WO2018185105A1 - Procédés de détection de micro-organismes - Google Patents

Procédés de détection de micro-organismes Download PDF

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Publication number
WO2018185105A1
WO2018185105A1 PCT/EP2018/058494 EP2018058494W WO2018185105A1 WO 2018185105 A1 WO2018185105 A1 WO 2018185105A1 EP 2018058494 W EP2018058494 W EP 2018058494W WO 2018185105 A1 WO2018185105 A1 WO 2018185105A1
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Prior art keywords
labelling
target
microorganisms
oligonucleotide probe
ddntp
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PCT/EP2018/058494
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English (en)
Inventor
Morten L ISAKSEN
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Bio-Me As
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Priority claimed from GBGB1705346.3A external-priority patent/GB201705346D0/en
Priority claimed from GBGB1715481.6A external-priority patent/GB201715481D0/en
Application filed by Bio-Me As filed Critical Bio-Me As
Priority to EP18714782.2A priority Critical patent/EP3607096A1/fr
Publication of WO2018185105A1 publication Critical patent/WO2018185105A1/fr

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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

  • the present invention relates to methods of determining the presence, absence or quantity of target microorganisms in a sample containing multiple microorganisms, wherein each target microorganism is a member of a group of related microorganisms and each target microorganism is a member of a pre-defined set of target
  • the present invention also relates to kits for performing the methods of the invention.
  • the microbiome is defined as the sum total of all genetic material from microorganisms that inhabits a particular environment, for example the human gut.
  • the role of the microbiome in health and disease is becoming increasingly recognized, and affects many different disease states.
  • GI tract gastrointestinal tract
  • gut variations in the bacterial culture (also called the microbiota) are associated with various health conditions.
  • NGS Next Generation DNA Sequencing
  • 16S amplicon sequencing is the preferred method to date because it is a relatively low cost and quick technique and involves simpler processes than WGS.
  • WGS Wired GAA
  • there is a limited number of sequence variations within the 16S rRNA gene it is, in most instances, not possible to determine bacterial specificity beyond genus level.
  • Ruminococcus e.g. Ruminococcus lactaris
  • bacteria of the genus Coprococcus increased.
  • WGS limits the ability to process samples comprising large numbers of different microorganisms and typically limits processing of samples to those comprising a maximum of a few hundred different microorganisms.
  • DNA and RNA microarrays with overlapping sequences have also been used to detect various bacteria in culture (Rajilic-Stojanovic et al. (2009), Environ Microbiol. Jul; 11(7): pp 1736-1751).
  • the use of such microarrays is laboursome as well as involving high cost and it can also be prone to errors.
  • GA-map Another approach, called GA-map, has been used to identify groups of bacteria, at the phylum, class, family, and in some instances genus level (Casen et al. (2015) Aliment Pharmacol Ther. Jul; 42(1): pp71-83).
  • the GA-map method fails to give detailed information about the composition of the bacterial culture at the species and strain level. This limitation is due to biological constraints in identifying and defining DNA probes within the 16S rRNA amplicon that is used.
  • the present invention provides a method of determining the presence, absence or quantity of target microorganisms in a sample containing multiple microorganisms, wherein each target microorganism is a member of a group of related microorganisms and each target microorganism is a member of a pre-defined set of target
  • each target microorganism in the set of target microorganisms can be distinguished from the other target microorganisms in the set, the method comprising:
  • each amplicon is specific for a different group of related target microorganisms of the predefined set; wherein amplification products within each amplicon comprise first and second conserved regions of DNA sequence interspersed with a target DNA sequence region, wherein both first and second conserved regions are conserved across members of the group of related
  • a target DNA sequence region comprises a target DNA sequence that is a sequence within an amplicon which is unique to each target microorganism within the pre-defined set of microorganisms;
  • the step of generating a plurality of amplicons comprises performing amplification reactions using forward and reverse primers designed to anneal to DNA sequence regions comprising respectively the sequences of first and second conserved regions.
  • amplification reactions may be performed by multiplex amplification in the same reaction vessel, preferably wherein multiplex amplification is performed by PC ; or (b) DNA from the sample may be divided into aliquots and added to different reaction vessels, and wherein amplification reactions are performed by multiplex amplification in each reaction vessel, preferably wherein multiplex amplification is performed by PCR.
  • the step of determining the presence, absence or quantity of target DNA sequences of amplification products may comprise detecting the presence of target DNA sequences using probes, wherein each probe is specific for a target DNA sequence and wherein the sequence of each probe is designed to distinguish a given target DNA sequence from all other target DNA sequences of target microorganisms in the pre-defined set.
  • each probe may be a labelling oligonucleotide probe, as defined herein, conjugated to a labelling entity and the step of determining the presence, absence or quantity of target DNA sequences of amplification products (step 4) comprises measuring a signal from the labelling entity, for example the labelling entity may be a fluorophore and the step of determining the presence, absence or quantity of target DNA sequences of amplification products (step 4) comprises measuring the energy (fluorescence) emitted by the fluorophore.
  • each probe may be a labelling oligonucleotide probe, as defined herein, conjugated to a labelling entity which is a quencher and the step of determining the presence, absence or quantity of target DNA sequences of amplification products (step 4) comprises measuring a signal from a labelling entity which is a fluorophore by measuring the energy (fluorescence) emitted by the fluorophore, wherein the fluorophore is conjugated to a detection oligonucleotide probe, as defined herein, and wherein the energy
  • fluorescence emitted by the fluorophore is measured when the nucleic acid strands of a labelling oligonucleotide-detection oligonucleotide probe hybrid are separated.
  • each labelling oligonucleotide probe comprises DNA and is designed to hybridize under stringent conditions to a target DNA sequence from the given target microorganism of the pre-defined set and not to hybridize under stringent conditions to target DNA sequences of all other target microorganisms in the pre-de fined set.
  • the probe-based methods described above may be implemented in a variety of different ways. Certain embodiments involve the use of probes in conjunction with the performance of single nucleotide extension (SNE) reactions.
  • SNE single nucleotide extension
  • each probe is designed to act as a pimer in an enzymatic extension reaction and wherein the method further comprises performing a single nucleotide extension reaction (SNE) to add a single ddNTP to a terminal end of the probe, wherein each probe is designed to hybridise to the target DNA sequence in such a way that the next nucleotide to be added to the probe in the SNE is complementary to a nucleotide which is unique to the target DNA sequence, and wherein the SNE is performed in a reaction mixture which comprises only ddNTPs which are complementary to said nucleotide which is unique to the target DNA sequence.
  • SNE single nucleotide extension reaction
  • the ddNTPs provided in the reaction mixture may be conjugated to a quencher of a signal, a labelling entity or a binding entity.
  • the step of determining the presence, absence or quantity of each target DNA sequence within each amplicon may comprise the steps of:
  • microorganism in the set (step 4) based on the amount of the at least one labelling oligonucleotide probe- ddNTP-conugate.
  • a labelling oligonucleotide probe-ddNTP-conjugate purification step may be performed before step (c), preferably wherein step (b) comprises providing ddNTPs conjugated to a binding entity and wherein the binding entity can bind to a capture entity.
  • step (c) may comprise hybridising the at least one labelling oligonucleotide probe-ddNTP-conjugate of step (b) to at least one detection oligonucleotide probe to form a reaction mixture comprising at least one labelling oligonucleotide probe-ddNTP-conjugate-detection oligonucleotide probe hybrid, and wherein in step (c) a signal can be generated when the nucleic acid strands of the at least one labelling oligonucleotide probe-ddNTP- conjugate-detection oligonucleotide probe hybrid are separated.
  • the detection oligonucleotide probe may be coupled to at least one labelling entity and the step of measuring the amount of the at least one labelling oligonucleotide probe- ddNTP-conjugate comprises quantifying a signal generated via the labelling entity.
  • the labelling entity may be a quencher and the signal is an increase in energy emitted by a fluorophore coupled to the detection oligonucleotide when the nucleic acid strands of a labelling oligonucleotide probe-ddNTP-conjugate-detection oligonucleotide probe hybrid are separated.
  • the labelling entity coupled to a probe for the detection of a given target microorganism may differ from the the labelling entities coupled to probes for the detection of every other target microorganism in the pre-defined set; or (b) the labelling entity coupled to a probe for the detection of a given target microorganism may be the same as the labelling entities coupled to probes for the detection of every other target microorganism in the predefined set.
  • Each labelling oligonucleotide probe-ddNTP-conjugate-detection oligonucleotide probe with the same labelling entity may have a different melting temperature. In such methods different detection oligonucleotide probes may have different labelling entities.
  • the step of measuring the amount of the at least one detection oligonucleotide coupled fluorophore may comprise:
  • a labelling oligonucleotide probe may have a labelling entity coupled to the 5 ' end of the probe.
  • a ddNTP may be conjugated to a binding moiety and the detection
  • oligonucleotide probe may be coupled to at least one quencher, and the step of measuring the amount of the at least one labelling oligonucleotide probe-ddNTP- conjugate comprises quantifying a signal generated via the labelling entity.
  • each detection oligonucleotide probe may be conjugated to a labelling entity, and wherein each detection oligonucleotide probe corresponding to a specific target DNA sequence can be distinguished from the other detection oligonucleotide probes corresponding to all other specific target DNA sequences in the reaction vessel by different lengths.
  • different labelling entities may be conjugated to different detection oligonucleotide probes.
  • any combination of labelling entities and length of detection oligonucleotide probes may be used to uniquely determining the presence, absence or quantity of the target
  • microorganism in the set (step 4) based on the amount of the at least one detection oligonucleotide probe.
  • the ddNTP may be conjugated to a labelling moiety and wherein each labelling oligonucleotide probe corresponding to a specific target DNA sequence can be distinguished from the other labelling oligonucleotide probes corresponding to all other specific target DNA sequences in the reaction vessel by different lengths.
  • different labelling entities may be conjugated to different labelling oligonucleotide probes. Any combination of labelling entities and length of labelling oligonucleotide probes may be used to uniquely determine the presence, absence or quantity of the target microorganism in the set (step 4) based on the amount of the at least one labelling oligonucleotide probes.
  • the labelling moiety may be a fluorophore.
  • a binding moiety/entity may be able to be captured by an antibody or other capture entity.
  • a binding moiety/entity may be biotin and a capture entity may be streptavidin.
  • a labelling entity may be a quencher.
  • a quencher may be selected from the group consisting of BHQO, BHQ1, BHQ2, BHQ3, BHQ10, TAMRA, QXL520, EDQ, QXL570, EDQ1, QXL610, DDQ-II, QXL670, QXL, DDQ-1, Dabcyl, Eclipse, Iowa
  • a ddNTP is selected from the group consisting of ddC, ddT, ddA, ddG, ddl or ddU.
  • the methods of the invention may be implemented in a variety of different ways.
  • the methods of the invention may be implemented by sequencing amplicons.
  • step 4 the step of determining the presence, absence or quantity of target DNA sequences of amplification products within each amplicon and thus determining the presence, absence or quantity of each target microorganism in the pre-defined set (step 4) comprises:
  • sequence in the target DNA sequence region to identify the sequence and quantifying the amount thereof
  • step B comparing the sequence results of step A against known DNA sequences of all of the target microorganisms in the set;
  • Amplicons may be sequenced by next generation sequencing (NGS) methods.
  • NGS next generation sequencing
  • adaptor sequences may be attached to amplicons as part of the amplification steps to facilitate DNA sequencing.
  • To facilitate DNA sequencing index sequences may be attached to amplicons.
  • To facilitate DNA sequencing index sequences may be attached to amplicons in the same amplification reaction performed to attach adaptor sequences, or wherein index sequences may be attached to amplicons in an amplification reaction which is separate from the reaction performed to attach adaptor sequences.
  • the presence, absence or quantity of at least 2, at least 4, at least 6, at least 8, at least 10 or at least 20 different target microorganisms in the sample may be determined, optionally wherein at least 50-200 different target microorganisms in the sample is determined.
  • the target DNA sequence of any or all target microorganisms has a length of between about 50 to about 1500 bases.
  • Any of the methods of the invention further comprise quantifying the target microorganisms in the sample. Such methods may comprise determining the proportions of target microorganisms present within the sample.
  • target microorganisms may include bacteria, including gram-negative and gram-positive bacteria; and/or wherein target microorganisms may include fungi; and/or wherein target microorganisms include algae; and/or wherein target microorganisms may include viruses, including eukaryotic viruses and prokaryotic viruses.
  • the set of target microorganisms may be a set of microorganisms which inhabit a body region of an individual.
  • the body region may be a region of the gastrointestinal tract.
  • the sample may be from a mucosal layer of a region of the gastrointestinal tract or wherein the sample may be from the lumen of a region of the gastrointestinal tract.
  • the region of the gastrointestinal tract may be the mouth, toungue, throat, oesophagus, stomach, small intestine, large intestine, colon or rectum.
  • the body region may be the eye, ear, nasal cavity, skin, vagina or urethra.
  • the body region may be a region of biofilm on a surface of the individual.
  • the sample may be nasal mucous, saliva, sputum, oesophageal mucus, vomit, faeces, urine, vaginal mucous or skin.
  • the sample may be a sample from an individual, and wherein the individual is an animal, preferably a mammal such as an equine animal, a bovine animal, a porcine animal, a canine animal, a feline animal, an ovine animal, a rodent animal such as a murine animal including species of the genus mus and species of the genus rattus, preferably the individual is human.
  • the individual is an animal, preferably a mammal such as an equine animal, a bovine animal, a porcine animal, a canine animal, a feline animal, an ovine animal, a rodent animal such as a murine animal including species of the genus mus and species of the genus rattus, preferably the individual is human.
  • the set of target microorganisms may be a set of microorganisms which inhabit an environment or medium, and wherein the sample is a sample from the environment or medium.
  • the medium may be soil.
  • the group of microorganisms may be a microorganism genus and the target microorganism may be a microorganism species or strain.
  • the target microorganisms may be bacteria.
  • the target DNA sequence region may be outside of a ribosomal 16S sequence region.
  • the first and second DNA sequence conserved regions may be outside of a ribosomal 16S sequence region.
  • the target microorganisms may comprise bifidobacteria, enterobacteria, archebacteria, lactobacilli or chlostridia.
  • the target microorganisms may comprise are bifidobacterium adolescentis, bifidobacterium angulatum, bifidobacterium animalis, bifidobacterium bifidum, bifidobacterium breve, bifidobacterium catenulatum, bifidobacterium dentium, bifidobacterium gallicum, bifidobacterium kashiwanohense, bifidobacterium longum subsp. longum,
  • bifidobacterium longum subsp. infantis bifidobacterium pseudocatenulatum and/or bifidobacterium stercoris.
  • kits adapted for use in the method of any one of the methods of the invention.
  • a kit of the invention may comprise:
  • the at least one labelling oligonucleotide probe is complementary to a target DNA sequence in at least one genomic region and the at least one labelling oligonucleotide probe is complementary to the at least one detection oligonucleotide probe.
  • the kit may comprise at least two, at least three, at least four, at least five, at least ten or at least twenty labelling oligonucleotide probes.
  • the at least one labelling entity may be a quencher or a molecule to which a quencher may be attached.
  • the at least one labelling moiety may be a molecule to which a quencher may be attached which is a first binding moiety
  • the method comprises attaching the ddNTP conjugated to the first binding moiety to a quencher by exposing the ddNTP conjugated to the first binding moiety to a quencher-second binding moiety conjugate, wherein the first binding moiety and the second binding moiety have affinity for one another.
  • the quencher may be selected from the group consisting of BHQO, BHQ1, BHQ2, BHQ3, BHQ10, TAMRA, QXL520, EDQ,
  • QXL570 EDQ1, QXL610, DDQ-II, QXL670, QXL, DDQ-1, Dabcyl, Eclipse, Iowa Black FQ, QSY-7, QS-9, Iowa Black RQ, malachite green, blackberry quencher 650, ElleQuencher and QSY-21.
  • the kit may comprise at least 2, at least 4, at least 6, at least 8, at least 10 or at least 20 different labelling oligonucleotide probes.
  • the at least one detection oligonucleotide probe may be conjugated to a fluorophore.
  • the kit comprises at least 2 at least 4, at least 6, at least 8, at least 10 or at least 20 different detection oligonucleotide probes.
  • each detection oligonucleotide probe may be unique in that it has either a different fluorophore or a different melting temperature compared to other detection
  • kits of the invention may further comprise at least one affinity chromatography column.
  • kits of the invention may further comprise at least one buffer.
  • kits of the invention may further comprise enzymes for performing reactions required in methods of the invention.
  • the enzymes may comprise Taq polymerase.
  • the enzymes may comprise two different Taq polymerases.
  • the enzymes may comprise Exonuclease I.
  • the enzymes may comprise Shrimp Alkaline
  • a kit of the invention may also be a kit comprising forward and reverse primer pairs for generating a plurality of amplicons, wherein each amplicon is specific for a different group of related target microorganisms of the pre-defined set as defined herein, and wherein forward and reverse primer pairs are designed to anneal to DNA sequence regions comprising respectively the sequences of first and second conserved regions as defined herein.
  • Any of these kits of the invention may also further comprise at least one buffer and may further comprise enzymes for performing reactions required in methods of the invention.
  • the enzymes may comprise Taq polymerase.
  • the enzymes may comprise two different Taq polymerases.
  • the enzymes may comprise Exonuclease I.
  • the enzymes may comprise Shrimp Alkaline Phosphatase. BRIEF DESCRIPTION OF THE FIGURES
  • Figure 1 provides an example cartoon depiction of sections of the genomes of six different microorganisms.
  • Microorganisms A, B and C are target microorganisms which are all members of a group of related microorganisms.
  • Microorganisms D, E and F are different microorganisms from different groups of microorganisms.
  • Figure 2 (A to C) provides example cartoon depictions of four example non- limiting alternative method schematics for detecting probes specific for target DNA sequences using methods described herein for the incorporation of ddNTPs into labelling oligonucleotide probes.
  • Figure 2 (D) provides an example cartoon depiction of an example non-limiting alternative method schematic for detecting probes specific for target DNA sequences using methods described herein using bead-based reagents.
  • Figure 2 (E and F) provides example cartoon depictions of two example non-limiting alternative method schematics for detecting probes specific for target DNA sequences using methods described herein for the incorporation of ddNTPs into labelling oligonucleotide probes and wherein probes may be separated and detectibly resolved on the basis of their unique sizes.
  • Figure 3 shows genomic sequences of several exemplary Bifidobacteria species in an alignment. Species and strain specific sequence areas are shown in the boxed area together with example putative forward and reverse primer sequence areas. Each Bifidobacterium species corresponds to a target microorganism. Forward and reverse primer binding sequence areas correspond to first and second conserved regions of DNA sequence, wherein conserved regions are regions which are common to the group of Bifidobacteria microorganisms of which each target microorganism is a member and unique to that particular group.
  • Species and strain specific sequence areas shown between first and second conserved regions correspond to DNA sequences of target DNA sequence regions, each target DNA sequence region comprising DNA sequence that is unique to each target microorganism of the group within the group-specific amplicon generated via the first and second conserved regions.
  • Figure 4 shows a gel containing the result of a 16S rRNA amplification of genomic DNA. Lanes 1-12 indicate the 12 different bacteria amplified. The lanes headed BM1-BM6 designate the mock libraries. The lane headed Control contains a non-template control (containing no genomic DNA, but otherwise containing everything else for the PCR to occur).
  • Figure 5 provides a gel containing the result of a Bifidobacteria amplification of genomic DNA. Lanes 1-12 indicate the 12 different bacteria that were amplified. The lanes entitled BM1-7 designate the mock libraries. The Control is a non-template control (containing no genomic DNA, but otherwise containing everything else for the PCR to occur).
  • Figures 6 and 7 provide the results from LightCycler analysis.
  • the lines marked with a star represent ISsang PCR product, ISsangL (LP) and ISsangD (DP).
  • the lines identified with a square represent 2CramsD (DP).
  • Table 8 provides 6 Tables.
  • Table 1 describes the whole genome bacterial sequences that were used in Example 3.
  • Tables 2 and 3 lists the specific PCR primers and labelling oligonucleotide probes (LP) and detection oligonucleotide probes (DP) used in Example 4.
  • Table 4 describes the set-up of analysis of specificity of labelling oligonucleotide probes (LP) and detection oligonucleotide probes (DP).
  • Table 5 describes the specificity of each of the LP and DP.
  • Table 6 lists mock libraries of different compositions of the 12 genomic bacterial DNA listed in Table 1.
  • Figure 9 shows the separation of labelled probes on a Urea-PAGE gel on the basis of size.
  • the current invention applies a unique combination of steps which result in the desired outcome, i.e., a method to determine the presence, absence or quantity of target microorganisms in a sample containing multiple microorganisms at species and strain level in a convenient, fast, high-throughput and low-cost manner.
  • a first step in this process is to pre-define the target microorganisms of interest.
  • pre-defining the target microorganisms of interest the detection of those microorganisms becomes much faster and more accurate, and eliminates the occurrence of false positives and false negatives. This is because only the target microorganisms of interest in a sample will be analysed, and the analysis will involve the use of sequences which are unique to each target microorganism of interest.
  • a further step is to identify sequences in the DNA of groups of the pre-defined target microorganisms that can give rise to common amplicons (i.e. a part of the DNA sequence that is amenable to amplification, e.g. by PC ) wherein each amplicon is unique to a given group.
  • This is a critical step in order to detect only those target microorganisms within the selected groups, and leads to the high specificity of the methods of the invention.
  • the methods of the current invention instead limit the number of microbial sequences that are amplified in an amplicon. This leads to simpler and more specific detection, as only the target microorganisms that are amplified in the amplicon will need to be considered when determining the specific target microorganisms present in a complex mixture.
  • a unique sequence within a narrow amplicon can unequivocally be assigned to a specific microbial species or strain.
  • DNA sequences relative to a given target DNA sequence will not be represented in the same amplicon, and will therefore not be detected and will therefore not interfere with subsequent analysis. This is depicted visually in Figure 1.
  • the methods of the invention provide a high degree of specificity and selectivity.
  • the methods of the invention will require a plurality of several specific amplicons in order to detect a wide range of microorganisms, multiplex amplification of the specific amplicons may be performed. These amplicons can later optionally be combined, since it is possible to identify each amplicon in a multiplex sequencing reaction, for example by using specific probes.
  • test DNA is divided into different aliquots/reaction volumes, and perform single or multiplex amplification in each aliquot. This leads to higher flexibility in obtaining species and strain level identification of the pre-selected microorganisms.
  • these steps of the invention are new and different from the common methods used to identify microorganisms in a sample, and facilitate in determining the presence, absence or quantity of target microorganisms down to species and strain level in a sample containing multiple microorganisms.
  • the present methods therefore, result in detailed and accurate information about the composition of a microbiome, such as the gut microbiome, in a simplified and high- throughput format.
  • a target DNA sequence, as defined herein, derived from a target microorganism is considered to be "present” in a sample, if it is detected using a method of the invention.
  • a target microorganism is considered to be "absent” from a sample if the target DNA sequence, as defined herein, derived from the target microorganism is not detected using the methods of the invention.
  • Quantify should be understood to have its normal meaning in the art, i.e. that the stated feature or group of features is quantified, either absolutely or relatively to other microorganism or to previous analysis of similar samples, but that the term does not exclude any other stated feature or group of features from also being present.
  • a nucleic acid sequence such as an oligonucleotide sequence
  • TAGG is exactly complementary to ATCC
  • TAGG is exactly complementary to ATCC
  • a nucleic acid sequence such as a labelling oligonucleotide probe as described and defined herein, may be 80% or more, 90%> or more, 95% or more, 99% or 100%) identical to a sequence that is exactly complementary to another nucleic acid sequence, such as a target DNA sequence as described and defined herein.
  • complementary refers to a sequence that is exactly complementary (100% identical).
  • a moderately stringent hybridisation condition uses a prewashing solution containing 5x sodium chloride/sodium citrate (SSC), 0.5% SDS, 1.0 mM EDTA (pH 8.0), hybridisation buffer of about 50%> formamide, 6xSSC, and a hybridisation temperature of 55° C (or other similar hybridisation solutions, such as one containing about 50% formamide, with a hybridisation temperature of 42° C), and washing conditions of 60° C, in 0.5xSSC, 0.1 % SDS.
  • a stringent hybridisation condition hybridises in 6xSSC at 45° C, followed by one or more washes in O. lxSSC, 0.2% SDS at 68° C.
  • the methods of the invention involve determining the presence, absence or quantity of target DNA sequences from a sample which can uniquely identify a target microorganism and distinguish that target microorganism from all other target microorganisms in a pre-defined set of target microorganisms for the sample.
  • first and second DNA sequence conserved regions are identified which flank a target DNA sequence region.
  • a target DNA sequence region comprises a target DNA sequence.
  • the first and second conserved DNA sequence regions are selected such that they are converved across a group of microorganisms of which the target
  • microorganism is a member, but not across members of any other group.
  • a given first and second conserved DNA sequence region pair is unique to a particular group of microorganisms.
  • the first and second conserved DNA sequence regions are selected such that they may give rise to an amplicon under appropriate amplification reaction conditions.
  • a first and second conserved DNA sequence region pair can define an amplicon which is unique to that particular group of microorganisms.
  • the target DNA sequences interspersed between first and second conserved
  • DNA sequence regions are selected such that within the context of the group-specific amplicon each target DNA sequence is unique to a species of microorganism of the group or to a strain of microorganism of the group.
  • first and second conserved DNA sequence regions are selected such that a given pair of first and second conserved DNA sequence regions is converved across a group of microorganisms and is unique to that particular group of
  • an amplicon arising from that particular first and second conserved DNA sequence region pair will define an amplicon unique to a particular group of microorganisms and which amplicon comprises amplified products comprising target DNA sequences which can uniquely identify all species or strains of microorganism of the group which have been selected for analysis in the pre-defined set of
  • a target DNA sequence of a given target microorganism to be detected may not necessarily be a sequence that is unique to the genome of that given target
  • microorganism compared to all other microorganisms.
  • that specific target DNA sequence is represented in the genome of any other different species or any other different strain of microorganism (i.e. a non-target microorganism, or a target microorganism from a different group) it will not be present in amplified products arising from such other microorganisms because it will not contain the two conserved DNA regions of the target DNA sequence. Consequently, it will not interfere with detection of the target DNA sequence from the given target microorganism.
  • a given target DNA sequence is pre-selected such that it is a sequence which is unique to a given target microorganism of a group of target microorganisms within a pre-selected amplicon which is unique for the group of target microorganisms to which the given target microorganism belongs (see Figure 1).
  • the methods of the invention provide improvements over pre-existing methods by reducing or eliminating false positives, since the same or similar target DNA sequences which might be represented in the genomes of such other microorganisms will in contrast not be represented in amplicons generated for detection.
  • the present methods involve determining the presence, absence or quantity of target microorganisms in a sample containing multiple microorganisms.
  • the sample may be any suitable sample which will contain multiple amino acids
  • microorganisms and wherein the preceise number, composition and proportion of microorganisms within the sample may vary depending upon the origin of the sample.
  • the sample may for example be a specific environment or medium such as soil. Soil is known to contain many different microorganisms.
  • the present methods enable the establishment of a set of target microorganisms, i.e. the set of target microorganisms is pre-defined.
  • the set of target microorganisms represents groups of microorganisms, wherein each target microorganism in the set is known to be potentially present in any given test sample.
  • the preceise number, composition and proportion of known target microorganisms within the sample may vary.
  • the methods allow the identity of target microorganisms of the set of potential target microorganisms to be determined for any given sample.
  • the proportion of target microorganisms within the set of potential target microorganisms can also be determined for any given sample, thus allowing for quantification of target microorganisms within the set of potential target microorganisms.
  • the sample is typically a sample from an individual, such as a sample from a body region of an individual.
  • the individual is typically an animal, preferably a mammal such as an equine animal, a bovine animal, a porcine animal, a canine animal, a feline animal, an ovine animal, a rodent animal such as a murine animal including species of the genus mus and species of the genus rattus, preferably the individual is human.
  • the sample may for example be a sputum sample, skin swab or a fecal or stool sample.
  • Identification of the target microorganisms of a set of target microorganisms known to be potentially present in such samples will assist in the diagnosis of conditions relevant to the gastrointestinal tract, such as irritable bowel syndrome.
  • Identification of the target microorganisms present in such samples may also help in determining the most appropriate treatment options for individuals suffering from particular disorders.
  • the methods of the present invention also allow the determination of target microorganisms which might be lacking in a body region of an individual. For example, the identification of the absence or low abundance of a particular group, species or strain of target microorganisms in a body region of an individual, e.g.
  • bifidobacteria in the stomach or intestine may allow the individual to supplement that body region with the target microorganisms that are absent or present in low abundance.
  • the sample may be from a body region and/or may represent target microorganisms which inhabit a body region.
  • the body region may be a region of the gastrointestinal tract such as the mouth, toungue, throat, oesophagus, stomach, small intestine, large intestine, colon or rectum.
  • the body region may be the eye, ear, nasal cavity, skin, vagina or urethra.
  • the body region may be a region of biofilm on a surface of the individual.
  • the sample may be a sample of for example nasal mucous, saliva, sputum, oesophageal mucus, vomit, faeces, urine, vaginal mucous or skin.
  • any suitable target microorganism may be detected using the methods of the present invention. Since a set of target microorganisms to be detected is pre-defined, the methods allow a determination to be made as the presence, absence or quantity of a given target microorganism of the set.
  • Target microorganisms which may be detected using the methods of the invention include bacteria.
  • bacteria that can be detected include but are not limited to Staphylococcus aureus, Streptococcus pyogenes (group A), Streptococcus sp. (viridans group), Streptococcus agalactiae (group B), S.
  • Streptococcus anaerobic species
  • Streptococcus pneumoniae and Enterococcus sp.
  • gram-negative cocci such as, for example, Neisseria gonorrhoeae, Neisseria meningitidis, and Branhamella catarrhalis
  • gram-positive bacilli such as Bacillus anthracis, Bacillus subtilis, Propionibacterium acnes
  • Infection with one or more of these bacteria can result in diseases such as bacteremia, pneumonia, meningitis, osteomyelitis, endocarditis, sinusitis, arthritis, urinary tract infections, tetanus, gangrene, colitis, acute gastroenteritis, impetigo, acne, acne posacue, wound infections, born infections, fascitis, bronchitis, and a variety of abscesses, nosocomial infections, and opportunistic infections.
  • diseases such as bacteremia, pneumonia, meningitis, osteomyelitis, endocarditis, sinusitis, arthritis, urinary tract infections, tetanus, gangrene, colitis, acute gastroenteritis, impetigo, acne, acne posacue, wound infections, born infections, fascitis, bronchitis, and a variety of abscesses, nosocomial infections, and opportunistic infections.
  • target bacteria include but are not limited to Achrombacter sp.,
  • Acinetobacter sp. including Aerobacter aerogeus; Alcaligenes sp.; Bacillus sp.
  • Bacillus cerius Bacillus subtilus
  • Beggiatoa sp. Brevibacterium sp.
  • Burkholderia cepacia Citrobacter sp.
  • Clostridium sp. Clostridium sp.
  • Corynebacterium sp. Burkholderia cepacia, Citrobacter sp.;
  • Pseudomonas aeruginosa Pseudomonas alcaligenes, Pseudomonas cepacia,
  • Funsi Target fungal microorganisms which may detected using the methods described herein include but are not limited to dermatophytes (e.g., Microsporum canis and other Microsporum sp.; and Trichophyton sp. such as T. rubrum, and T. mentagrophytes), yeasts (e.g., Candida albicans, C. tropicalis, or other Candida species), Saccharomyces cerevisiae, Torulopsis glabrata, Epidermophyton floccosum, Malassezia furfur
  • Rhizopus, Mucor Paracoccidioides brasiliensis , Blastomyces dermatitides ,
  • Histoplasma capsulatum Histoplasma capsulatum, Coccidioides immitis, and Sporothrix schenckii.
  • Target fungi include but are not limited to Alternaria sp.; Amorphotheca sp.; Aspergillus niger, Aureobasidium sp.; Cephalosporium sp.; Chaetomium globosum, Cladosporium sp.; Fungi imperfecti; Fusarium sp.; Geotricum sp.; Gloeophyllum sp.; Lentinus sp.; Mucro sp.; Penicillium sp.; Phoma sp.; Rhizopus sp.; Saccharomyces sp.; Trichoderma sp.; Tricophyton sp.; Trichosporon sp.
  • Target algal microorganisms which may detected using the methods described herein include but are not limited to Anabaena sp.; Anacystis sp.; Ankistrodesmus sp.; Ascomycetes; Basidomycetes; Chlorella sp.; Calothrix sp.; Chlorococcum sp.;
  • Phormidium sp. Phordium luridum; Scenedesmus sp.; Schizothrix sp.; Selenastrum sp.; Spirogyra sp.; Ulothrix sp.
  • Target microorganisms which may be detected using any of the methods of the invention described herein include viruses.
  • Viruses which may be detected using any of the methods described herein include eukaryotic viruses and prokaryotic viruses.
  • Target eukaryotic viruses which may be detected using any of the methods described herein include, but are not limited to, the following.
  • Viruses of the Reoviridae family such as rotavirus and reovirus
  • viruses of the Orthomyxoviridae family such as influenza virus
  • viruses of the Arenaviridae family such as Lymphocytic Choriomeningitis Virus (LCMV)
  • viruses of the Flaviviridae family such as denge virus
  • viruses of the Picornaviridae family such as Theiler's Murine Encephalomyelitis Virus (TMEV), poliovirus and Coxsackievirus B3 (CVB3)
  • viruses of the Retroviridae family such as Mouse Mammary Tumor Virus (MMTV), Murine Leukemia Virus (MLV) and Human Immunodeficiency Virus (HIV); viruses of the Adenoviridae family; viruses of the Caliciviridae family, such as norovirus; viruses of the Papov
  • Prokaryotic viruses Target prokaryotic viruses which may be detected using any of the methods described herein include, but are not limited to, archeal viruses and bacterial viruses (bacteriophage).
  • Target prokaryotic viruses which may be detected using any of the methods described herein include lysogenic bacteriophages and lytic bacteriophages.
  • Target prokaryotic viruses which may be detected using any of the methods described herein include viruses of the Microviridae family.
  • Target prokaryotic viruses which may be detected using any of the methods described herein include viruses of the Caudovirales order, such as viruses of the Podoviridae family, viruses of the Siphoviridae family and viruses of the Myoviridae family.
  • Non-limiting examples of target microorganisms which can colonise specifc body regions are described as follow.
  • Target microorganisms which can colonise the nasopharynx and which can be detected using the methods described herein include but are not limited to the following. Haemophilus, Neisseria, Staph, aureus, Staph, epidermidis, Strep, viridans and Strep, pneumoniae.
  • Target microorganisms which can colonise the outer ear and which can be detected using the methods described herein include but are not limited to the following. Enterobacteriaceae, Pseudomonas, Staph, epidermidis and Strep, pneumoniae.
  • Target microorganisms which can colonise the eye and which can be detected using the methods described herein include but are not limited to the following.
  • Target microorganisms which can colonise the stomach and which can be detected using the methods described herein include but are not limited to the following. Helicobacter pylori, Lactobacillus, Bifidobacteria and Streptococcus .
  • Target microorganisms which can colonise the small intestine and which can be detected using the methods described herein include but are not limited to the following. Bacteroides, Candida, Clostridium, Enterobacteriaceae, Enterococcus, Fusobacterium, Lactobacillus, Peptostreptococcus, Staphylococcus, and Streptococcus .
  • Target microorganisms which can colonise the large intestine and which can be detected using the methods described herein include but are not limited to the following. Bacteroides, Candida, Clostridium, Corynebacterium, Enterobacteriaceae,
  • Enterococcus Fusobacterium, Lactobacillus, Mycobacterium, Peptostreptococcus, Pseudomonas, Staphylococcus and Streptococcus .
  • Target microorganisms which can colonise the anterior urethra and which can be detected using the methods described herein include but are not limited to the following.
  • Candida Corynebacterium, Enterobacteriaceae, Enterococcus, Gardneralla vaginalis, Lactobacillus, Mycoplasma, Staph, epidermidis, Streptococcus and Ureaplasma.
  • Target microorganisms which can colonise the vagina and which can be detected using the methods described herein include but are not limited to the following.
  • Actinomyces Bacteroides, Candida, Clostridium, Enterobacteriaceae, Enterococcus, Fusobacterium, Gardnerella vaginalis, Lactobacillus, Mobiluncus, Mycoplasma, Staphylococcus, Streptococcus, Torulopsis and Ureaplasma.
  • Target microorganisms which can colonise the skin and which can be detected using the methods described herein include but are not limited to the following.
  • Candida Clostridium, Corynebacterium, Proprionibacterium, Staph, aureus, Staph, epidermidis and Strep, pyogenes.
  • the present methods allow for determining the presence, absence or quantity of target microorganisms in a sample containing multiple microorganisms, wherein each target microorganism is a member of a pre-defined set of target microorganisms for the sample.
  • the step of pre-defining the set of target microorganisms which may be present within a given sample is performed before the procedural steps of analysing DNA in a given sample to determine the presence or absence of specific target microorganisms within that sample.
  • Pre-defining a set of target microorganisms for a sample, each of which may potentially be present in any given subsequent test sample may be achieved by suitable techniques such as described in the Examples herein.
  • the likelihood of one or more given target microorganisms to be present in a given sample from a given environment or medium may be known, e.g. from the literature. It is therefore possible to arrive at a pre-defined set of
  • microorganisms for a given environment, medium or sample therefrom, wherein the set represents a set of microorganisms any of which may be present in a test sample.
  • the analysis methods of the present invention it is possible to determine whether any given target microorganism is indeed present or absent in a test sample.
  • the "set" of target microorganisms is the sum total of all "target microorganisms" which are pre-defined to be potentially present in a given sample.
  • any given target microorganism in the "set” is a member of a “group” of related microorganisms within the set.
  • related it is meant that any given target microorganism may be for example a species or strain belonging to a particular group, e.g. a genus, of microorganisms.
  • the set of target microorganisms may comprise several species and/or strains from the group of bifidobacteria, several species and/or strains from the group of enterobacteria, several species and/or strains from the group of archebacteria, several species and/or strains from the group of lactobacilli and several species and/or strains from the group of chlostridia.
  • each species and/or strains from the group of bifidobacteria may comprise several species and/or strains from the group of enterobacteria, several species and/or strains from the group of archebacteria, several species and/or strains from the group of lactobacilli and several species and/or strains from the group of chlostridia.
  • species/strain whose presence, absence or quantity is to be determined is a "target microorganism".
  • Each species/strain is a target microorganism belonging to a particular "group" of related target microorganisms, the groups being bifidobacteria,
  • the pre-defined set comprises the sum total of all target microorganisms of all groups of related microorganisms which may be potentially present in a given sample.
  • the target DNA sequence regions of the target microorganism to be detected are interspersed between two conserved regions of DNA sequence, wherein the conserved regions are conserved across the group of
  • microorganisms of which the target microorganism is a member are a member.
  • the two conserved regions of DNA sequence may be referred to herein as first and second conserved regions of DNA sequence. These sequences are selected such that they are capable of promoting the formation of an amplicon under appropriate amplification conditions.
  • the first and second conserved regions of DNA sequence are selected such that they may promote the formation of a group-specific amplicon under appropriate amplification conditions wherein the group-specific amplicon comprises amplification products which are unique to a given group of target microorganisms, and wherein the amplification products of that group-specific amplicon do not comprise amplification products derived from any other group of target microorganisms.
  • the methods of the invention involve the generation of a plurality of group-specific amplicons wherein each group-specific amplicon may comprise amplification products derived from multiple different individual members of that particular group of microorganisms.
  • first and second "conserved" regions of DNA sequence it is meant regions of sequence both of which are represented in each member of a particular group of microorganisms and in a positional configuration in the genome which may allow the formation of an amplicon under appropriate amplification reaction conditions; and wherein one or both sequences are not represented in any member of another group of microorganisms, or cannot give rise to an amplicon derived from any member of another group of microorganisms under appropriate amplification reaction conditions.
  • Enterobacteria genus it will be necessary to pre-select first and second conserved regions of DNA sequence which are represented in all members of the Lactobacillus genus whose detection is desired, but absent from all members of the Clostridia and Enterobacteria genuses or which cannot promote the formation of amplicons from members of the Clostridia and Enterobacteria genuses.
  • first and second conserved regions of DNA sequence which are represented in all members of the Clostridia genus whose detection is desired, but absent from all members of the Lactobacillus and Enterobacteria genuses or which cannot promote the formation of amplicons from members of the Lactobacillus and Enterobacteria genuses.
  • first and second conserved regions of DNA sequence which are represented in all members of the Enterobacteria genus whose detection is desired, but absent from all members of the Clostridia and Lactobacillus genuses or which cannot promote the formation of amplicons from members of the Clostridia and Lactobacillus genuses.
  • sequences within a pre-selected first conserved region of DNA sequence are 100% identical across all members of a given group whose detection is desired.
  • sequences within the associated preselected second conserved region of DNA sequence are 100% identical across all members of that given group whose detection is desired. Degeneracy in both sequence regions may be permitted, provided that the pre-selected sequences, when used together under appropriate amplification reaction conditions, are capable of promoting the formation of an amplicon from the desired group of target microorganisms but are not capable of promoting the formation of an amplicon from any other different group of target microorganisms.
  • Amplicons can be generated using degenerate sequence regions which are conserved across members of a group of target microorganisms using techniques known in the art, such as by using the same forward and reverse primers which are designed to take into account such degeneracy and which can thus specifically anneal to first and second conserved regions notwithstanding any sequence degeneracy that may exist between different members of the same group.
  • a “conserved” region may be a region of DNA having a sequence which has e.g. 85% or more, 90% or more, 95% or more, 98% or more, 99% or 100%) sequence identity to the sequence in a corresponding region in a different
  • a conserved region could be one where the sequence was 90%> identical between at least two sequences of Ruminococcus.
  • the methods of the invention involve the generation of a plurality of group-specific amplicons.
  • the methods may allow the generation of two or more amplicons. This would mean the method would allow the identification of target microorganism members of two different groups of target microorganism.
  • the methods of the invention are not limited by the number of amplicons that can be generated for any given sample, particularly since multiplex amplification of DNA from the same sample in multiple aliquots allows versatility in the number of primers that can be used in separate parallel reactions.
  • the methods may allow the generation of 5 or more amplicons, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, 35 or more, 40 or more, 45 or more, 50 or more amplicons from a given sample.
  • DNA from a given sample is split into aliquots and added to separate reactions for amplicon generation.
  • Multiple different amplicons may be generated in each aliquot. Typically 5 or more amplicons may be generated in each aliquot, 10 or more amplicons may be generated in each aliquot, 15 or more amplicons may be generated in each aliquot, 20 or more amplicons may be generated in each aliquot, 25 or more amplicons may be generated in each aliquot, 30 amplicons may be generated in each aliquot.
  • First and second conserved DNA regions may comprise any appropriate length, provided that the above-noted principles are applied.
  • first and second conserved DNA regions will comprise binding sites for primers to be used in amplification reactions, and therefore the length of these regions may be dictated by primer binding requirements, and further to take into account any possible sequence degeneracy that may exist.
  • first and second conserved DNA regions may be from about 15 to about 50 bases in length, preferably about 25 bases in length.
  • target DNA sequence regions of target microorganisms to be detected in any group-specific amplicon are interspersed between the two conserved regions of DNA sequence.
  • a target DNA sequence region comprises a target DNA sequence.
  • a target DNA sequence is a variable sequence region within amplification products of the amplicon, wherein the target DNA sequence comprises DNA sequence that is unique sequence to a specific target microorganism within the amplicon. Examplary methods for determining the presence, absence or quantity of such target DNA sequence regions in test samples are described in more detail herein, including in the Examples herein.
  • a target DNA sequence may have less than 90%, less than 95%, less than 98% or less than 100% identity compared to corresponding sequence areas in other bacteria.
  • a target DNA sequence of a given target microorganism to be detected may not necessarily be a sequence that is unique to the genome of that given target microorganism compared to all other microorganisms. However, if that specific target DNA sequence is represented in the genome of any other different species or any other different strain of microorganism (i.e. a non-target microorganism, or a target microorganism from a different group) it will not be present in amplified products arising from such other microorganisms. This is because the first and second converved regions noted above will have been pre-selected in such a way that any such DNA sequence which might be represented in the genome of any other different species or any other different strain of microorganism will not form part of any amplicon.
  • Target DNA sequences may comprise any appropriate length, provided that the above-noted principles are applied. Typically target DNA sequences will be about 50 to about 1500 bases in length.
  • FIG. 1 provides an example cartoon depiction of sections of the genomes of six different microorganisms.
  • Microorganisms A, B and C are target microorganisms which are all members of a group of related microorganisms. For example,
  • microorganism A could be bifidobacterium bifidum
  • microorganism B could be bifidobacterium breve
  • microorganism C could be bifidobacterium catenulatum.
  • the boxes depicted with vertical line shade represent first conserved DNA regions (C 1) which are conserved across all members of the group of related target microorganisms.
  • the boxes depicted with horizontal line shade represent second conserved DNA regions (CR2) which are also conserved across all members of the group of related target microorganisms.
  • CR1 and CR2 are pre-selected so that they can promote the formation of an amplicon under appropriate amplification reaction conditions, e.g. by acting as binding sites for amplification primers such as forward and reverse PCR primers.
  • CR1 and CR2 are deliberately pre-selected such that they are in an appropriate positional configuration in the genomes of all members of a specific group of target microorganisms so that they will promote the generation of an amplicon under appropriate amplification conditions, and such that they will not promote the generation of amplicons derived from microorganisms which are members of a different group of microorganisms.
  • the amplicon derived from CR1 and CR2 will be a group-specific amplicon.
  • CR1 and CR2 of each of target microorganisms A, B and C are interspersed with target DNA sequences, each of which are, within the group-specific amplicon, unique to the target microorganism. Therefore, specific detection of each target DNA sequence within the group-specific amplicon provides a means to identify a target microorganism and distinguish it from all other target microorganisms in a pre-defined set of target microorganisms.
  • Detection of a given target DNA sequence in an amplicon generated from DNA from a sample indicates that the given target microorganism is present in the sample because the target DNA sequence was amplified. Conversely, if a given target DNA sequence is not detected in an amplicon generated from DNA from a sample this indicates that the given target microorganism is absent from the sample as the target DNA sequence was not amplified.
  • microorganism D of Figure 1 harbours a sequence within its genome which is identical to the target DNA sequence of microorganism C. However, this sequence of microorganism D will not be amplified because it is not interspersed between sequences CRl and CR2. Sequences CRl and CR2 are deliberately preselected such that they promote the formation of amplicons only from the group of microorganisms of which microorganism C is a member.
  • microorganism E of Figure 1 harbours a sequence within its genome which is identical to the target DNA sequence of microorganism C and thus sequence is juxtaposed next to a DNA sequence which is identical to CRl .
  • this sequence of microorganism E will not be amplified because it is not interspersed between sequences CRl and CR2.
  • C 1 and CR2 comprise sequences that are 100% unique to microorganism members of the specific group of microorganisms.
  • microorganism F depicted in Figure 1 may be a different microorganism from a different group of microorganisms compared to microorganisms A, B and C.
  • Microorganism F harbours a sequence which is 100% identical to the sequence of CR2.
  • CR2 is not positioned next to a sequence corresponding to CR1, and consequently no amplicon will be generated from microorganism F.
  • a group-specific amplicon of appropriate length by pre-selecting sequences for CR1 and CR2 that are both represented in and conserved across the genomes of all target microorganisms in a group of related microorganisms, and such that their relative positioning in the genomes of microorganisms of that group allows the formation of an amplicon of appropriate length; and conversely by also ensuring that such sequences are not both represented in the genomes of different microorganism members of different groups, or that the positioning of sequences in the genomes of such other microorganisms is such that no amplicon can form.
  • microorganisms from all other target microorganisms in a pre-defined set of target microorganisms. At the same time the occurrence of false positive results and false negative results is significantly reduced or eliminated completely. Furthermore, by generating a plurality of group-specific amplicons the amount of sequence information that needs to be detected and processed is dramatically reduced.
  • the core methodology of the invention provides for extremely sensitivity, specificity and efficiency in the detection of target microorganisms within complex mixtures.
  • Amplicons can be generated from pre-selected first and second conserved regions of DNA sequence using any standard amplification technique known in the art. Typically, amplification may be performed by PCR using forward and reverse primers wherein a forward primer may bind to a first conserved region and a reverse primer may bind to a second conserved region.
  • Amplification may be performed by any suitable method, such as polymerase chain reaction (PCR), polymerase spiral reaction (PSR), loop mediated isothermal amplification (LAMP), nucleic acid sequence based amplification (NASBA), self- sustained sequence replication (3SR), rolling circle amplification (RCA), strand displacement amplification (SDA), multiple displacement amplification (MDA), ligase chain reaction (LCR), helicase dependant amplification (HDA), ramification
  • PCR polymerase chain reaction
  • PSR polymerase spiral reaction
  • LAMP loop mediated isothermal amplification
  • NASBA nucleic acid sequence based amplification
  • NASBA self- sustained sequence replication
  • RCA rolling circle amplification
  • SDA strand displacement amplification
  • MDA multiple displacement amplification
  • HDA helicase dependant amplification
  • amplification is performed by polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • a variation of PCR may be used, for instance Real Time PCR (also known as quantitative PCR, qPCR), hot-start PCR, competitive PCR, or any other.
  • the PCR reaction used may comprise 20-40 repeats of the following steps:
  • a denaturation step which may comprise subjecting the oligonucleotides in the aliquot or in the sample to a temperature between 90-100 °C for 10- 40 seconds;
  • an annealing step which may comprise subjecting the oligonucleotides in the aliquot or in the sample to a temperature between 50-65 °C for 10-40 seconds;
  • an extension step which may comprise subjecting the oligonucleotides in the aliquot or in the sample to a temperature in which the DNA polymerase has optimum activity.
  • a suitable DNA polymerase is Taq polymerase, which has its optimum activity between 70-85 °C. Since the methods of the invention may require multiple parallel amplification reactions, the reactions may be performed separately in parallel using multiplex approaches. For these purposes the sample may be divided into one or more aliquots. In such embodiments, the method may comprise a step of preparing one or more aliquots from the sample. In the context of the present invention, an "aliquot" is a portion of the sample. It is possible to use any number of aliquots in the method of the invention.
  • the presence or absence of a target DNA sequence is detected using detection methods described herein. Such detection methods may be used to quantify the amount of target DNA sequence that is present.
  • the number of these which hybridise will be proportional to the amount of the target DNA sequence that is present in the original sample.
  • a sample is probed with multiple copies of a first probe that hybridises to a first target DNA sequence and multiple copies of a second probe that hybridises to a second oligonucleotide, it will be possible to determine the amount of the first target DNA sequence relative to the amount of the second target DNA sequence.
  • the term “quanity” may be used interchangeably with “relative amount” and both terms may refer to the amount of one target DNA sequence in a sample compared to the amount of a second target DNA sequence in a sample.
  • the second target DNA sequence may be a reference
  • oligonucleotide For example, a known quantity of a known oligonucleotide may be added to the sample. The user can then compare the relative amount of a target DNA sequence to the amount of the known oligonucleotide to estimate the absolute concentration of the target DNA sequence in the sample.
  • concentration may also be used interchangeably with "absolute amount”.
  • the methods of the invention provide a means to determine the absolute amount of a given microorganism in a sample. A skilled person will appreciate that such determination may be achieved e.g. relative to a calibrated control. Preparing Target DNA Sequences for Detection
  • any of the methods described and defined herein may comprise a step of preparing the target DNA sequences for the step of detecting the presence, absence or quantity of the target DNA sequences.
  • the step of preparing the target DNA sequences occurs prior to the step of detecting the presence, absence or quantity of the target DNA sequences.
  • the step of preparing the target DNA sequences removes or inactivates any DNA/RNA polymerase and/or PCR primers and/or free nucleotides that may be present.
  • the step of preparing the target DNA sequences may be used to remove DNA/RNA polymerase and/or primers and/or free nucleotides that were used in a step of amplifying the target DNA sequences.
  • the step of preparing the target DNA sequences may comprise a step of purifying the target DNA sequences.
  • the step of preparing the target DNA sequences may comprise a chromatography step, such as an affinity chromatography step.
  • the target DNA sequences may be purified by application to a column comprising glass beads under conditions in which the target DNA sequences bind to the glass beads. Suitable conditions include the addition of a chaotropic agent. Suitable chaotropic agents include sodium iodide or sodium perchlorate.
  • the purification step may comprise a filtration step, such as filtration using a filter having a pore size of 0.2 ⁇ . Purification may be used to remove primers and free nucleotides.
  • primers may be removed using one or more washing steps (e.g. with water or a buffered solution which may contain formamide and/or a detergent), or by electrophoresis, centrifugation, capture onto solid supports, chromatography or any combination thereof.
  • washing steps e.g. with water or a buffered solution which may contain formamide and/or a detergent
  • the step of preparing the target DNA sequences may also comprise an enzymatic treatment step.
  • an enzyme that inactivates DNA/RNA polymerase include proteinase K.
  • surplus primers can be inactivated with an exonuclease that digests any free single stranded oligonucleotides in solution.
  • Suitable enzymes that can be used for this purpose include for example, an enzyme with a 3' ⁇ 5' single strand exonuclease activity such as Exonuclease I and an enzyme that catalyzes the dephosphorylation of 5 ' and 3 ' ends of deoxyribonucleoside triphosphates and inactivates free nucleotides, for example Shrimp Alkaline Phosphatase.
  • the preparation step may further comprising a heat exposure step, such as exposure of the target DNA sequences to a temperature greater than 40°C, 50°C, 60°C, 70°C or 80°C.
  • a heat exposure step such as exposure of the target DNA sequences to a temperature greater than 40°C, 50°C, 60°C, 70°C or 80°C.
  • the target DNA sequences could be exposed to a temperature between 60°C and 120°C, between 70°C and 100°C, or around 80°C for a period of time of greater than 30 minutes, greater than 40 minutes, between 30 minutes and 2 hours, between 20 minutes and 1 hour, or around 50 minutes.
  • Such a heat treatment step can be used to inactivate enzymes and avoids the need for a further purification step.
  • Methods of the invention may also comprise a step of measuring the amount of total target DNA sequences before the step of detecting the presence, absence or relative amount of the target DNA sequences, i.e. if three target DNA sequences are present in the sample, the amount of all three target DNA sequences can be measured using a technique such as gel electrophoresis or spectrophotometry.
  • the absorbance at 260 nm can provide a measure of total oligonucleotides present in a sample, and so measuring the absorbance at 260 nm after the step of preparing the target DNA sequences will provide a measure of the total amount of target DNA sequences in the sample.
  • Other suitable methods includes quantitative PC methods such as using the FentoTM bacterial DNA quantification kit (Zymo Research).
  • the amount of target DNA sequences may be compared to a reference, for example a sample having a known amount of DNA present.
  • the methods of the invention may use at least one labelling oligonucleotide probe, and the kits of the invention may comprise at least one labelling oligonucleotide probe.
  • the at least one labelling oligonucleotide probe is an oligonucleotide that is complementary to at least one of the target DNA sequences.
  • three different labelling oligonucleotide probes will be used and each of the three different labelling oligonucleotide probes will hybridise with one of the three different target DNA sequences and each of the three different labelling oligonucleotide probes will hybridise with a different target DNA sequence.
  • kits of the invention may comprise at least 2, at least 4, at least 6, at least 8, at least 10, at least 20, at least 30, at least 50, between 2 and 75, between 4 and 50, between 6 and 30, between 8 and 25, between 10 and 20 and between 50-200 different labelling oligonucleotide probes.
  • the at least one labelling oligonucleotide probe hybridises to at least one of the target DNA sequences under a moderately stringent hybridisation condition.
  • the at least one labelling oligonucleotide probe hybridises to at least one of the target DNA sequences under a stringent hybridisation condition.
  • Methods of the invention may comprise a step of hybridising the target DNA sequences to at least one labelling oligonucleotide probe to form hybridised labelling oligonucleotide probes.
  • hybridising the target DNA sequences to at least one labelling oligonucleotide probe
  • hybridising is intended to refer to exposing the target DNA sequences to at least one labelling oligonucleotide probe under conditions in which hybridisation of the target DNA sequence to the at least one labelling
  • the at least one labelling oligonucleotide probe can occur. If the target DNA sequence is present in the sample, then the at least one labelling oligonucleotide probe will anneal to the target DNA sequence under suitable conditions.
  • conditions suitable for hybridisation to occur include cool temperatures and high salt conditions.
  • the at least one labelling oligonucleotide probe and the target DNA sequences are mixed together and exposed to temperatures between 40°C and 70°C or between 50°C and 65 °C for at least 10 seconds (for example at least 20 seconds, at least 30 seconds, between 10 seconds and 5 minutes, between 20 seconds and 2 minutes, or between 20 seconds and 1 minute).
  • methods of the invention may comprise a step of performing a single nucleotide extension (SNE) reaction to add a ddNTP to the at least one labelling oligonucleotide probe-amplification product hybrid to form at least one ddNTP-labelling oligonucleotide probe-amplification product hybrid.
  • SNE single nucleotide extension
  • the ddNTP that is used may be selected from ddC (dideoxycytosine), ddT (dideoxythymine), ddG (dideoxy guanine), ddA (dideoxyadenine), ddl (dideoxyinosine) or ddU (dideoxyuracil).
  • ddC is more preferred because it will bind to guanine more strongly than ddA or ddT will bind to adenine or thymine (adenine binds to thymine with three hydrogen bonds whilst guanine binds to cytosine with two hydrogen bonds).
  • the sample may be divided into multiple aliquots.
  • different ddNTPs may be used in different aliquots.
  • the labelling oligonucleotide probe is designed in such a way that it hybridises to the target DNA sequence in such a way that the next nucleotide to be added to the probe in an enzymatic nucleic acid extension reaction is the specific nucleotide of the ddNTP that is present in the reaction mixture.
  • single nucleotide extension reaction is a reaction in which a single ddNTP is added to an oligonucleotide.
  • the ddNTP is added to hybridised labelling oligonucleotide probes, as the hybridised labelling oligonucleotide probes will comprise an overhang region.
  • the at least one labelling oligonucleotide probe is shorter than the target DNA sequence to which it hybridised, meaning that the hybridised labelling oligonucleotide probes will comprise a double-stranded region and a single stranded (overhang) region.
  • a ddNTP may be added that is complementary to the first nucleotide of the overhang region and this is a "single nucleotide extension reaction" of the invention. Since ddNTPs lack the 3'-hydroxyl group of normal deoxynucleotides, only a single ddNTP can be added to the chain. This provides an additional degree of specificity to probe-based detection methods of the invention in addition to only hybridization.
  • the single nucleotide extension reaction may be performed by any suitable method
  • a suitable polymerase for use in the step of performing a single nucleotide extension reaction is a DNA polymerase that has enhanced efficiency for incorporating unconventional nucleotides.
  • the DNA polymerase has 5 ' to 3' polymerase activity.
  • the DNA polymerase used in the step of performing a single nucleotide extension reaction may be HotTERMIPol DNA Polymerase (Solis).
  • the single nucleotide extension reaction is carried out in a polymerase chain reaction (PCR) machine.
  • the PCR machine may be used to provide suitable conditions to add the ddNTP to the hybridised labelling oligonucleotide probe.
  • suitable conditions include at least one cycle of high temperature (for example a temperature higher than 80°C) followed by at least one cycle at a lower temperature (for example a temperature lower than 70 °C).
  • the suitable conditions include at least one cycle at a temperature between 80°C and 100°C, between 80°C and 95°C or between 85 °C and 95 °C, followed by at least one cycle at a temperature between 30°C and 70°C, between 40°C and 65 °C, or between 50°C and 65 °C. 2 or more cycles may be used. For example, 3, 4, 5, 6, 7, 8, 9 or 10 or more cycles may be used.
  • Methods of the invention may comprise a step of performing a single nucleotide extension reaction to add a ddNTP to a hybridised labelling oligonucleotide probe, as described above, optionally wherein the ddNTP is conjugated to at least one further labelling moiety.
  • Kits of the invention may comprise a ddNTP conjugated to at least one labelling moiety.
  • oligonucleotide probe that successfully hybridised to a target DNA sequence, since a ddNTP can only be added to a terminal end of the probe in an oligonucleotide overhang region wherein the ddNTP to be incorporated is complimentary to the nucleotide in the target DNA sequence at the corresponding position to the position which will be occupied by the incorporated ddNTP.
  • the single nucleotide extension reaction may be performed by adding free ddNTP conjugated to at least one labelling moiety and a DNA polymerase in conditions that are suitable for the DNA polymerase to work. Since ddNTPs lack the 3'-hydroxyl group of normal deoxynucleotides, only a single ddNTP can be added to the chain. This is useful as it means that only a single labelling moiety will be added to the hybridised labelling oligonucleotide probes.
  • the single nucleotide extension reaction to add a ddNTP conjugated to at least one labelling moiety is carried out in a polymerase chain reaction (PC ) machine.
  • the PCR machine may be used to provide suitable conditions to add the conjugated ddNTP to the hybridised labelling
  • oligonucleotide probe Suitable conditions are described above.
  • the step is preferably performed after the step of performing a single nucleotide extension reaction and before the step of hybridising the ddNTP-labelling oligonucleotide probe to the at least one detection probe.
  • the method may comprise an affinity chromatography step.
  • the labelling moiety is a molecule to which a labelling entity or quencher is to be attached
  • the method may comprise affinity chromatography using a column comprising beads that have affinity for the molecule to which the labelling entity or quencher is to be attached.
  • the molecule to which the labelling entity or quencher is to be attached is a binding entity such as biotin
  • the affinity chromatography column could comprise beads comprising avidin or streptavidin.
  • the methods may comprise a step of adding proteinase K, in order to inactivate DNA polymerase.
  • the step of removing unlabelled labelling oligonucleotide probe and free ddNTP is performed before the step of measuring the amount of the at least one hybridised ddNTP-labelling oligonucleotide probe.
  • An advantage of incorporating a ddNTP conjugated to a specific labelling moiety is that it can allow a bias towards the detection of a labelling oligonucleotide probe only when the labelling oligonucleotide probe is hybridised to the correct specific target DNA sequence.
  • the sequence of the labelling oligonucleotide probe can be designed so that it will hybridise to a specifically-selected target DNA sequence.
  • the terminal 3' nucleobase of the labelling oligonucleotide probe will hybridise to a complementary nucleobase in the specific target DNA sequence region and such that the next nucleobase in the strand comprising the specific target DNA sequence region overhangs the terminal 3' nucleobase of the labelling oligonucleotide probe.
  • the region of compementarity between target sequence and probe, and their specific sequences, are specifically selected so that this overhanging nucleobase is unique for the specific target DNA sequence (e.g.
  • nucleobases at this position relative to non-target sequence regions which may be very similar to target sequence regions will be different (i.e. in this example C, T or G).
  • ddTTP single nucleotide extension reaction
  • the single nucleotide extension reaction will not proceed to incorporate the ddTTP into the labelling oligonucleotide probe if the labelling oligonucleotide probe is hybridised to a non-specific non-target DNA sequence region.
  • the nucleobase overhanging the 3' terminal nucleobase of the labelling oligonucleotide probe will be either C, T or G, i.e. non-complimentary with respect to the ddTTP provided in that specific aliquot.
  • the onligonucleotide probe so as to provide a target DNA sequence region-specific overhang one can bias the single nucleotide extension reaction to incorporate a ddNTP only into labelling onligonucleotide probes that have hybridised to the correct target DNA sequence region.
  • the further provision of a labelling moiety conjugated to the ddNTP means that only labelling onligonucleotide probes that have hybridised to the correct target DNA sequence region will become labelled.
  • a "labelling moiety” is a generic term which may be used to describe a quencher or a molecule or conjugate which may be attached to a probe and which may act to tether a quencher to the probe.
  • the term “labelling moiety” may also be used to describe a "labelling entity” or a molecule or conjugate which may be attached to a probe and which may act to tether a labelling entity to the probe.
  • a "labelling entity” may be any compound/molecule or group of compounds/molecules which can give rise to a detectable signal.
  • a labelling entity will typically be a fluorophore.
  • a labelling entity may be a radioactive isotope or may be a molecule which may comprise a radioactive isotope.
  • a labelling entity may be a colorimetric molecule.
  • a labelling entity may be a a luminescent molecule, for example luciferase.
  • a molecule or conjugate which may be attached to a probe and which may act to tether a quencher or a labelling entity to the probe may be any suitable molecule or conjugate.
  • a conjugate may be two or more molecules or a pair of molecules which form an affinity interaction.
  • the molecule biotin may be attached to a probe and a quencher or labelling entity may be attached to streptavidin. When contacted together biotin will bind to streptavidin thus tethering the quencher or labelling entity to the probe.
  • a "conjugate" may be two or more molecules which can form an affinity interaction such as biotin and streptavidin so as to tether the quencher or labelling entity to the probe.
  • Other examples include an antibody and an antigen to which the antibody binds.
  • a ddNTP may itself act as a labelling moiety which tethers the quencher or a labelling entity (i.e. further labelling moieties) to the probe.
  • a quencher or a labelling entity could be tethered to a probe via a conjugate e.g.
  • biotin could be attached to a ddNTP and incorporated into the probe via a single nucleotide extension reaction and then the quencher or the labelling entity could be attached to streptavidin and subsequently tethered to the probe.
  • all of the ddNTPs may be conjugated to the same labelling moiety.
  • the sample may be separated into multiple aliquots, and in these embodiments all of the ddNTPs used in the same aliquot are conjugated to the same labelling moiety.
  • the labelling moiety is attached at the 3 ' end of the at least one labelling oligonucleotide probe as part of the single nucleotide extension reaction.
  • the labelling entity may be a quencher.
  • quencher refers to a substance that absorbs energy from a fluorophore and dissipates the energy as heat. Thus, if a quencher is placed in proximity to a quencher.
  • the labelling entity is a broad spectrum quencher, i.e. a quencher that is able to absorb fluorescence energy across a large range of the visible light spectrum.
  • a quencher that is able to absorb fluorescence energy across a large range of the visible light spectrum.
  • all of the labelling entities used are the same quencher.
  • all of the ddNTPs may be conjugated to the same quencher.
  • the ddNTPs may be conjugated to first binding moieties and the first binding moieties may be associated with second binding moieties wherein all of the second binding moieties are conjugated to the same quencher.
  • the quencher may be selected from the group consisting of BHQO (Black Hole Quencher 0), BHQl (Black Hole Quencher 1), BHQ2 (Black Hole Quencher 2), BHQ3 (Black Hole Quencher 3), BHQ10 (Black Hole Quencher 10), TAM A
  • the quencher is a black hole quencher (such as BHQO, BHQl, BHQ2 and BHQ3).
  • the quencher is selected from the group consisting of BHQO, BHQl, BHQ2 and BHQ3.
  • Labelling oligonucleotide probes or detection oligonucleotide probes may be attached to a fluorophore.
  • Suitable fluorophores include the following, 7-AAD (7- Aminoactinomycin D), Acridine Orange (+DNA), Acridine Organe (+RNA), Alexa Fluor® 350, Alexa Fluor® 430, Alexa Fluor® 488, Alexa Fluor® 532, Alexa Fluor® 546, Alexa Fluor® 555, Alexa Fluor® 568, Alexa Fluor® 594, Alexa Fluor® 633, Alexa Fluor® 647, Alexa Fluor® 660, Alexa Fluor® 680, Alexa Fluor® 700, Alexa Fluor® 750, Allophycocyanin (APC), AMCA / AMCA-X, 7-Aminoactinomycin D (7- AAD), 7- Amino-4-methylcoumarin, 6-Aminoquinoline, Aniline Blue, ANS, APC-Cy7
  • a labelling moiety is a molecule to which a labelling entity or a quencher may be attached
  • the methods of the invention may comprise a step of attaching a labelling entity or a quencher to the at least one labelling moiety.
  • the molecule to which a labelling entity or a quencher may be attached can be a first binding moiety.
  • the method comprises attaching a ddNTP conjugated to a first binding moiety to a labelling entity or a quencher.
  • the method may comprise exposing the ddNTP conjugated to the first binding moiety to a labelling entity-second binding moiety conjugate or to a quencher-second binding moiety conjugate, wherein the first binding moiety and the second binding moiety have affinity for one another.
  • the step of exposing the ddNTP conjugated to the first binding moiety to a labelling entity-second binding moiety conjugate or to a quencher-second binding moiety conjugate takes place under conditions suitable for the first binding moiety and the second binding moiety to associate with one another.
  • the labelling moiety may be biotin, and, if this is the case, a labelling entity or quencher may be attached to the labelling moiety by exposing a ddNTP-biotin conjugate to an avidin/streptavidin-labelling entity conjugate.
  • the labelling moiety may be avidin or streptavidin and a labelling entity may be attached to the labelling moiety by exposing a ddNTP-avidin/streptavidin conjugate to a biotin-labelling entity conjugate.
  • the method comprises a step of attaching a labelling entity or a quencher to the labelling moiety, the step preferably takes place before the step of hybridising the ddNTP-labelling oligonucleotide probes to the at least one detection oligonucleotide probe.
  • Methods of the invention may comprise a step of hybridising the ddNTP- labelling oligonucleotide probes to at least one detection oligonucleotide probe to form a reaction mixture comprising at least one hybridised ddNTP-labelling oligonucleotide probe.
  • a kit of the invention may comprise at least one detection oligonucleotide probe.
  • the at least one detection oligonucleotide probe is an oligonucleotide that is complementary to at least one of the ddNTP-labelling oligonucleotide probes.
  • three different labelling oligonucleotide probes will be used and will detect the presence of these three different labelling oligonucleotide probes using three different detection oligonucleotide probes.
  • Each of the three different labelling oligonucleotide probes will hybridise to one of the three different detection
  • oligonucleotide probes For the same number of detection oligonucleotide probes as labelling oligonucleotide probes can be used, and each of the detection oligonucleotide probes will hybridise with a different labelling oligonucleotide probe.
  • the at least one detection oligonucleotide probe hybridises to at least one of the labelling oligonucleotide probes under a moderately stringent hybridisation condition.
  • the at least one detection oligonucleotide probe hybridises to at least one of the at least one labelling oligonucleotide probes under a stringent hybridisation condition. Hybridising a ddNTP-labelling oligonucleotide probe to a detection oligonucleotide probe.
  • Methods of the invention may comprise a step of hybridising the ddNTP- labelling oligonucleotide probes to at least one detection oligonucleotide probe to form a reaction mixture comprising at least one hybridised ddNTP-labelling oligonucleotide probe.
  • hybridising ddNTP-labelling oligonucleotide probes to detection oligonucleotide probes
  • hybridisation exposing the ddNTP-labelling oligonucleotide probe to a detection oligonucleotide probe under conditions in which hybridisation of the ddNTP-labelling oligonucleotide probe to the detection oligonucleotide probe can occur.
  • conditions suitable for hybridisation to occur include cool temperatures and high salt conditions.
  • the ddNTP-labelling oligonucleotide probe and the detection oligonucleotide probe are mixed together and exposed to temperatures between 40°C and 70°C or between 50°C and 65 °C for at least 10 seconds (for example at least 20 seconds, at least 30 seconds, between 10 seconds and 5 minutes, between 20 seconds and 2 minutes, or between 20 seconds and 1 minute).
  • the different detection oligonucleotide probes may have different melting temperatures.
  • the different detection oligonucleotide probes have at least 2, at least 4, at least 6, at least 8 or between 2 and 8 different melting temperatures.
  • the temperature at which the hybridised ddNTP-labelling-oligonucleotide probe melts will be different for one detection oligonucleotide probe compared to another detection oligonucleotide probe. As will be discussed below, this can help with determining the amount of the at least one hybridised ddNTP-labelling oligonucleotide probe.
  • the method may further comprise a step of purifying the at least one hybridised ddNTP-labelling oligonucleotide probe.
  • This step is preferably after the step of hybridising the ddNTP-labelling oligonucleotide probe and before the step of measuring the amount of the at least one hybridised ddNTP-labelling oligonucleotide probe.
  • Such a purification step will remove some of the excess detection oligonucleotide probe and will ensure that the fluorescence signal that is detected will be more accurate.
  • the purification step is optionally performed by affinity chromatography.
  • oligonucleotide probes to form single stranded oligonucleotides.
  • This melting step is preferably performed before the step of hybridising the ddNTP-labelling
  • oligonucleotide probes and may comprise heating the ddNTP-labelling oligonucleotide probes to between 70°C and 120°C or around 95°C.
  • the methods may comprise a labelling oligonucleotide probe purification step which is performed before the step of measuring the amount of the at least one ddNTP- labelling oligonucleotide probe.
  • a method may comprise providing a labelling oligonucleotide probe with a binding moiety.
  • binding moiety is a generic term which may be used to describe a binding entity which may be attached to a probe or a molecule or conjugate which may be attached to a probe and which may act to tether a binding entity to the probe.
  • a "binding entity” may be any compound/molecule or group of compounds/molecules which can be attached to a probe or tethered to a probe and which can bind to another compound/molecule or group of compounds/molecules to facilitate purification of the probe.
  • a binding entity will naturally bind to another molecule when brought into close proximity.
  • binding entities can include biotin, avidin, streptavidin, digoxiginin and anti- digoxiginin.
  • a molecule or conjugate which may be attached to a probe and which may act to tether a binding entity to the probe may be any suitable molecule or conjugate.
  • a binding entity will bind to a "capture entity" to facilitate purification of the probe.
  • a “capture entity” is any entity which will bind to a binding entity to facilitate purification of the probe.
  • a binding entity/capture entity may be two or more molecules or a pair of molecules which form an affinity interaction.
  • the binding entity may be biotin which is attached to or tethered to the probe and the capture entity may be streptavidin attached to a substrate. When contacted together biotin will bind to streptavidin thus tethering the probe to the substrate. Probes which are not tethered to the substrate can then be washed away. After a wash step an elution step may be performed to elute the tethered probes from the sutbstrate.
  • a binding entity and capture entity may be two or more molecules which can form an affinity interaction such as biotin and streptavidin so as to facilitate purification of the probe.
  • Other examples include an antibody and an antigen to which the antibody binds.
  • a binding entity such as biotin may be tethered to a probe is by attaching the binding entity to a ddNTP and incorporating the ddNTP into the probe via a single nucleotide extension reaction.
  • a ddNTP may itself act as a binding moiety which tethers the binding entity to the probe.
  • the methods may involve the incorporation into a labelling oligonucleotide probe of a ddNTP if the labelling oligonucleotide probe is hybridised to a target DNA sequence.
  • Such methods may further comprise providing the ddNTP with a binding entity and wherein the binding entity allows the labelling oligonucleotide probe to be purified.
  • Such a method may comprise providing the ddNTP with a binding entity and wherein a labelling oligonucleotide probe purification step is performed before the step of measuring the amount of the labelling oligonucleotide probe, the purification step comprising:
  • step (i) optionally, dissociating the at least one ddNTP-labelling oligonucleotide probe of step (b) from the amplification product;
  • Such a method may further optionally comprise a dissociation step (step iv), wherein after step (iii) the dissociation step (step iv) is performed comprising dissociating the binding entity from the capture entity to release the at least one ddNTP-labelling oligonucleotide probe from the substrate.
  • the methods may comprise methods wherein the labelling oligonucleotide probe for detection of amplicons generated from the DNA of a given target microorganism differ in length from the labelling oligonucleotide probes for detection of amplicons generated from the DNA of every other target microorganism in the pre-defined set, and wherein a step of measuring the amount of the labelling oligonucleotide probe comprises performing the dissociation step (step iv) and separating all labelling oligonucleotide probes for the pre-defined set of target microorganisms on the basis of their length before the step of quantifying a signal generated via the labelling entity, optionally wherein separation is performed by electrophoresis, e.g. capilliary electrophoresis.
  • the detection oligonucleotide probe for detection of amplicons generated from the DNA of a given target microorganism may differ in length from the detection oligonucleotide probes for detection of amplicons generated from the DNA of every other target microorganism in the pre-defined set, and wherein the step of measuring the amount of the at least one ddNTP-labelling oligonucleotide probe comprises separating all detection
  • oligonucleotide probes for the pre-defined set of target microorganisms on the basis of their length before the step of quantifying a signal generated via the labelling entity, optionally wherein separation is performed by electrophoresis, e.g. capilliary electrophoresis.
  • more than one labelling oligonucleotide probe or detection oligonucleotide probe will be used, more than one fluorophore may be used.
  • at least 2, at least 4, at least 6, at least 8, at least 10, or between 2 and 20 different fluorophores may be used.
  • oligonucleotide probes may be conjugated to a different fluorophore.
  • more than one of the labelling oligonucleotide probes or detection oligonucleotide probes may be conjugated to the same nuorophore.
  • each of the five different detection oligonucleotide probes may be conjugated to a different fluorophore, or two of the detection oligonucleotide probes could be conjugated to the same fluorophore and three of the detection oligonucleotide probes could be conjugated to a different fluorophore.
  • the same fluorophores may be used in different aliquots.
  • 5 different detection oligonucleotide probes conjugated to 5 different fluorophores may be used in a first aliquot and those same 5 fluorophores may be conjugated to 5 different detection oligonucleotide probes used in a second separate aliquot.
  • a fluorophore may be attached to either the 5 ' end or 3 ' end of a detection oligonucleotide probe or labelling oligonucleotide probe.
  • a labelling moiety is at the 3' end of a labelling oligonucleotide probe, and a fluorophore is attached to the 5' end of a detection oligonucleotide probe.
  • a fluorophore could also be attached to the 3 ' end or somewhere along the length of the detection oligonucleotide probe or labelling oligonucleotide probe, provided that the distance from the labelling moiety to the fluorophore is such that quenching occurs when the two probes are hybridised, typically 10-100 A.
  • the at least one detection oligonucleotide probe may optionally be shortened by 1 - 5, at least 2 - 3 nucleotides at the 5' end, in order for the fluorophore not to be too close to the labelling moiety, to avoid any steric hinderance.
  • the methods comprise a step of measuring the amount of the at least one hybridised ddNTP-labelling oligonucleotide probe.
  • ddNTPs will only be added to labelling oligonucleotide probes that successfully hybridised with a target DNA sequence.
  • the amount of one of the different at least one hybridised ddNTP-labelling oligonucleotide probes will be proportional to the amount of the target DNA sequence that was complementary to that at least one labelling oligonucleotide probe in the sample.
  • Each of the different detection oligonucleotide probes is conjugated to a detection moiety, for example a fluorophore.
  • the ddNTP-labelling oligonucleotide probe can be conjugated to a quencher.
  • the fluorescence from a fluorophore will be quenched.
  • the hybridised ddNTP- labelling oligonucleotide probes are melted, fluorescence from the fluorophore can be detected.
  • the difference in fluorescence before and after a step of melting the hybridised ddNTP-labelling oligonucleotide probe provides the user with information on the amount of labelling oligonucleotide probe comprising a fluorophore that is present in the sample.
  • the user uses several detection oligonucleotide probes and these are unique in that they each have a different fluorophore or a different melting temperature compared to other detection oligonucleotide probes present, i.e. each detection oligonucleotide probe has a different combination of melting temperature and fluorophore to each other detection oligonucleotide.
  • detection oligonucleotide probes each have a different fluorophore or a different melting temperature compared to other detection oligonucleotide probes present, i.e. each detection oligonucleotide probe has a different combination of melting temperature and fluorophore to each other detection oligonucleotide.
  • each different detection oligonucleotide probe and thereby determine the amount of the at least one hybridised ddNTP-labelling oligonucleotide probe that is present. For example, if three different detection oligonucleotide probes are present and two of these have the same melting temperature and the third has a different melting temperature, the user could firstly apply the first melting temperature and thereby melt two of the detection oligonucleotide probes. Assuming that the two detection oligonucleotide probes have fluorophores fluorophores and melting temperatures to detect a wide variety of detection oligonucleotides and therefore target DNA sequences.
  • the user may, therefore, use combinations of these should fluoresce at different wavelengths so that the user can establish the amount of each fluorophore that is present. Since the amount of ddNTP-labelling oligonucleotide probe that is present is proportional to the amount of at least one target DNA sequence, this can be used to detect the presence, absence or relative amount of the at least one target DNA sequence. Optionally the fluorescence may be compared to reference levels. A suitable reference is a sample containing a known amount of a fluorophore.
  • steps of measuring the amount of the at least one hybridised ddNTP- labelling oligonucleotide probe can be carried out by:
  • Steps (i) and (iii) may be performed in a standard qPCT machine.
  • the fluorescence signal is detected by flow cytometry or spectrofiuorometry.
  • altering the conditions of the reaction mixture is meant that the user changes any condition which leads to melting of the hybridised ddNTP-labelling oligonucleotide probes.
  • Suitable conditions include increasing the temperature of the reaction mixture or increase the salt content.
  • the step of "altering the conditions of the reaction mixture” comprises increasing the temperature of the reaction mixture, for example the temperature may be increased by between 2°C and 120°C, between 20°C and 100°C, between 50°C and 100°C or around 60°C.
  • RNA microarrays with overlapping sequences have also been used to detect various bacteria in culture (Rajilic-Stojanovic et al. (2009), Environ Microbiol. Jul; 11(7): 1736-1751).
  • the method uses Agilent Technologies microarrays, and involves both labour-some and error-prone RNA technology, as well as high cost microarrays.
  • NGS Next generation sequencing
  • Illumina for example MiSeq, HiSeq and NextSeq
  • Roche 454 sequencing Ion Torrent PGM sequencing
  • SOLiD sequencing SOLiD sequencing
  • PacMan and Nanopore to name a few.
  • NGS can be used to sequence DNA actually present in the sample, or DNA amplified from DNA actually present in the sample so as to determine whether DNA from a given target microorganism of the set is actually present in the sample.
  • NGS is the preferred way of analysing the gut microbiome since it has the potential to provide the most complete information about the microbiome.
  • the most relevant equipment are: Illumina MiSeq, HiSeq, MiniSeq and NextSeq (see https://www.illumina.com/).
  • Amplicon sequencing can for example be performed according to the Illumina MiSeq protocol '"''Overview of tailed amplicon sequencing approach with MiSeq" (http://www.Illumina.com).
  • This protocol provides a two-step PC method utilising sequence-specific primers and a specific DNA index kit. Primers are designed according to low diversity amplicon specifications. Adapter overhang sequences are added to the 5' end of both the forward and reverse primers. These 5 '-primer regions are complementary to index sequences and thus permit the addition to the template of a unique sample index (barcode). The 5 '-primer regions also allow the addition of adapters to make the template compatible for hybridisation to the MiSeq flow cell.
  • PPi pyrophosphate
  • nanopore technologies DNA molecules are passed through or positioned next to nanopores, and the identities of individual bases are determined following movement of the DNA molecule relative to the nanopore. Systems of this type are available commercially e.g. from Oxford Nanopore (https://www.nanoporetech.com/).
  • a DNA polymerase enzyme is confined in a "zero-mode waveguide" and the identity of incorporated bases are determined with florescence detection of gamma-labeled phosphonucleotides (see e.g. Pacific Biosciences; http://www.pacificbiosciences.com/).
  • NGS techniques which can be used to implement methods of the present invention may be facilitated by various techniques known in the art. For example, a variety of techniques have been developed for the targeted enrichment of genomic regions of interest for the purposes of subsequently performing next generation sequencing (see for example Mertes et al. (2011, Breifings in Functional Genomics 10(6), 374-386) for a review of some specific techniques which may be used to implement the methods of the invention).
  • the current invention thus uses a targeted approach to microbiome sequencing.
  • the invention for the first time makes it possible to obtain detailed information about the microbiome down to species and strain level, while reducing costs, time and complexity associated with sequencing and data analysis. This makes it possible to conduct larger studies, involving more subjects and more time-points compared to what is practically possible with current technologies. This will lead to increased
  • the present methods also allow quantification of a target microorganism in a sample.
  • the number of sequence reads within each amplicon with sequence identity to the target microorganism will provide a measure of the abundance of each target micoorganism relative to the other target micoorganisms in that group, and it will provide a measure the abundance of each target micoorganism relative to the other target micoorganisms in the set of target micoorganisms, i.e. in the sample as a whole.
  • the present methods allow a user to rapidly and accurately detect
  • microorganisms in a sample involve detecting whether target DNA sequences are present, absent and at what relative amount in a sample. As discussed above, detecting whether target DNA sequences are present, absent and at what relative amount in a sample can provide the user with information on whether that certain microorganism is present in the sample, and therefore allows the user to analyse the microorganisms present in the sample. Accordingly, the invention provides methods for analysis of a plurality of microorganisms in a sample.
  • microoganisms that are analysed may be any suitable microorganism.
  • the microorganisms may comprise bacteria, viruses, fungi or a combination of all three.
  • the plurality of microorganisms is a collection of bacteria or yeast.
  • the plurality of microorganisms is a collection of bacteria.
  • the method is a method for analysing a microbiome.
  • the term "microbiome" is intended to refer to the genetic material of microorganisms present in a particular environment.
  • a microbiome of the invention may be genetic material from microorganisms present in the gut of an animal, the
  • microorganisms present in a source of water or the microorganisms present in the soil of a particular area are present in a source of water or the microorganisms present in the soil of a particular area.
  • the sample may be sourced from an animal, water, air, food, forensic sites, buildings or biofilms.
  • a sample may be a sample from an animal, for example a mammal.
  • the sample is from a human, cat, dog, horse, cow, mouse, rat, guinea pig, zebrafish or bee.
  • the sample is from a human.
  • the sample is a sample from an animal
  • the sample may be a faecal, gut mucosal, skin, vaginal excretion, mouth, spit or toe sample from the animal.
  • the sample is from the gut of an animal.
  • the method can be used to analyse the gut microflora and to detect imbalances in the gut microflora. If the method is used to detect imbalances in the gut microflora it may comprise an additional step of determining a treatment that may be used to correct the gut microflora imbalance, for example the administration of a probiotic, prebiotic, specific diet or drug.
  • the method further comprises a step of purifying the sample, i.e., if the sample is derived from the gut of a human animal it may comprise faecal matter that has been purified to enrich for DNA.
  • the step of purifying the sample is before the step of amplifying target DNA sequences. Any method known in the art for purifying total genomic DNA can be used. Suitable purification methods may comprise addition of a detergent or surfactant to lyse any cells in the sample, addition of a protease and/or an RNase to break down proteins and/or RNA, centrifugation of the sample to remove cell debris or isolation of DNA using minicolumn purification.
  • the sample could be exposed to a detergent such as polysorbate 80 or sodium dodecylsulphate, the sample could be centrifuged and the supernatant applied to a column comprising glass beads in the presence of a chaotropic agent such as sodium iodide or sodium perchlorate.
  • a detergent such as polysorbate 80 or sodium dodecylsulphate
  • the sample could be centrifuged and the supernatant applied to a column comprising glass beads in the presence of a chaotropic agent such as sodium iodide or sodium perchlorate.
  • the method further comprises a step of measuring the amount of total genomic DNA, for example bacterial DNA, in the sample. This step can take place before the step of amplifying target DNA sequences.
  • the step of measuring the total amount of genomic bacterial DNA in the sample is performed after a step of purifying the sample.
  • the amount of total genomic bacterial DNA may be measured using any conventional means.
  • the amount of total genomic bacterial DNA may be measured using a spectrophotometer.
  • the absorbance at 260 nm can provide a measure of total genomic bacterial DNA.
  • other suitable methods includes quantitative PC methods such as using the FentoTM bacterial DNA quantification kit (Zymo Research).
  • the amount of total genomic DNA may be compared to a reference, for example a sample having a known amount of DNA present.
  • the sample may be divided into one or more aliquots.
  • the method may comprise a step of preparing one or more aliquots from the sample.
  • an "aliquot" is a portion of the sample. It is possible to use any number of aliquots in the method of the invention. Preferably around 4 aliquots are used. Suitably, the total number of aliquots is between 2 and 50, between 2 and 25, between 2 and 10 or around 4.
  • the method of the invention involves detecting the presence, absence, or relative amount of target DNA sequences. This step may involve hybridising the target DNA sequence to a labelling oligonucleotide probe.
  • oligonucleotide probes may be used at the same time and, for example, their binding to a target DNA sequence could be measured using a multiplex fluorescence detection system.
  • probes when different probes are present in the same aliquot, they may interact with each other, for example by binding to one another or to the same target sequence (albeit with different affinity), and this reduces the accuracy of the assay.
  • a smaller number of probes By separating the sample into more than one aliquot, a smaller number of probes may be used in each aliquot, and this reduces unwanted interactions.
  • a first ddNTP selected from the group consisting of ddC, ddT, ddA, ddG, ddl and ddU is used in some of the one or more aliquots
  • a second ddNTP selected from the group consisting of ddC, ddT, ddA, ddG, ddl and ddU is used in some of the one or more aliquots
  • the first ddNTP and the second ddNTP are not the same ddNTP.
  • a first ddNTP selected from the group consisting of ddC, ddT, ddA, ddG, ddl and ddU is used in some of the one or more aliquots,
  • a second ddNTP selected from the group consisting of ddC, ddT, ddA, ddG, ddl and ddU is used in some of the one or more aliquots,
  • a third ddNTP selected from the group consisting of ddC, ddT, ddA, ddG, ddl and ddU is used in some of the one or more aliquots, and
  • the first ddNTP is not the same as the second ddNTP or the third ddNTP and the second ddNTP is not the same as the third ddNTP.
  • a first ddNTP selected from the group consisting of ddC, ddT, ddA, ddG, ddl and ddU is used in some of the one or more aliquots,
  • a second ddNTP selected from the group consisting of ddC, ddT, ddA, ddG, ddl and ddU is used in some of the one or more aliquots,
  • a third ddNTP selected from the group consisting of ddC, ddT, ddA, ddG, ddl and ddU is used in some of the one or more aliquots,
  • a fourth ddNTP selected from the group consisting of ddC, ddT, ddA, ddG, ddl and ddU is used in some of the one or more aliquots and
  • the first ddNTP is not the same as the second ddNTP, the third ddNTP or the fourth ddNTP, the second ddNTP is not the same as the third ddNTP or the fourth ddNTP and the third ddNTP is not the same as the fourth ddNTP.
  • any suitable detection platform may be used to implement the core amplicon-based approaches described herein, and as such the methods are not intended to be limited to probe-based approaches involving single nucleotide extension and DNA sequencing as described herein.
  • One particular platform may utilise specific bead comprising e.g. polystyrene microspheres/beads or paramagnetic microspheres/beads.
  • Such beads possess internal dyes e.g. comprising red and infrared fluorophores of differing intensities, or beads may possess different distinguishable shapes.
  • Each bead may be provided with a unique number/barcode, colour or other distinguishing feature, allowing one type of bead to be differented from another.
  • Detection oligonucleotide probes may be applied to the surfaces of such beads, wherein the detection oligonucleotide probes are complementary to each labelling oligonucleotide probe which has incorporated a labelling entity, such as a fluorophore, during the single nucletotide extension reaction.
  • a labelling entity such as a fluorophore
  • instruments may be provided that are capable of detecting e.g. a specific coloured bead plus the hybridised labelling oligonucleotide probe-specific fluorophore, or e.g. the specific shape of the bead plus the labelling oligonucleotide probe-specific fluorophore.
  • the present invention is directed towards kits suitable for use in any of the methods described above.
  • the present invention provides a kit that is adapted for use in of the methods described above.
  • the present invention also provides a kit comprising:
  • kits comprise primers.
  • the kit may be used to detect at least 3 target DNA sequences from a genomic region.
  • the primers are complementary to conserved regions of the genomic region of interest.
  • primers complementary to conserved regions of more than one genomic region of interest are included.
  • the kit further comprises a manual containing instructions for performing a method of the invention.
  • the kit further comprises components that can be used to purify a reaction mixture, such as an affinity chromatography column.
  • the kit further comprises at least one buffer.
  • the kit may comprise buffer components that can be used at different stages of the methods of the invention. Suitable buffers include TAPS
  • (Tris(hydroxymethyl)methylaminopropanesulphonic acid) buffer bicine buffer, tris buffer, tricine buffer, TAPSO (3-[[l,3-dihydroxy-2-(hydroxymethyl)propan-2- yl]amino-2-hydroxypropane-l-sulphonic acid) buffer, HEPES (2-[4-(2- hydroxyethyl ⁇ piperazin-l-yl]ethanesulphonic acid) buffer, TES (2-[l,3-dihydroxy-2- (hydroxymethyl)propan-2-yl] amino] ethanesulphonic acid) buffer, PIPES (1,4- piperazinediethanesulphonic acid) buffer, cacodylate buffer and MES (2-morpholin-4- ykethanesulphonic acid) buffer.
  • the buffer is a Tris-EDTA or Tris buffer.
  • the kit comprises enzymes.
  • the methods of the invention comprise steps which may use enzymes and any of these enzymes may be included in a kit of the invention.
  • the kit of the invention may comprise a DNA polymerase such as Taq polymerase, two polymerases,
  • Example outline methodology for defining genomic sequence regions for use in microorganism identification assays is described below.
  • Microorganisms typically bacteria, can be divided into groups dependent on their sequence similarities. For example, it is difficult to distinguish the different Bifidobacteria that are present in the human gut just based on their 16S r NA sequence. Bifidobacteria are important for good health. Ingestion of Bifidobacteria compositions, and other mechanisms of promoting Bifidobacteria in gut flora, have been suggested as a probiotic treatment for many different diseases, such as Irritable Bowel Syndrome (IBS), Ulcerative Colitis (UC) and Necrotizing Enterocolitis (NEC) (Tojo et ah (2014) Nov 7, 20(41): 15163-15176). Correct detection of the different bacteria can be helpful in optimizing diets and treatments options, and for monitoring the effect of treatments.
  • IBS Irritable Bowel Syndrome
  • UC Ulcerative Colitis
  • NEC Necrotizing Enterocolitis
  • bacteria such as Lactobacillus, Clostridia and Enterobacteria may also be difficult to detect down to species and strain level just based on 16S rRNA sequence.
  • these and other groups of bacteria have distinct features, and therefore also unique gene sequences, which can be utilized for the purposes of detection in microbiome analysis.
  • the present methods involve determining the presence, absence or quantity of target DNA sequences from a sample which can uniquely identify a target microorganism and distinguish that target microorganism from all other target microorganisms in a pre-defined set of target microorganisms for the sample.
  • the group of microorganisms may be, for example, a microorganism genus and the target microorganism may be, for example, a microorganism species or strain within the genus.
  • the methods involve pre-defining a set of target microorganisms to be detected, wherein the set of target microorganisms comprises groups of related target
  • the methods further involve pre-defining first and second conserved regions of DNA sequence which are conserved across members of a specific group of related microorganisms, which are unique to the group of related microorganisms, and which can give rise to an amplicon speficic for that group.
  • pre-defining the first and second conserved regions of DNA sequence target DNA sequence regions are predefined, wherein the target DNA sequence regions are located between the first and second conserved regions and wherein each target DNA sequence region comprises sequences which are unique for any given species or strain of a group, within the group- specific amplicon.
  • sequence regions within a group of bacteria can be identified that are characteristic of that group. These regions can be used as a basis for applying methods, such as those described and defined herein, to detect and quantify all bacterial species and strains within that group in any test assay.
  • An example method for pre-defining a set of candidate microorganisms in an environment may consist of the steps outlined below.
  • Predefining a set of candidate microorganisms from other environments of interest can be achieved using the same principles. Obtain whole genome bacterial sequences for all bacteria expected to be present in the human gut. This can be obtained for example from
  • Clean up the sequences This may be performed as a two step approach as follows.
  • a search algorithm can be set up that searches for groups of bacteria without any pre-existing input.
  • the search criteria may include two sequence regions with conserved sequences within the group, interspersed with a sequence region with variations between the target bacteria within that group.
  • a sequence region with conserved sequences within the group it is meant either a sequence region of defined length (i.e. 10 - 30 bases), or that is able to hybridise a homologous oligonucleotide at a defined melting temperature (Tm).
  • Tm melting temperature
  • close to homologous sequence a sequence with a defined length containing a limited number of mismatch sequences (typically 1 to 3 mismatches), or is defined by an oligonucleotide that can hybridise at a defined Tm within a given range (for example +/- 5 degrees Celsius).
  • the distance between the two homologous sequence regions containing the variable region is typically from 50 - 1500 bases (optionally 75 - 500 bases).
  • FIG. 3 An example sequence analysis is shown in Figure 3. elevent genomic sequences of several Bifidobacteria species are shown in an alignment. Species and strain specific sequence areas (target DNA sequence regions) are shown in the boxed area together with example putative forward and reverse primer areas (conserved regions) for generating a Bifidobacteria-specific genus amplicon containing
  • amplification products comprising species- and strain-specific target DNA sequences.
  • Example 2 Preparing a Sample & Amplifying Sample DNA.
  • Biological samples are collected. Samples can be processed immediately following collection or stored for future use.
  • DNA is extracted and purified from samples according to standard procedures known in the technical field. Since the methods of the invention are applicable to any suitable sample, the precise protocol for DNA extraction will vary. For example, the precise protocol for DNA extraction from soil will differ from the precise protocol for
  • DNA extraction from a biological sample such as saliva. DNA extraction protocols will therefore be optimized using routine procedures known in the art depending on the particular sample to be studied.
  • a biological sample will comprise nasal mucous, saliva, sputum, oesophageal mucus, vomit, faeces, urine, vaginal mucous or skin.
  • DNA sequence regions identified are added to a solution of the purified DNA and other reagents in order to carry out an amplification reaction, preferably a PCR amplification reaction.
  • Primers may be designed to include degenerative sequences if necessary.
  • the number of cycles, the type of amplification (for example: linear PC followed by cyclic PCR, ligase chain reaction, loop mediated isothermal amplification, multiple displacement amplification) and other conditions may vary or may be adapted.
  • DNA sequence regions are amplified by PCR.
  • the methods will typically involve determining the presence or absence of many microorganism species within an environment, a number of DNA sequence regions may be amplified in parallel, and this will require multiplex amplification techniques.
  • the number of primer pairs that can be used in the same reaction vessel in a singleplex or multiplex manner, and therefore the number of amplification reations that can be perfomed in the same reaction vessel can be determined empirically using routine optimization tests.
  • the methods of the present invention allow the sample to be divided into several aliquots and singleplex or multiplex PCR performed on the same sample material in different reaction vessels, with optionally multiple sets of primer pairs added to each reaction vessel. If required, samples from completed reactions in separate reaction vessels can then be combined back into one or more combined aliquot before being further processed.
  • the methods of the present invention allow different conditions to be used in each aliquot (e.g. temperature, time, salt concentrations, etc.). This may provide greater flexibility in designing amplification primers. Alternatively, the same conditions may be applied for all aliquots. After a clean-up of amplified DNA to remove primers, template, etc., a further amplification may be performed with instrument-specific sequence tags and indexes as required.
  • Example 3 Identification of Bifidobacteria amplicons.
  • LP containing an additional C and ddCTP-Black-Hole-Quencher2 (Jena Sciences) and DP containing a fluorophore and specific melting temperature ('Tm') as listed in Table 3 and 4 was used to assess the feasibility of using FRET technology to detect labelled probes.
  • the samples were mixed in a LightCycler® 480 Multiwell Plate 96, white (Roche) and run on a LightCycler 480 II using a melting point programme with the following parameters:
  • Example 5 Mock library of genomic bacterial DNA.
  • Example 6 PCR of 16S rRNA and Bifidobacteria amplicon.
  • Total genomic DNA listed in Table 1 ( Figure 8) was amplified using either primer pair 16SV3V9F and 16SV3V9 for 16S rRNA amplicon amplification, and Bifi4F and BIFI4R for Bifidobacteria amplicon 4 amplification.
  • the amplification included a 15-min activation stage at 95°C, followed by 30 cycles with 30 sec denaturation at 95°C, 30 sec annealing at 55°C for 16S rRNA amplification and 65°C for Bifidobacteria amplification, and 90 sec extension at 72°C. A final elongation for 7 min at 72°C was included for completion of all the PCR products. 3 ⁇ 1 of each PCR reaction was analysed by gel electrophoresis, and visualized by UV light. Example 7: Test of labelling and detection on LightCycler.
  • PCR products made according to Example 4 were treated with 3 U exonuclease I (New England BioLabs, Ipswich, MA) and 8 U shrimp alkaline phosphatase (New England BioLabs, Ipswich, MA) at 37°C for 2 h and inactivated at 80°C for 15 min, and quantified by gel electrophoresis.
  • the PCR products were purified using MinElute (Qiagen) according to manufacturers recommendation, and the amount of purified PCR product was measured using NanoDrop.
  • 100 ng was used in the following labelling reaction mixture: in a total reaction volume of 20 ⁇ , 2.5 U Hot TermiPol (Solis Biodyne), 2 ⁇ buffer C (Solis Biodyne), 4 mM MgCl 2 (Solis Biodyne), 0.4 ⁇ ddCTP- Black-Hole-Quencher2 (Jena Bioscience, Jena, Germany) and 0.2 ⁇ of each labelling probe (LP).
  • the labelling protocol included a 12-min activation stage at 95°C, followed by 10 cycles with 20 sec denaturation at 96°C and 35 sec combined annealing and extension at 60°C.
  • the 20 ⁇ 1 sample was transferred to LightCycler® 480 Multiwell Plate 96, white (Roche), and 2 ⁇ of a 0.1 ⁇ solution of DP for each LP was added to each well.
  • the samples were run on a LightCycler 480 II using a melting point programme with the following parameters:
  • Example 8 PCR of specific Bifidobacteria amplicon.
  • the amplification included a 15-min activation stage at 95°C, followed by 30 cycles with 30 sec denaturation at 95°C, 30 sec annealing at 55°C for 16S rRNA amplification and 65°C for Bifidobacteria amplification, and 90 sec extension at 72°C. A final elongation for 7 min at 72°C was included for completion of all the PCR products. 3 ⁇ 1 of each PCR reaction was analysed by gel electrophoresis, and visualized by UV light. Table A
  • Example 9 Labelling and detection of probes based on size.
  • PCR products made according to Example 8 were purified using MinElute (Qiagen) according to manufacturers recommendation, and the amount of purified PCR product was measured using NanoDrop .
  • 100 ng was used in the following labelling reaction mixture: in a total reaction volume of 20 ⁇ , 2.5 U Hot TermiPol (Solis Biodyne), 2 ⁇ buffer C (Solis Biodyne), 4 mM MgCl 2 (Solis Biodyne), 0.4 ⁇ ddCTP- biotin (Jena Bioscience, Jena, Germany) and 0.2 ⁇ of each labelling probe (LP).
  • the labelling protocol included a 12-min activation stage at 95°C, followed by 10 cycles with 20 sec denaturation at 96°C and 35 sec combined annealing and extension at 60°C.
  • NGS next generation sequencing
  • a preferred method involves a single read length of around 100 bases (Ilumina has 75 and 150 as standard length) in only one direction (i.e. unpaired reads).
  • the result of the run will be compared to the known DNA sequences of the target microorganisms, i.e. sequences specific for target microorganisms within the set of target microorganisms to be detected.
  • This will be further simplified by only comparing DNA sequences within the particular DNA sequence region/amplicon that the target microorganism belongs to. For example, in the exemplary case of Bifidobacteria, in the Bifidobacteria DNA sequence region, the reads from the sequence run will only be compared to the 13 Bifidobacteria known to be present in human gut microbiome.
  • Bifidobacteria are: Bifidobacterium adolescentis, Bifidobacterium angulatum, Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium catenulatum, Bifidobacterium dentium, Bifidobacterium gallicum, Bifidobacterium kashiwanohense, Bifidobacterium longum subsp. longum, Bifidobacterium longum subsp. infantis, Bifidobacterium pseudocatenulatum and Bifidobacterium stercoris.
  • a sequence homology of 98% - 100% (or even down to 95%) will be used to identify the correct bacteria.
  • DNA sequence regions can be analysed in the same manner.
  • the commonly used 16S sequence region will be used to detecting unique microbial species and strains that can be adequately distinguished within the 16S sequence region.
  • Examples of such bacteria that can be adequately distinguished within the 16S sequence region are Akkermansia muciniphila, Faecalibacterium prausnitzii, Methanobrevibacter smithii and Bacteroides thetaiotaomicron.
  • one of the benefits of the methods of the present invention is that they can exclude all other sequences. This overcomes a main challenge with 16S amplicon sequencing (or any amplification-based sequencing approach), namely the presence of chimeras, that leads to incorrect identification of bacteria that may not be present in the sample at all. Since the methods of the present invention do not "see” these sequences, it is not necessary to consider them.
  • the method was essentially as outlined in Illumina manual for 16S amplicon sequencing, expect that MinElute was used to clean up the PCR reaction, and a final Exo I treatment was used in the end to eliminate large primer-dimers that would interfere with the sequencing reaction.
  • a first PC reaction were performed for each mock library and amplicon in separate tubes. Each tube contained 2.5 ng of bacterial DNA, 0.2uM each of forward and reverse primer med adaptor from Table C, 1,5U HotFirePol (Solis BioDyne), 2.5 mM MgC12, 1 x B2 buffer (Solis BioDyne) and 0.2 uM dNTP in a 20ul reaction.
  • the amplification reaction consisted of 15 min at 95°C, followed by 25 cycles of: 30 sec each at 95°C, 55°C and 72°C; 5 min 72°C, then 4°C.
  • MinElute Qiagen
  • the Nextera XT DNA Library Preparation Kit (24 samples) (FC-131-1024) was used. 5ul of purified sample from the first PCR reaction was added to 5ul each of Nextera Indexes N7 and N5 as shown in Table D, as well as 1,5U HotFirePol (Solis BioDyne), 2.5 mM MgCb, 1 x B2 buffer (Solis BioDyne) and 0.2 uM dNTP in a final reaction volume of 50ul.
  • the amplification reaction consisted of 15 min at 95°C, followed by 8 cycles of: 30 sec each at 95°C, 55°C and 72°C; 5 min 72°C, then 4°C. Two ul from each reaction was analyzed on 1% agarose gel w/TBE buffer.

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Abstract

La présente invention concerne des procédés de détermination de la présence, de l'absence ou de la quantité de micro-organismes cibles dans un échantillon contenant de multiples micro-organismes, chaque micro-organisme cible étant un membre d'un groupe de micro-organismes apparentés et chaque micro-organisme cible étant un membre d'un ensemble prédéfini de micro-organismes cibles pour l'échantillon, et chaque micro-organisme cible de l'ensemble de micro-organismes cibles pouvant être distingué des autres micro-organismes cibles de l'ensemble. La présente invention concerne également des kits de mise en œuvre des procédés selon l'invention.
PCT/EP2018/058494 2017-04-03 2018-04-03 Procédés de détection de micro-organismes WO2018185105A1 (fr)

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GB201817889D0 (en) 2018-11-01 2018-12-19 Bio Me As Microorganism detection methods
WO2021074087A1 (fr) * 2019-10-16 2021-04-22 Illumina, Inc. Systèmes et procédés pour détecter de multiples analytes
CN111304354A (zh) * 2020-03-31 2020-06-19 中国人民解放军陆军勤务学院 检测喷气燃料中球毛壳菌引物、应用及检测方法
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CN113444803A (zh) * 2021-07-14 2021-09-28 武汉大学中南医院 宫颈癌预后标志微生物及其在制备宫颈癌预后预测诊断产品中的应用
CN113444803B (zh) * 2021-07-14 2022-03-15 武汉大学中南医院 宫颈癌预后标志微生物及其在制备宫颈癌预后预测诊断产品中的应用
CN115267033A (zh) * 2022-08-05 2022-11-01 西湖大学 基于质谱数据的宏蛋白质组学分析方法及电子设备

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