples was tested by conducting assays using filter-sterilized bay water amended with fresh feces or raw sewage. Feces and sewage were used rather than cultured organisms because fecal organisms were the intended targets.
- the limit of detection of host-specific markers varied, with the 222-bp cow-specific marker being the least sensitive (2.8 x 10 "5 g dry feces/L), followed by the 119-bp human-specific marker (6.8 x 10 "7 g dry sewage/L) and with the 227-bp cow-specific marker being the most sensitive (2.8 x 10 "8 g dry feces/L).
- Species were identified by composition differences in the Bacteroides/Prevotella and Bifidobacterium populations between human and cow feces. These differences are useful for, inter alia, identifying fecal-pollution non-point sources in coastal waters.
- the human-specific genetic markers within the Bacteroides/Prevotella group were closely related, but not identical, to
- Gene clusters are sets of gene sequences more closely related to each other than to any characterized species; they have been found in many diverse natural bacterial populations (e.g. Giovannoni et al, Nature 345:60-3, 1990; Hales et al, Appl. Environ. Microbiol. 62:668-75, 1996; Ohkuma et al, Appl. Environ. Microbiol. 62:461-8, 1996; and Field et al, Appl. Environ. Microbiol. 63:63-70, 1997)
- the Bacteroides/Prevotella group were better indicators than Bifidobacterium in coastal waters; however refinement of the primers used in the Bifidobacterium samples may improve the results.
- Results from the sensitivity assays are comparable to results of other studies that used PCR to detect single Bacteroides species in feces (Kreader, Appl. Environ. Microbiol. 62:1102-6, 1996; Wang et al, Appl. Environ. Microbiol. 62:1242-7, 1996; and Kreader, Appl. Environ. Microbiol.
- the 119-bp human-specific marker and the 227-bp cow-specific marker appear to be more sensitive, as assayed by T-RFLP using general Bacteroides/Prevotella PCR primers. By designing primers specific to these markers, the sensitivity may be increased.
- the 222-bp cow- specific marker which represents the same sequences as the 276-bp marker, was the least sensitive by 3-4 orders of magnitude. Some samples were also found to test positive for one, but not the other (Table 4). Since the sensitivity for these markers is much lower, it is possible that the source contamination was at or near the limit of detection; therefore, inconsistent detection of these two markers in the same water sample is not surprising.
- the Bacteroides/Prevotella group is a good indicator for source identification of fecal contamination in water samples.
- the data provided shows: 1) human-specific and 2) cow-specific gene clusters of fecal markers from Bacteroidesi 'Prevotella. The data disclosed herein demonstrates that these markers can be recovered from natural fresh-water and saltwater samples.
- the marker genes also have been identified phylogenetically as members of the Bacteroides/Prevotella group, but representing uncharacterized species. Using the species-specific PCR assay described above, additional tests for the presence of human- and ruminant-specific genetic markers in 55 water samples collected from Tillamook Bay and its tributaries on five different sampling dates were performed.
- At least one host-specific fecal marker was detected in 27 of the 55 water samples (Table 8). Sixteen samples were positive for the human-specific marker HF8. For the ruminant-specific markers, eleven were positive for CF123, and twenty were positive for CF151. CF123 was not detected in any bay samples; HF8 and CF151 were found in both bay and river samples.
- Table 8 Detection of human- and ruminant-specific markers in water samples.
- CF123 never was detected in samples more than 2.7 km from the nearest CAFO permitted site, and was found in only three of the samples downstream of the nearest CAFO site. CF151 was not detected in any samples over 5.9 km from the nearest CAFO site, and was found in five of the samples collected downstream of the nearest CAFO site.
- Table 9 Distances (km) from each sampling site to the nearest permitted pollution source.
- Precipitation during sampling varied from 0 to almost 3 inches on the day of sampling (Table 10). Total precipitation during the five days prior to the sampling date ranged from 0.8 to over 10 inches. Rainfall was heaviest during the February sampling. Table 10: Precipitation on sampling dates and five days prior to the sampling date.
- #Precipitation data (in inches) was obtained from the Oregon climate Service website and was measured at station Tillamook 1W, 45'27" W and 123'52"N. * Includes the day of sampling and the five days prior to sampling.
- PCR assay is a rapid method for identifying non-point source pollution in coastal waters.
- Fecal markers were detected in at least one sample on all sampling dates, except during November. Precipitation on the sampling date during November and the five days prior was quite high, averaging more than an inch per day. Heavier rains earlier in the week may have washed significant portions of fecal bacteria from the fields, thus leaving low numbers on the day of sampling. Additionally, the large amount of rain may have created a dilution effect, so that the concentrations of the markers were below the detection limits. The same pattern was not observed in February, however, when rains were even higher.
- CF123 One of the ruminant markers, CF123, was found only in freshwater locations and river mouths, where salinity was less than 1 ppt. The sensitivity of detection of this marker under laboratory conditions (Table 7) was greater than the sensitivity of detection of the other ruminant marker, CF151. Salinity effects, however, were not investigated. CF151 was detected in nine samples that were negative for CF123. Most of these samples were located in the bay, which is affected by saline ocean waters. Samples were collected during high tides when the influence of saltwater would be greatest. It is possible that the species or strains that comprise CF123 die off more rapidly upon exposure to saline waters, as has been observed with other fecal bacteria (Sinton et al, Appl.
- CF123 and CF151 are useful for detecting ruminant-specific fecal pollution, so it is possible that deer and elk feces might have contributed to the signal.
- prevailing land-use patterns suggest that these sources would be insignificant in most cases.
- Most of the sampling sites were located in rural areas with a high density of agricultural operations. Additional evidence for the lack of wildlife contribution is that in sites upriver, such as Rl and Wl, where wildlife would be most concentrated, no fecal markers ever were detected.
- nucleotide sequences of probes and primers facilitates the creation of DNA molecules derived from those disclosed but which vary in their precise nucleotide sequence from those disclosed. Such variants can be obtained through standard molecular biology laboratory techniques and the sequence information disclosed herein.
- Probe and primer variants, fragments, extensions, and polymorphisms will retain the ability to be used to detect fecal contamination.
- probe and primer variants retain at least 70%, 80%, 85%, 90%, 92%, 95%, 98%, 99%, or greater sequence identity to the primer sequences disclosed herein, and in particular embodiments at least this much identity to SEQ ED NOS: 1, 3, and 5-13.
- Variant and fragment sequences maintain the functional activity of the probes and primers as defined herein. Such activity can be readily determined using the assays disclosed herein.
- Variant DNA molecules include those created by standard DNA mutagenesis techniques, for example, M13 primer mutagenesis. Details of these techniques are provided in Sambrook et al. (In: Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, New York, 1989, Ch. 15). By the use of such techniques, variants may be created which differ in minor ways from those disclosed. DNA molecules and nucleotide sequences which are derivatives of those specifically disclosed herein and which differ from those disclosed by the deletion, addition or substitution of nucleotides while still possessing the functional characteristics of the disclosed probes and primers, are comprehended by this disclosure.
- Probes and primers derived from those disclosed can also be defined as sequences which hybridize under stringent conditions to the sequences disclosed, or fragments thereof.
- Hybridization conditions resulting in particular degrees of stringency vary depending upon the nature of the hybridization method and the composition and length of the hybridizing DNA used. Generally, the temperature of hybridization and the ionic strength (especially the Na + concentration) of the hybridization buffer determines hybridization stringency. Calculations regarding hybridization conditions required for attaining particular amounts of stringency are discussed by Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, New York, 1989, Chapters 9 and 11), herein incorporated by reference.
- a hybridization experiment can be performed by hybridization of a DNA molecule (for example, a variant of a nucleic acid sequence shown in SEQ ED NOS: 1, 3, and 5-13) to a target DNA molecule (for example, a nucleic acid sequence shown in SEQ ID NOS: 1, 3, and 5-13) which has been electrophoresed in an agarose gel and transferred to a nitrocellulose membrane by Southern blotting (Southern, J. Mol. Biol. 98:503, 1975), a technique well known in the art.
- a DNA molecule for example, a variant of a nucleic acid sequence shown in SEQ ED NOS: 1, 3, and 5-13
- target DNA molecule for example, a nucleic acid sequence shown in SEQ ID NOS: 1, 3, and 5-13
- Specific hybridization refers to the binding, duplexing, or hybridizing of a molecule only or substantially only to a particular nucleotide sequence when that sequence is present in a complex mixture (e.g. total cellular DNA or RNA). Specific hybridization may also occur under conditions of varying stringency.
- Hybridization with a target probe labeled with [ 32 P]-dCTP is generally carried out in a solution of high ionic strength such as 6xSSC at a temperature that is about 5-25°C below the melting temperature, T m .
- hybridization is typically carried out for 6-8 hours using 1-2 ng/ml radiolabeled probe (specific activity equal to 10 9 CPM/ ⁇ g or greater).
- the nitrocellulose filter is washed to remove background hybridization. Washing conditions should be as stringent as possible to remove background hybridization but retain a specific hybridization signal.
- T m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence remains hybridized to a perfectly matched probe or complementary strand.
- washing the filter in 0.3 xSSC at 59.4-64.4°C will produce a stringency of hybridization equivalent to 90%; that is, DNA molecules with more than 10% sequence variation relative to the target cDNA (for example a nucleic acid sequence shown in SEQ ID NOS: 1, 3, and 5-13) will not hybridize.
- washing the hybridized filter in 0.3 xSSC at 65.4-68.4°C yields a hybridization stringency of 94%; that is, DNA with more than 6% sequence variation relative to the target cDNA will not hybridize.
- the above example is given by way of theoretical illustration. One skilled in the art will appreciate that other hybridization techniques can be utilized and that variations in experimental conditions will necessitate alternative calculations for stringency.
- stringent conditions are those under which DNA molecules with more than 25%, 15%, 10%, 6% or 2% sequence variation (also termed "mismatch") will not hybridize. Longer sequences hybridize specifically at higher temperatures.
- An example of stringent conditions is a salt concentration of at least about 0.01 to 1.0 M Na ion concentration (or other salts) atpH 7.0 to 8.3 and a temperature of at least about 30°C for short probes (e.g. 10 to 50 nucleotides).
- Stringent conditions can also be achieved with the addition of destabilizing agents such as formamide.
- 5X SSPE 750 mM NaCl, 50 mM Na phosphate, 5 mM EDTA, pH 7.4 at 25-30°C are suitable for allele-specific probe hybridizations.
- a perfectly matched probe or primer has a sequence perfectly complementary to a particular target sequence.
- the test probe or primer is typically perfectly complementary to a portion (subsequence) of the target sequence.
- the term "mismatch probe” refers to probes whose sequence is deliberately selected not to be perfectly complementary to a particular target sequence.
- the degeneracy of the genetic code further widens the scope of the present disclosure as it enables major variations in the nucleotide sequence of a DNA molecule while mamtaining the amino acid sequence of the encoded protein. Because of the degeneracy of the genetic code, four nucleotide codon triplets, GCT, GCG, GCC and GCA, code for Ala.
- variant DNA molecules may be derived from the DNA molecules disclosed herein using standard DNA mutagenesis techniques as described above, or by synthesis of DNA sequences. DNA sequences which do not hybridize under stringent conditions to the probe and primer sequences disclosed by virtue of sequence variation based on the degeneracy of the genetic code are also comprehended by this disclosure.
- nucleic acid consists of nitrogenous bases that are either pyrimidines (cytosine (C), uracil (U), and tl ⁇ ymine(T)) or purines (adenine (A) and guanine (G)). These nitrogenous bases form hydrogen bonds with the bonding of a p rimidine to a purine, and the bonding of a pyrimidine to a purine is referred to as "base pairing.” More specifically, A will bond to T or U, and G will bond to C.
- “Complementary” refers to base pairing that occurs between two distinct nucleic acid sequences or two distinct regions of the same nucleic acid sequence.
- oligonucleotide and “specifically complementary” are terms that indicate a sufficient degree of complementarity, such that stable and specific binding occurs between the oligonucleotide (or its analog) and the DNA or RNA target.
- the oligonucleotide or oligonucleotide analog need not be 100% complementary to its target sequence to be specifically hybridizable.
- An oligonucleotide or analog is specifically hybridizable when such binding of the oligonucleotide or analog to the target DNA or RNA molecule allows for the detection of a complementary strand.
- oligonucleotide For example, if 10 nucleotides of a 15-nucleotide oligonucleotide form base pairs with a targeted region of a DNA molecule, then the oligonucleotide is said to have 66.67% complementarity to the region of DNA targeted.
- Sufficient complementarity occurs when a sufficient number of base pairs exist between the oligonucleotide and the target sequence to achieve detectable binding.
- the percentage complementarity that fulfills this goal can range from as little as about 50% complementarity to full (100%) complementary. In general, sufficient complementarity is about 50%, such as about 75% complementarity, such as about 90% or 95% complementarity, such as about 98% or 100% complementarity.
- Variant oligonucleotides are oligonucleotides that have one more base substitutions compared to the sequences shown in SEQ ED NOS: (i.e., naturally occurring bases such as A, T, C, G, or U, or synthetic bases such as those described below), one or more base deletions, and/or one or more base insertions, so long as the variant oligonucleotide substantially retains the activity of the original oligonucleotide, or has sufficient complementarity to a target sequence.
- a variant of the oligonucleotide shown in SEQ ID NO: 1 substantially would retain the activity of SEQ ID NO: 1, such that it would be useful for detecting the presence of fecal matter.
- the probe or primer should be sufficiently stable to resist degradation during storage, and during temperature fluctuations, and to avoid degradation from potential contaminating nucleases caused by the repeated opening and closing of stock solutions containing the primer and/or the probe.
- Tliis can be done, for example, by substituting the normally occurring phosphodiester linkage, which connects the individual bases, with modified linkages.
- modified linkages may, for example, be aphosphorothioate, methylphosphonate, phosphodithioate, or phosphoselenate.
- a single probe or primer molecule may contain multiple substitutions in various combinations.
- a probe or primer molecule also can be designed to contain different sugar molecules.
- the molecule may contain the sugars ribose, deoxyribose or mixtures thereof, linked to a base.
- the bases give rise to the molecules' ability to bind complementarity to the target RNA and DNA.
- probes and primers need not be 100% complementary to the target RNA or DNA to detect the target.
- the probes and/or primers can vary in length. Generally, a longer complementary region will give rise to a molecule with higher specificity.
- the probes and or primers can be DNA or RNA, or chimeric mixtures or derivatives or modified versions thereof.
- the oligonucleotide can be modified at the base moiety, sugar moiety, or phosphate backbone.
- the probes and/or primers are single-stranded DNA (ssDNA) molecules, although the disclosure is not limited to such primers.
- ssDNA single-stranded DNA
- An oligonucleotide can be modified at any position on its structure with substitutes generally known in the art.
- a modified base moiety may be 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylarnmomethyl-2-tMouridine, 5- carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N ⁇ 6- sopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2- methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5- methylaminomethyluracil, methoxyarninomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'- methoxycarboxymethyluracil, 5-
- the polynucleotide includes at least one modified sugar moiety such as arabinose, 2-fluoroarabinose, xylose, and hexose, or a modified component of the phosphate backbone, such as phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl phosphotriester, or a formacetal, or analog thereof.
- modified sugar moiety such as arabinose, 2-fluoroarabinose, xylose, and hexose
- a modified component of the phosphate backbone such as phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl phosphotriester, or a formacetal, or analog thereof
- the relative ability of an oligomer such as a polynucleotide to bind to a complementary strand is compared by deterrr ⁇ ning the melting temperature of a hybridization complex of a polypeptide and its complementary strand.
- Base stacking which occurs during hybridization, is accompanied by a reduction in UV absorption (hypochromicity).
- a reduction in UV absorption indicates a higher T m .
- the higher the T m the greater the strength of the binding of the hybridized strands.
- 100% complementarity between two nucleic acid sequences achieves optimal hybridization of a polynucleotide to its target RNA and/or DNA.
- Nucleic acid hybridization analysis generally involves the detection (using probes or primers) of very small numbers of specific target nucleic acids (DNA or RNA) among a large amount of non-target nucleic acids.
- hybridization normally is carried out under the most stringent conditions, achieved through various combinations of temperature, salts, detergents, solvents, chaotropic agents, and denaturants.
- nucleic acid hybridization analysis has been conducted on a variety of filter and solid support formats (see Beltz et al, in Wu et al. (eds.), Methods in Enzymology, Vol. 100, Part B, Academic Press, New York, Chapter 19, pp. 266-308, 1985).
- One format, the so-called "dot blot" hybridization involves non-covalent attachment of target DNAs to a filter. The dots are hybridized with a radioisotope-labeled probe(s). Dot blot hybridization gained wide-spread use, and many versions now have been developed (see Anderson et al, in Hames et al.
- Sandwich hybridization involves attaching oligonucleotide probes covalently to a solid support and using them to capture and detect multiple nucleic acid targets.
- a distinctive exception to the general difficulty in detecting low-copy-number target nucleic acids using a direct probe is the in-situ hybridization technique. This technique allows low ⁇ copy- number unique nucleic acid sequences to be detected in individual cells.
- target nucleic acid is naturally confined to the area of a cell or a nucleus at a relatively high local concentration.
- the probe/target hybridization signal is confined to a microscopic and morphologically distinct area; this makes it easier to distinguish a positive signal from artificial or non-specific signals than hybridization on a solid support.
- FISH fluorescence in-situ hybridization
- a similar technique could be used to detect target nucleic acid sequences in a sample.
- a sample could be concentrated such that the bacteria were trapped on a filter or other support media. The concentrated immobilized bacteria could then be probed with a label probes.
- Mimicking the in-situ hybridization in some aspects, new techniques are being developed for carrying out multiple sample nucleic acid hybridization analysis on micro-formatted multiplex or matrix devices (e.g., DNA chips) (see Barinaga, Science, 253:1489, 1991; and Bains, Bio/Technology 10:757-8, 1992). These methods usually attach specific DNA sequences to very small specific areas of a solid support, such as micro-wells of a DNA chip. These hybridization formats are micro-scale versions of the conventional reverse dot blot and sandwich hybridization systems.
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WO2006050479A2 (en) * | 2004-11-01 | 2006-05-11 | George Mason University | Compositions and methods for diagnosing colon disorders |
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WO2000009686A1 (en) * | 1998-08-12 | 2000-02-24 | Medical Research Council | Gene |
WO2000029615A2 (en) * | 1998-11-13 | 2000-05-25 | Centre National De La Recherche Scientifique (Cnrs) | A whole-genome radiation hybrid map of the dog genome and use thereof for identifying genes of interest |
-
2001
- 2001-09-28 WO PCT/US2001/030399 patent/WO2002027014A2/en active Application Filing
- 2001-09-28 AU AU2001293164A patent/AU2001293164A1/en not_active Abandoned
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2000009686A1 (en) * | 1998-08-12 | 2000-02-24 | Medical Research Council | Gene |
WO2000029615A2 (en) * | 1998-11-13 | 2000-05-25 | Centre National De La Recherche Scientifique (Cnrs) | A whole-genome radiation hybrid map of the dog genome and use thereof for identifying genes of interest |
Non-Patent Citations (1)
Title |
---|
ZOETENDAL ET AL.: 'Temperature gradient gel electrophoresis analysis of 16S rRNA from human fecal samples reveals stable and host-specific communities of active bacteria' APPLIED AND ENVIRONMENTAL MICROBIOLOGY vol. 64, no. 10, October 1998, pages 3854 - 3859, XP002909919 * |
Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP1323835A2 (en) * | 2001-12-27 | 2003-07-02 | Nisshinbo Industries, Inc. | Method for determining biospecies contained in test specimen and kit used for the same |
EP1323835A3 (en) * | 2001-12-27 | 2004-04-21 | Nisshinbo Industries, Inc. | Method for determining biospecies contained in test specimen and kit used for the same |
WO2006050479A2 (en) * | 2004-11-01 | 2006-05-11 | George Mason University | Compositions and methods for diagnosing colon disorders |
WO2006050479A3 (en) * | 2004-11-01 | 2007-01-04 | Univ George Mason | Compositions and methods for diagnosing colon disorders |
EP2280085A3 (en) * | 2004-11-01 | 2011-02-23 | George Mason University | Compositions and methods for diagnosing colon disorders |
WO2007115590A1 (en) * | 2006-04-11 | 2007-10-18 | Technische Universität Wien | Detection and quantification of faecal pollution in an environmental sample |
Also Published As
Publication number | Publication date |
---|---|
AU2001293164A1 (en) | 2002-04-08 |
WO2002027014A3 (en) | 2002-07-18 |
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