Methods for identifying microbes in clinical and non-clinical situations

Abstract

The present invention provides a method for detecting and identifying microorganisms in a biological sample at the species or strain level by polymerase chain reaction (PCR), comprising: a) providing a biological sample suspected of containing microbes, and optionally isolating a nucleic acid sequence contained in the biological sample; b) PCR-amplifying an internal transcribed spacer (ITS) region of at least one microbial rRNA contained within the optionally isolated nucleic acid sequence using a set of broad taxonomic range amplification primers for amplifying the at least one microbial rRNA ITS region, thereby generating a PCR amplicon; c) recording high-resolution melting curves for the PCR amplicons generated in step b) and recording the lengths of the PCR amplicons generated in step b) by capillary electrophoresis or sequencing; d) comparing the high-resolution melting curve recorded in step c) to a database containing high-resolution melting curves of reference amplicons generated from reference microbial species or strains of known taxonomic identity using the same set of amplification primers, thereby identifying the microorganisms present in the biological sample. e) comparing the length of each PCR amplicon having a distinct length recorded in step c) with a database comprising PCR amplicon lengths of reference amplicons generated from a reference microbial species or strain of known taxonomic identity using the same set of amplification primers, thereby obtaining a second taxonomic identity of the microorganism present in the biological sample; f) identifying the microorganism present in the biological sample to the species or strain level if the first taxonomic identity and the second taxonomic identity match.

Description

[Technical field]

[0001] The present invention is in the field of microbial diagnostics, in particular for the rapid identification of microbes in a sample, in particular bacteria in clinical and non-clinical samples, at species or strain level. The present invention relates to a method for rapid microbial identification based on DNA analysis and a system for carrying out such a method. [Background technology]

[0002] As populations grow, urbanization and global connectivity increase, the effectiveness of infectious disease pathogen transmission increases significantly. This is clearly illustrated by the very recent and very rapid global spread of high-risk clones of multidrug-resistant bacteria that now represent a major public health threat. Early detection protocols for dangerous bacteria are therefore needed more than ever. In addition, in line with the World Health Organization's "One Health" approach, laboratory diagnosis of infections in livestock, or microbial contamination in non-clinical situations, such as microbiological testing of sterile products in the food sector or pharmaceutical industry, should include improved protocols for accurate detection and identification of pathogenic or contaminating microorganisms while reducing turnaround times and costs.

[0003] Because classical culture techniques are insufficiently sensitive and time-consuming (>24 hours), in recent years, the focus has shifted to culture-independent methods, including DNA-based molecular methods, to detect, characterize, and test for epidemiological and clinical impact of such microbes. Many DNA-based diagnostic approaches are currently available. Although this progress is promising, no single diagnostic platform can adequately address the need for actionable data with short turnaround times in all settings.

[0004] The development and application of species- and strain-specific polymerase chain reaction (PCR) procedures to amplify and detect with high specificity even minimal amounts of microbial DNA from a sample has revolutionized the detection of microbes, but such methods require predictable prediction of the presence of microbes in clinical samples. A negative test result is limited in information since it indicates only the absence of one or a few specific species. Furthermore, in situations where the causative infectious agent is difficult to predict, evades detection by mutation, or is not in fact a single species or type of microbe, specific DNA-based diagnostic approaches have limited applicability. It is clear that the high specificity of PCR amplification has both advantages and disadvantages for clinical and non-clinical diagnostics.

[0005] Also, in such methods, despite the high specificity of the amplification, verification of amplicon identity is still necessary to exclude the amplification of non-target sequences. Amplicon verification may involve the use of hybridization probes, detection of restriction sites within the amplicon, confirmation of the predicted length of the amplicon in number of nucleotides, or sequencing of the amplicon.

[0006] Currently, bacterial identification methods by next-generation DNA sequencing are not suitable for clinical application because treatment decisions need to be made within the shortest possible time frame, whereas these sequencing methods require at least about 24 hours for results to be available. Furthermore, common sequencing methods can only analyze relatively short DNA fragments (e.g., about 300 bp), which are too short to allow sufficient resolution at the species or strain level, while sequencing techniques that allow sequencing of longer fragments are too expensive, require large sample batches to be affordable, or are too inaccurate to be routinely implemented in the clinic.

[0007] To summarize the situation in clinical diagnostics, there are five key issues that define the applicability of any assay: (1) scope, (2) speed, (3) sensitivity, (4) specificity, and (5) scalability. Classical culture-based microbial diagnostics score highly in three of the five above: scope (many different micro-organisms can be detected), sensitivity (down to single bacterial cells can be detected), and specificity (determination of bacterial species / strain is accurate). However, culture does not score highly in speed and scalability (high throughput).

[0008] Current qPCR-based diagnostics score highly in 4 out of 5 categories: speed, sensitivity, specificity, and scalability. However, these assays are extremely limited in scope: a single qPCR can only detect single or a few target micro-organisms, which means that an endless array of qPCRs is required to achieve a range somewhat comparable to that of culture.

[0009] Moreover, even in such situations, negative results (absence of any kind of micro-organisms) may not be obtained. Negative results are very important in clinical microbiology, since some of the most important clinical decisions are made based on negative results. Examples of such clinical decisions include finding an alternative cause of disease to infection, stopping antibiotics, discharging the patient, removing indwelling devices (post-operative drains, and iv catheters), and decisions regarding keeping critical foreign bodies in the patient (artificial joints, osteosynthesis plates, and others). Due to the importance of negative results and the inability of qPCR to accurately produce negative results, culture remains the mainstay of microbiological diagnosis, despite its obvious limitations. Summary of the Invention [Problem to be solved by the invention]

[0010] It is therefore evident that it is highly desirable to provide a cost-effective and efficient PCR-based microbial detection system that scores highly in all the above five key objectives, especially for clinical applicability. [Means for solving the problem]

[0011] The inventors have now unexpectedly discovered a method for broad microbial identification that can also be applied in the clinic. The inventors realized that the development of even more species- and strain-specific PCR amplification tests to add to the existing array of available diagnostic tests for specific pathogens does not go far enough to solve the problem. The inventors have adopted an approach that combines a low specificity amplification protocol as a first part and a highly specific detection / verification procedure as a second part, where both parts can be performed in high throughput.

[0012] Instead of amplifying species- or strain-specific sequences, the method of the present invention uses a more "universal" amplification of the internal transcribed spacer (ITS) region of microbial rRNA from genomic DNA. The identity of the microbe in which this ITS region is amplified is then determined by combining information on the amplicon length with the DNA melting curve of the amplicon. Surprisingly, it was found that this method provides a very high level of resolution at the species and strain level, can be universally applied to a large number of microbial organisms, and results in the construction of a database with data on amplicon length and melting curves annotated to species and strains, which improves the accuracy of subsequent identification of microbes in samples. Most surprisingly, this relatively simple method was able to provide clinically relevant information on which clinicians can make treatment decisions, even in critically negative samples (where no microorganisms were detected).

[0013] In the method of the present invention, the amplification of the internal transcribed spacer (ITS) region of the rRNA of the microbial genomic DNA is substantially neither species-specific nor strain-specific. Although the highest resolution at the strain level can be achieved by using a species-specific amplification protocol, in the method of the present invention, the amplification procedure is intended to target the rRNA ITS region of a large number of microbial species (including several taxonomic genera, families, orders, classes, phyla, or even kingdoms of a broader range), and thus the amplicons are potentially derived from a group of large microbes. Therefore, the "universal" DNA amplification is preferably performed using higher taxon level or group-specific primers, such as genus-specific, family-specific, order-specific, class-specific, phylum-specific, kingdom-specific and / or domain-specific amplification primers, to amplify the rRNA ITS region of the microbial genomic DNA suspected to be present in the sample. By choosing a universal amplification approach, all bacterial species can be detected, eliminating the need for many separate qPCRs to achieve broad scope. Moreover, since all bacterial species should be amplified, a negative result means that no bacteria are present in the sample and can be used as a well-founded basis for clinical decisions. Up to now, such universal PCR approaches have not been applied for microbial identification in clinical practice. The reason is that such broad PCR amplification can result in the amplification of many PCR amplicons originating from many species, each with different lengths and different nucleotide sequences, which would not result in actual identification in all situations, since the sensitivity level would be considered too low for clinical purposes. Furthermore, the nature of clinical specimens is such that human DNA is often present in much higher abundance than bacterial DNA. These concentration differences can be extremely large (the number of human DNA is 10 times the number of potential bacterial DNAs), so that the results are not very accurate. 10Even with highly specific bacterial amplification primers (it is not uncommon for the primers to be 2-fold higher than normal), cross-reactivity is common, leading to non-specific amplification and false positive results.

[0014] Unexpectedly, the inventors have found a way to create a universal PCR procedure that is accessible for clinical use despite the above mentioned problems by combining in said universal PCR procedure two techniques that together provide clinically sufficient sensitivity and specificity: (i) high resolution melting curve analysis (hrMCA) of the amplified PCR products and (ii) PCR amplicon length analysis, e.g. by separation of the amplified PCR products (i.e. amplicons) by capillary electrophoresis and subsequent fragment length analysis.

[0015] The present invention relates to a method for detecting and identifying microorganisms in a biological sample at the species or strain level by polymerase chain reaction (PCR), comprising the steps of: a) providing a biological sample suspected of containing microbes and isolating a nucleic acid sequence from said biological sample; b) PCR amplifying an internal transcribed spacer (ITS) region of at least one rRNA contained within the isolated nucleic acid sequence using a set of broad-taxonomic range amplification primers, thereby generating a PCR amplicon; c) recording high-resolution melting curves for the PCR amplicons generated in step b) and recording the lengths of the PCR amplicons generated in step b) by capillary electrophoresis or sequencing; d) comparing the high-resolution melting curve recorded in step c) to a database comprising high-resolution melting curves of reference amplicons generated using the same primer set from reference microbial species or strains of known taxonomic identity, thereby obtaining a first indication of taxonomic identity of the microorganisms present in said sample; e) comparing each PCR amplicon length having a distinct length recorded in step c) to a database comprising PCR amplicon lengths of reference amplicons generated using the same primer set from a reference microbial species or strain of known taxonomic identity, thereby obtaining a second indication of identity of the microorganism present in said sample; f) identifying the microorganism present in the sample to the species or strain level if the first taxonomic index of identity and the second index of identity match. The above method is now provided, comprising the steps of:

[0016] The methods of the invention can be used to identify, at the species or strain level, microorganisms present in a sample, where the microorganism can be, for example, a microorganism capable of causing disease or a microorganism that contaminates a product that should be sterile, such as a bacterium, virus, protozoan, or fungus.

[0017] If no signal is detected in any of the steps of the method of the present invention, it is concluded that the biological sample is negative for the presence of microbes.

[0018] The claimed method uses a combination of high resolution melting curve (hrMC) analysis, which discriminates based on DNA sequence, and PCR amplicon length analysis, which discriminates based on amplicon length, with confidence in the accuracy of each respective outcome (hrMC features vs. amplicon length) being interpreted based on the other outcomes, providing an assay that has a wide range (i.e., many different microorganisms can be detected; e.g., universal bacterial detection), can potentially be performed very quickly, is scalable, and has clinically relevant (i.e., extremely high) sensitivity and specificity. Although each separate outcome may not usually be sufficient to reach a clear species assignment or may mistake contaminating human DNA for microbial DNA, the hrMC outcome is uniquely found and combined or matched with the amplicon length measurement, providing an extremely accurate identification of the amplicon, and therefore the microbial origin of the microbes present in the sample. Each outcome is compared to a database containing hrMC data annotated to microbial strains or species, and amplicon length data annotated to microbial strains or species. A microbial species or strain is then positively identified only if both outcomes are annotated to the same species or strain in the database. High-resolution melting curves depend on amplicon length and sequence, but may show overlap between different amplicons, since they may lead to comparable melting curves despite different lengths and different sequence combinations. Combining mrMC and amplicon length measurements solves this problem. If amplicons have the same melting curves and the same length, amplicons of the same length with the same melting curves must be nearly identical in sequence, and therefore originate from the same species (the sequence of the rRNA ITS region is highly variable between different species, and therefore small variations can only be found within the same species), so the origin of the amplicons must be the same microorganism.Therefore, amplicons not derived from the same microorganism that still show comparable melting curves must have different lengths, and conversely, amplicons not derived from the same microorganism that still show comparable lengths must have different melting curves. Differentiation of rRNA ITS amplicons based on melting curve data and amplicon length thereby provides a highly robust clinical method for microbial identification.

[0019] One current problem in clinical microbial identification is that human DNA is often co-amplified when microbial amplification primers are used. The proportion of human DNA in clinical samples can exceed the proportion of bacterial DNA by up to 10%. 10 It is well known that the amplification rate of microbial DNA can exceed 10 times that of the human target DNA. Although the primers for amplifying microbial DNA are specific for the microbial DNA, there is always a chance that non-target human DNA will be amplified because of the presence of regions that show homology to parts of the bacterial primers. Even when the chance of such non-specific amplification is very small (e.g., 10 9 1 in 10 chance of being the real target 10In an environment where the probability of such an event becomes very real. Considering the high proportion of human DNA in clinical samples, this means that in principle, every PCR-based microbial detection assay using samples obtained from humans has a high probability of forming false-positive amplification products. When using classical qPCR approaches, this challenge is mitigated by using highly specific probes in addition to the amplification primers. These probes can only bind to very specific target regions within the amplicon. These probes do not bind to non-specific products, thus mitigating the challenge of non-specific amplification of human DNA. However, such approaches also hinder the possibility of developing an assay with a broad scope. If the aim is to develop a broad scope assay, such non-specific DNA needs to be dealt with in an alternative way. When only amplicon length analysis is used, such false positives cannot be adequately identified with sufficient clinical certainty, since the non-specific (human) amplicon length may overlap, be similar to, or even be identical to the amplicon length of the micro-organism. If only hrMC is used, this problem remains: non-specific amplicons may produce hrMC curves similar to those of a particular microbial strain or species. Again, by combining hrMC with length analysis, this problem can be solved. The combination of hrMC and amplicon length is specific so that the assignment (e.g., specific microbial amplicon or non-specific amplicon) can be made with certainty. If the length and hrMC match the microbial signature, the amplicon must come from the microorganism (since the length and hrMC are the same, the sequence must be nearly identical). If the combination does not match the microbial signature, the amplicon is not of microbial origin.Since non-specific human amplicons are generally similar between different patients, a library of hrMC and human amplicon lengths can be constructed, allowing the non-specific human amplicons to be easily positively identified as non-specific.By combining detection of a broad range of microorganisms with accurate identification of false positives, an assay can be made that can accurately provide positive as well as negative results for clinical specimens, both of which can be used to make clinical decisions.

[0020] In the context of the present invention, the database comprises high-resolution melting curves and PCR amplicon lengths of reference amplicons generated from reference microbial species or strains of known taxonomic identity as described herein, and the database may further comprise high-resolution melting curves and PCR amplicon lengths of reference amplicons generated from human sequences using a set of amplification primers of the broad taxonomic range described above as a control for non-specific amplicon generation. The term "non-specific amplicon generation" should be understood herein to refer to non-microbial organisms, where non-specific binding of the primers to non-target DNA templates occurs.

[0021] Finally, combining hrMC with amplicon length detection opens the possibility of miniaturization of detection devices, which paves the way for point-of-care (POC) applications of a wide range of microbial detection assays. Currently, the mechanisms required to measure hrMC or amplicon length are typically large and expensive. However, it is envisioned that these measurements can be performed on very small and relatively inexpensive devices, such as lab-on-a-chip (LOC) devices. A current problem with these devices is their lack of precision relative to their larger counterparts. Amplicon length may not be measured with one nucleotide accuracy, for example, it may be measured with an accuracy of 5 nucleotides. To determine bacterial species using hrMC or length measurements alone, high resolution is essential (and even then, lacking specificity). However, by combining hrMC information with length measurements, the requirement to resolve each of these measurements separately can be less stringent. For example, if three different bacterial species have 16S-23S ITS amplicon lengths that are separated by only two nucleotides, they can only be distinguished by very precise measurements. However, the fact that these amplicons are from different bacterial species implies that the sequences of the 16S-23S ITS region must be different (the sequences of ribosomal DNA and its spacer are unique for each bacterial species). Since the melting curve depends only on length and sequence, if the lengths are similar, the sequence difference will necessarily lead to a significantly different hrMC profile. Therefore, even if three different bacterial species cannot be separated by amplicon length alone, the bacteria will always be separated by hrMC. As illustrated before, this is not a matter of luck, because different bacterial species have different 16S-23S ITS amplicon sequences. Then, if the lengths are similar, the different sequences will necessarily lead to a different hrMC profile.

[0022] It has now been found that the combination of high-resolution melting curve analysis and amplicon length analysis makes it feasible to detect and identify a wide range of microorganisms for clinical diagnosis.The method described herein is unique in that it is able to meet all five essential requirements for optimal clinical diagnosis: very broad in scope, including, for example, all bacterial species; it is a molecular method, and therefore does not require culture, so speed is guaranteed; it is a PCR-based method, so sensitivity is high; by combining hrMC with amplicon length analysis, the specificity of bacterial identification is extremely good (while the specificity from a clinical point of view, i.e., the correct identification of true negative samples is also very good due to the ability to identify contaminating human DNA); and finally, scalability is already good with the current setup and will be further enhanced in the future by miniaturization.

[0023] In preferred embodiments of this aspect of the invention, the biological sample is a sample from a human body, an animal body, a plant, a foodstuff, a pharmaceutical or chemical product, a laboratory culture, or an environmental sample, such as a sample from soil or a body of water. In other preferred embodiments, the biological sample is a bodily sample of a patient selected from a bodily fluid or exudate, including but not limited to uterine fluid, whole blood, serum, plasma, lymph, mucus, saliva, sputum, stool / feces, sweat, wound fluid, pus / purulence, stomach contents, ascites / ascitic fluid, bile, urine, semen, cerebrospinal fluid / liquor, and breast milk; or a body sample in the form of a swab, biopsy, lavage, or paper point sample, including but not limited to samples from the skin, organs, tissues, oral cavity, urogenital, vaginal tract, gastrointestinal tract, respiratory or pulmonary system, and cardiovascular system. The present invention relates to a bodily sample of a patient, the bodily sample being selected from the group consisting of a

[0024] In a preferred embodiment of this aspect of the invention, the microorganism is selected from archaea, bacteria, viruses, protozoa, and fungi, preferably said microorganism is a pathogenic microorganism capable of causing disease, preferably a pathogenic microorganism selected from pathogenic bacteria, viruses, protozoa, or fungi, most preferably a bacteria.

[0025] It will be understood that the microorganism or microbial nucleic acid sequence in the context of the present invention can be prokaryotic or eukaryotic. The fact that the internal transcribed spacer (ITS) in the genome of these microorganisms is a spacer DNA located between the small subunit ribosomal RNA (rRNA) gene and the large subunit rRNA gene in the corresponding transcribed region in the chromosome or in the polycistronic rRNA precursor transcript is known to those skilled in the art. Bacteria and archaea have two ITS regions. The first ITS is located between the 16S rRNA gene and the 23S rRNA gene, referred to herein as the 16S-23S rRNA ITS region, and the second ITS is located between the 23S rRNA gene and the 5S rRNA gene, referred to herein as the 23S-5S rRNA ITS region. In eukaryotes, two ITSs also exist; ITS1 is located between the 18S rRNA gene and the 5.8S rRNA gene, referred to herein as the 18S-5.8S rRNA ITS region, while ITS2 is located between the 5.8S rRNA gene and the 26S (plant) or (other 28S eukaryotic) rRNA gene, referred to herein as the 5.8S-26S / 28S rRNA ITS region.

[0026] Thus, in preferred embodiments of this aspect of the invention, the at least one rRNA ITS region may be selected from the 16S-23S rRNA ITS region, the 23S-5S rRNA ITS region, the 18S-5.8S rRNA ITS region, and the 5.8S-26S / 28S rRNA ITS region. In the most preferred embodiments of this aspect of the invention, the at least one rRNA ITS region is the 16S-23S rRNA ITS region.

[0027] In another preferred embodiment of this aspect of the invention, the reference amplicons in the database comprise amplicons generated by in vivo and / or in silico PCR amplification reactions to amplify internal transcribed spacer (ITS) regions of corresponding rRNAs of corresponding reference microbial species or strains of known taxonomic identity. Preferably, the reference microbial species or strains are of the same phylum, more preferably of the same kingdom, and most preferably from the kingdom Bacteria.

[0028] In another preferred embodiment of this aspect of the invention, the databases in step d) and step e) are combined into a single database, wherein said database contains data relating to bacteria.

[0029] In yet another preferred embodiment of this aspect of the invention, the broad taxonomic range amplification primers are for amplifying microbial rRNA ITS regions of multiple, preferably substantially all, strains or species from a microbial genus, family, order, class, phylum, kingdom and / or domain, preferably substantially all strains or species from a microbial phylum, more preferably substantially all strains or species from a microbial kingdom, most preferably bacteria.

[0030] As used herein, the term "substantially all strains or species" preferably refers to the situation where more than 50%, preferably more than 60%, more than 70%, more than 80%, more than 90% of the microbial strains or species within the genus, family, order, class, phylum, kingdom and / or domain are amplified by amplification primers from the broad taxonomic region.

[0031] In yet another preferred embodiment of this aspect of the invention, the broad taxonomic region amplification primers for amplifying the internal transcribed spacer (ITS) region of at least one rRNA comprise forward and reverse primers for amplifying the 16S-23S rRNA ITS region, the 23S-5S rRNA ITS region, the 18S-5.8S rRNA ITS region, or the 5.8S-26S / 28S rRNA ITS region. In a most preferred embodiment of this aspect of the invention, the broad taxonomic region amplification primers for amplifying the internal transcribed spacer (ITS) region of at least one rRNA comprise forward and reverse primers for amplifying the 16S-23S rRNA ITS region.

[0032] As used herein, the term "at least one microbial rRNA ITS region" refers to an embodiment in which the microbial rRNA ITS region is amplified over its entire length. It will be readily understood by those skilled in the art that taxonomically broad amplification of a microbial rRNA ITS region requires identical sequences (or sequences with a high level of sequence similarity over the entire length of the sequence, e.g., greater than 90%, preferably greater than 95%, greater than 96%, greater than 97%, greater than 98%, or greater than 99%) in the genomic DNA of multiple taxonomically related strains or species to serve as primer annealing sites. Such identical sequences or sequences with a high level of sequence similarity are usually found in conserved regions of the coding rRNA sequence and not in the ITS region. Therefore, amplifying at least one microbial rRNA ITS region by taxonomically broad amplification of a microbial rRNA ITS region usually requires the identification of a conserved region in the opposite coding rRNA region of the rRNA ITS of interest. As a result, the ITS region is usually amplified in its entirety, and it will be understood by those skilled in the art that subsequent annotation of taxonomic identity can be done considering only a portion of the microbial rRNA ITS of interest or a portion of the PCR amplicon generated in the method of the invention, but is expected to be done preferably considering the microbial rRNA ITS amplified in its entirety.

[0033] In yet another preferred embodiment of this aspect of the invention, the set of amplification primers covering the broad taxonomic region comprises each of the amplification primers of SEQ ID NOs:1 and 3-5, or each of the amplification primers of SEQ ID NOs:2-5, or each of the amplification primers of SEQ ID NOs:1-5.

[0034] In yet another preferred embodiment of this aspect of the invention, said set of amplification primers covering said broad taxonomic region comprises amplification primers of SEQ ID NOs: 6 and 7-13, respectively.

[0035] In yet another preferred embodiment of this aspect of the invention, the set of broad taxonomic range amplification primers is a universal bacterial amplification primer set, said set preferably including each of the amplification primers of SEQ ID NOs:14-15.

[0036] In yet another preferred embodiment of this aspect of the invention, said step of amplifying PCR comprises qPCR.

[0037] In yet another preferred embodiment of this aspect of the invention, the PCR amplicon lengths are recorded by capillary electrophoresis.

[0038] In yet another preferred embodiment of this aspect of the invention, the PCR amplification reaction comprises a PCR calibrator system, the PCR calibrator system comprising: a set of a plurality of PCR amplification primers, wherein at least one of the plurality of PCR amplification primers comprises a label; and a set of at least two PCR calibrators, wherein each PCR calibrator consists of a DNA fragment comprising a spacer region having a DNA sequence of a given length flanked by an upstream adaptor DNA sequence and a downstream adaptor DNA sequence, the adaptor DNA sequence comprising a primer binding site for binding of the PCR amplification primers, wherein said set of PCR amplification primers comprises a label and a set of at least two PCR calibrators. and a PCR calibrator system in which the spacer region DNA sequences contained in each of said set of at least two PCR calibrators are of different lengths, and wherein each PCR calibrator in said set of at least two PCR calibrators is present in equal or known amounts compared to other PCR calibrators in said set, and wherein said PCR amplifying step b) further comprises PCR amplifying said at least two PCR calibrators using the PCR amplification primers of said PCR calibrator system.

[0039] In yet another preferred embodiment of this aspect of the invention, the broad taxonomic region amplification primers for amplifying the internal transcribed spacer (ITS) region of the at least one rRNA comprise a labeled forward primer and / or a labeled reverse primer, preferably a labeled forward primer, more preferably a fluorescently labeled forward primer. [Brief description of the drawings]

[0040] [Figure 1]Cycling schedule including melting curve analysis performed on a LightCycler 480 machine. Fluorescence acquisition was performed at the end of the primer extension step at 72° C. (dot) and throughout the melting indicated by the temperature ramp starting at 1:43:17. [Diagram 2] Example of melting curve analysis. Top panel: It can be seen that the fluorescence decreases with increasing temperature as the double-stranded DNA becomes increasingly single-stranded and EvaGreen loses fluorescence. Bottom panel: The first derivative of the top melting curve clearly highlights the region of sharp decrease in fluorescence: the melting temperature of the target amplicon. [Diagram 3] Melting curves (left column) and fragment length analysis of the 16S-23S fragments of eight different bacterial species from three different phyla. [Figure 4] Length of the 16S-23S fragment of Streptococcus pyogenes and Corynebacterium jeikeium. Both species show identical length profiles. However, the melting curves of each species are unique. By combining the length data with the melting curve data, both species can be unambiguously identified. [Diagram 5] Combination of fragment length analysis and melting curve analysis of 16S-23S IS profiles of Klebsiella pneumoniae and Enterobacter cloacae. Although the melting curves are nearly impossible to distinguish, each species can be identified by its unique fragment length signature. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0041] definition The terms "nucleic acid sequence" or "nucleotide sequence" may be used interchangeably herein and refer to the base sequence of a DNA or RNA molecule, in single- or double-stranded form, in particular a DNA sequence encoding the ITS region of a ribosomal RNA gene.

[0042] An "isolated nucleic acid sequence" refers to a nucleic acid sequence that is no longer in the natural environment from which it was isolated. The term particularly refers to a nucleic acid molecule that has been separated from at least about 50%, at least about 75%, at least about 90%, or more of the proteins, lipids, carbohydrates, or other materials with which it is naturally associated, e.g., in a microbial host.

[0043] As used herein, the term "microbial" generally refers to objects of microorganism or microbe origin, which refers to organisms that are microscopic, meaning too small to be seen by the naked human eye.

[0044] As used herein, the term "target microbial nucleic acid sequence" refers to a nucleic acid fragment that is targeted for replication (or amplification) and subsequent detection and is diagnostic for the particular microorganism whose presence is to be determined.

[0045] As used herein, the term "polymerase chain reaction (PCR)" refers to a well-known in vitro technique for making multiple copies of a specific segment of target DNA from a template DNA - i.e., the DNA containing the target region to be copied. During the reaction, a mixture containing the target DNA, primers, dNTPs, and a thermostable DNA polymerase is heated to 90-95°C to denature the strands of the target DNA. The solution is cooled to a temperature that allows the primers (single-stranded DNA molecules about 18-30 nucleotides long) to anneal to their complementary sequences on the target DNA and provide the 3'-OH required for DNA synthesis. Then, at a temperature usually around 72°C, the DNA polymerase synthesizes new DNA strands complementary to the target by extending the primers. The thermal cycling scheme of denaturation / primer annealing / primer extension is repeated multiple times, with the DNA synthesized during the previous cycle serving as a template for each subsequent cycle. The result is a doubling of the target DNA present with each cycle, and an exponential accumulation of the target DNA sequence over the course of 20-40 cycles. For strict temperature control, a heating block is used with an automated thermal cycler. A preferred method for use in the present invention is qPCR amplification (also known as real-time PCR), where typically the amplification of targeted DNA molecules is monitored during the PCR (i.e., in real time) using a non-specific fluorescent dye that intercalates into any double-stranded DNA or a sequence-specific DNA probe consisting of an oligonucleotide labeled with a fluorescent reporter to detect PCR products in real time.

[0046] As used herein, the term "template" refers to a nucleic acid from which a target sequence is amplified in a nucleic acid amplification reaction. As used herein, the term "amplifiable template" refers to a template that, when amplified, results in a single amplicon. An amplifiable template contains primer binding sites for hybridization of amplification primers.

[0047] The term "isolating" as used herein in connection with isolating a nucleic acid sequence from a biological sample refers to an in vitro method in which nucleic acid, preferably genomic DNA, is extracted from the sample of interest. The method may generally include, but is not limited to, lysis of the biological sample (cells in the sample) using a guanidine-detergent lysing solution that allows selective precipitation of DNA from the (cell) lysate, and precipitation of the genomic DNA from the lysate with ethanol. After washing with ethanol, the precipitated DNA may be solubilized in either water or 8 mM NaOH and used as a template in a PCR reaction. Analysis of genomic DNA samples for diagnostic purposes may be obtained by using commonly known techniques for DNA isolation. Total genomic DNA may be purified, for example, by using a combination of physical and chemical methods. Very preferably, commercially available systems for DNA isolation may be used, such as the NucliSENS™ easyMAG™ Nucleic Acid Extraction System (bioMerieux, Marcy l'Etoile, France) or the MagNA Pure 96 System (Roche Diagnostics GmbH, Mannheim, Germany).

[0048] As used herein, the term "PCR mixture" refers to a mixture of (genomic) template DNA containing one or more target DNA sequences, a set of at least two oligonucleotide primers that hybridize to opposite strands of the one or more target DNA sequences and flank the region to be amplified, a thermostable DNA polymerase, four deoxyribonucleoside triphosphates (dNTPs), and Mg. 2+ A small amount of biochemical reactants in an aqueous liquid that contains ions to carry out a PCR reaction.

[0049] As used herein, the term "amplification primers" refers to oligonucleotide primers that hybridize to opposite strands of the one or more target DNA sequences and flank the region to be amplified.

[0050] The terms "amplification product" and "amplicon," as used interchangeably herein, refer to a nucleic acid fragment that is the product of a nucleic acid amplification or replication event, e.g., formed in a polymerase chain reaction (PCR). As used herein, the term "PCR amplicon" refers to a PCR product or amplified target DNA.

[0051] As used herein, the term "high-resolution melting curve (hrMC) analysis" refers to a post-PCR analysis method used to identify variations in nucleic acid sequences. The method is based on detecting small differences in PCR melting (dissociation) curves. Temperature-dependent dissociation between two DNA strands can be measured using DNA-intercalating fluorophores, such as SYBR green, EvaGreen, or "saturation dyes" (dyes that do not inhibit PCR even when used at concentrations that result in maximum fluorescence (saturation)), such as LCGreen™ I, LCGreen Plus, or Cyto9, in conjunction with real-time PCR instrumentation with strict temperature ramp control and advanced (fluorescence) data capture capabilities. Data is analyzed and manipulated using software specifically designed for hrMC analysis. High-resolution melting curves are generated by slowly ramping a temperature gradient with a high level of precision (e.g., 0.1°C or less) and measuring the level of fluorescence of the intercalating dye at each step. The melting temperature of DNA molecules is determined by nucleic acid sequence and length, and the difference in these nucleotide sequences between samples results in melting profiles that are unique to a particular species, even when amplicons are isolated using universal primers.Details of hrMC analysis procedures are well known to those skilled in the art and are described, for example, in Reed GH, Kent JO, Wittwer CT (2007) High-resolution DNA melting analysis for simple and efficient molecular diagnostics.Pharmacogenomics,8, 597-608; US Patent No. 7,297,484; US Patent No. 7,387,887; US Patent No. 7,524,632; US Patent Publication No. 20090117553 and US Patent Publication No. 20100041044, the contents of which are incorporated herein by reference.

[0052] As used herein, the term "high-resolution melting curve (hrMC)" refers to a melting curve that depicts the temperature-dependent dissociation between two DNA strands, measured using a fluorophore that intercalates into DNA.The high-resolution melting curve refers to a graph of the negative first derivative of the melting curve, which makes it easy to pinpoint the temperature of dissociation (defined as 50% dissociation) based on the peak thus formed.

[0053] As used herein, the term "electrophoretic separation and amplicon length analysis" refers to a technique in which a mixture of charged molecules, preferably nucleic acids, particularly PCR amplified DNA fragments, loaded into a gel matrix migrates through the gel from a negative electrode (cathode) to a positive electrode (anode) based on size, charge, and structure when the gel is placed in an electric field, whereby shorter nucleic acid fragments migrate more rapidly than longer nucleic acid fragments, resulting in size-based separation. Electrophoretic separation and amplicon length analysis are preferably performed by capillary electrophoresis, where DNA is detected either by UV absorption or by fluorescent labeling. In the presence of appropriate standards, fragments can be accurately sized based on their relative electrophoretic mobility, for example, using an ABI Prism 3500 Genetic Analyzer (Applied Biosystems) or similar analytical instrument.

[0054] "Electrophoretic separation and amplicon length analysis" as defined herein may also be performed by DNA sequencing of the amplicons, which may provide accurate information regarding the amplicon length.

[0055] As used herein, the term "negative control reaction" refers to a post-PCR mixture that does not contain one or more PCR amplicons as a result of the intentional absence of a target nucleic acid sequence or template DNA in the pre-PCR mixture.

[0056] As used herein, the term "primer dimer (PD)" refers to a potential by-product in PCR, consisting of primer molecules annealed (hybridized) to each other due to a stretch of complementary bases in the primer. As a result, DNA polymerase amplifies PD, leading to competition for PCR reagents, thus potentially inhibiting the amplification of the DNA sequence targeted for PCR amplification. In quantitative PCR, PD can interfere with accurate quantification.

[0057] As used herein, the term "non-specific PCR amplicon" refers to a potential by-product in PCR consisting of amplified DNA that is not the target DNA, which usually results from non-specific annealing (hybridization) of primer molecules with other nucleic acid sequences in the template DNA, e.g., human DNA. Non-specific PCR amplicons result in hrMCs that are different from PCR amplicons generated from the target microbial DNA sequence.

[0058] As used herein, the term "human PCR amplicon" refers to a non-specific PCR amplicon in which a primer molecule anneals to a human nucleic acid sequence in the template DNA instead of a microbial DNA sequence. Human PCR amplicons result in a different hrMC than PCR amplicons generated from (microbial) target DNA.

[0059] As used herein, the term "quantification cycle" or "Cq" includes reference to measurements taken in a real-time PCR assay or qPCR assay, where a positive reaction is detected by the accumulation of a signal, e.g., a fluorescent signal. The Cq (quantification cycle) can be defined as the number of cycles required for the signal to exceed a threshold (i.e., above background level). The Cq level is inversely proportional to the amount of target nucleic acid in the sample (i.e., the lower the Cq level, the greater the amount of target nucleic acid in the sample).

[0060] As used herein, the term "sample" or "biological sample" includes reference to a sample derived from the human body, an animal body, a plant, a laboratory culture, an environmental sample or a foodstuff, pharmaceutical or chemical product, preferably said foodstuff, pharmaceutical or chemical product being intended to be free of microbes or microbial DNA.

[0061] As used herein, the term "subject" is intended to refer to any individual or patient on whom the methods described herein are performed. Generally, the subject is a human, but as will be appreciated by those skilled in the art, the subject may be an animal. Thus, other animals, including mammals and birds, are also included within the definition of a subject.

[0062] Performing PCR in the method of the present invention In the methods of the present invention, the step of amplifying DNA is carried out by polymerase chain reaction (PCR).

[0063] Preferably, in the method of the invention, the PCR is qPCR or real-time PCR.The present invention relates to taxonomically broad or universal amplification of at least one rRNA ITS region of microbial genomic DNA in a sample, i.e. taxon-specific amplification of genomic DNA across microbial genera, families, orders, classes, phyla or kingdoms.

[0064] Those skilled in the art are well aware of the methods and means for setting up such PCR reactions. Preferably, the method of the present invention is for detecting or identifying bacteria or bacterial DNA in a sample. Exemplary primer sets for amplifying 16S-23S rRNA ITS regions from genomic DNA of one or more of the bacterial phyla Firmicutes, Bacteriodetes or Proteobacteria are provided as SEQ ID NOs: 1-13. Exemplary primer sets for universally amplifying 16S-23S rRNA ITS regions from bacterial genomic DNA are provided as SEQ ID NOs: 14-15. It is within the routine capabilities of a person skilled in the art to further design primer sets that allow for taxonomically broad amplification of microorganisms, preferably bacterial DNA.

[0065] High resolution melting curve (hrMC) analysis in the method of the present invention Characterization of nucleic acids by high-resolution melting curve (hrMC) analysis is a powerful technique for identifying sequence variations during PCR or in post-PCR samples. By measuring the fluorescence of a saturable intercalating dye as PCR-amplified DNA fragments are heated and dissociated, sequence-defined melting curves can be generated with single-nucleotide resolution.

[0066] In a preferred embodiment of this aspect of the invention, the hrMC dye is selected from the group consisting of, but not limited to, LC Green, SYTO9, Eva Green, Chromofy, BEBO, or SYBR Green, preferably Eva Green. The saturating intercalating dye should preferably not inhibit PCR.

[0067] In the method of the present invention, the hrMC profile of the PCR amplicon can be generated by measuring the fluorescence of the saturating intercalating dye of the amplicon present in the post-PCR sample during or after the PCR amplification step. As used herein, "melting profile" refers to the profile generated by hrMC analysis. Those skilled in the art fully understand how hrMC should be performed. Sufficient guidance can be found, for example, in Reed et al. 2007 Pharmacogenomics 8 (6):597-608 and Wittwer et al. 2003 Clin Chem.49:853-860.

[0068] The high-resolution melting curve analysis in qPCR (i.e. real-time PCR) is not equivalent to that carried out by digital PCR.In digital PCR, each section contains only a single sample template, which means that all amplicons for each section originate from a single template.This uniformity allows easier interpretation of hrMC profile and allows direct comparison with other sections.In real-time PCR, the heterogeneous sample of multiple targets remains heterogeneous after amplification, and therefore the melting curve reflects its heterogeneity.This makes it difficult to distinguish multiple targets by hrMC analysis when multiple amplicons exist in a sample.

[0069] In the method of the present invention, a post-PCR hrMC profile of the sample is generated. Preferably, the PCR is qPCR or real-time PCR. The generated hrMC profile is compared to a (predetermined) reference hrMC profile of one or more corresponding 16S-23S rRNA ITS amplicons of known (i.e. taxonomically identified) strains or species of microbes. Preferably, said reference hrMC profiles of one or more corresponding 16S-23S rRNA ITS amplicons of known strains or species are included in a library or database of reference hrMC profiles. Such a library or database can be established by (i) generating in vitro 16S-23S rRNA ITS PCR amplicons of individual strains and / or species of microbes, (ii) performing hrMC analysis of said amplicons, and (iii) storing said reference hrMC profiles in a library or database of reference hrMC profiles, where the identity of said species or strains is annotated to said hrMC profiles. Alternatively, a database or library of reference hrMC profiles can be generated by in silico prediction of the hrMC profiles of one or more 16S-23S rRNA ITS PCR amplicons of individual strains and / or species of microbes.

[0070] From a clinical perspective, if the hrMC profile of the 16S-23S rRNA ITS PCR amplicon in a PCR sample indicates that a microbe, e.g., a bacterium, is present in the clinical sample, the clinician can already make an important first treatment decision: to initiate administration of a therapeutic amount of an antimicrobial agent if a microbe susceptible to the antimicrobial agent is present. For example, if it is determined based on the hrMC analysis that a bacterium is present, the clinician can make the treatment decision to initiate administration of an antibiotic.

[0071] However, in many cases, qPCR or real-time PCR does not lead to a definitive identification of the microbe present. hrMC analysis of PCR products can provide a definitive answer regarding the identity of a single microbe only if it is present in the sample and matches the reference hrMC profile. In other cases, such as when more than one microbe is present in the sample, hrMC analysis generally does not provide the clinician with actionable information regarding the identity of the one or more microbes.

[0072] The inventors have found that combining hrMC analysis, which discriminates sequences, with PCR fragment length analysis, which discriminates based on fragment length, in one clinical assay is highly beneficial in improving the sensitivity and specificity of PCR-based microbial identification assays. This combined approach allows the generated hrMC profile to be interpreted based on the PCR fragment length profile, and vice versa, the PCR fragment length profile to be interpreted based on the hrMC profile, which greatly improves specificity. This was not known in the art until now. Combining the two techniques provides the clinician with useful information regarding the identity of the microbe. Preferably, the specificity (of microbial identification) provided by the method of the present invention is at least 90%, more preferably at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%.

[0073] Analysis of PCR fragment length in the method of the present invention The method of the invention comprises a step of amplicon fragment length analysis performed on the amplicons in a PCR sample to generate a length profile of the fragments of the amplicons present in the PCR sample.

[0074] Methods and means for determining the length profile of PCR fragments are generally known in the art. One exemplary and highly useful method for the analysis of PCR fragment lengths is capillary gel electrophoresis. Preferably, the capillary gel electrophoresis method is the IS-pro method described in Budding et al., FASEB J., 24, 4556-4564 (2010), WO 2008 / 125365 and WO 2015 / 170979, which in a preferred embodiment may include the differentiation of bacterial species based on the length of the 16S-23S rDNA ITS region using taxonomic classification by phylum-specific fluorescent labeling of PCR primers.

[0075] The IS-pro methods described in the aforementioned publications are expressly incorporated herein by reference, for example the methods for analyzing populations of micro-organisms described in WO 2008 / 125365, e.g., on page 4, lines 11 et seq., are incorporated herein by reference, including the primer sets described therein. In addition, for example, the PCR calibrator systems and uses thereof described in WO 2015 / 170979 (see, e.g., p. 44 et seq.), the IS-pro analysis described in WO 2015 / 170979 (see, e.g., p. 53 et seq.), the primer sets and probes described in WO 2015 / 170979 (see, e.g., p. 65 and 66 of WO 2015 / 170979), and the patient sample types described in WO 2015 / 170979 (see, e.g., p. 11, lines 18 et seq.) are incorporated herein by reference.

[0076] Briefly, with the IS-pro method, sequences of conserved DNA regions contained in the 16S and 23S rRNA gene sequences flanking intergenic regions in the genomic DNA of the microorganisms are used as primer binding sites to amplify (polymorphic) ITS DNA regions.

[0077] The IS-pro taxonomic diversity analysis is based on the fact that prokaryotic microorganisms, including bacteria and archaea, contain one or more copies of the rrn operon in their genome, which contains genes for 5S, 16S and 23S ribosomal RNA. In most prokaryotes, the ribosomal genes in the operon are in the order 16S-23S-5S and are cotranscribed into a single polycistronic RNA, which is processed to yield the RNA species present in the mature ribosome. The spacer between the 16S and 23S genes contains regions with secondary structure and sometimes tRNA genes. The variation in the spacer of the rRNA operon found between relatively close taxa is extremely large. The extreme differences in the size and sequence of the spacer between different groups of prokaryotes make it ideally suited as a taxonomic marker.

[0078] In the IS-pro method, the 16S-23S rRNA intergenic spacer (ITS) region is amplified using primers directed to a conserved region within the ribosomal gene sequence. More preferably, the conserved DNA region is the region located closest to the 3' end of the 16S rRNA gene and closest to the 5' end of the 23S rRNA gene.

[0079] Depending on the microbiome to be investigated, phylum-specific primer sets can be used. Suitable phylum-specific primer sets in IS-pro include those that allow for simultaneous amplification of multiple sequences in a single reaction, in a method called multiplex PCR.

[0080] For the amplification of the intergenic spacer region of bacterial DNA from the phyla Firmicuta and Actinobacteria, a suitable primer set includes the following: FirISf: 5'-CTGGATCACCTCCTTTCTAWG-3' (SEQ ID NO:1) as a forward primer, and one of DUISrI: 5'-AGGCATCCACCGTGCGCCCT-3' (SEQ ID NO:3), DUISr2: 5'-AGGCATTCACCRTGCGCCCT-3' (SEQ ID NO:4) and DUISr3: 5'-AGGCATCCRCCATGCGCCCT-3' (SEQ ID NO:5) as a reverse primer. Preferably, the FirISf primer is labeled with a fluorescent label herein. Preferably, the reverse primer is not labeled herein.

[0081] Genera within the phylum Firmicutes include: class Bacilli, order Bacillales (genus Bacillus, Listeria, Staphylococcus); class Bacilli, order Lactobacillales (genus Enterococcus, Lactobacillus, Lactococcus, Leuconostoc, Pectinatus, Pediococcus, Streptococcus genus; Clostridia class (Acetobacterium, Clostridium, Eubacuterium, Heliobacterium, Heliospirillum, Sporomusa genus); Mollicutes class (Mycoplasma, Spiroplasma, Ureaplasma, Erysipelothrix genus).

[0082] For the amplification of the intergenic spacer region of bacteria from the phylum Bacteroidetes, a suitable primer set includes the following: as forward primer, BacISf: 5'-CTGGAACACCTCCTTTCTGGA-3' (SEQ ID NO:2), and as reverse primer, one or more, preferably all of DUISrI: 5'-AGGCATCCACCGTGCGCCCT-3' (SEQ ID NO:3), DUISr2: 5'-AGGCATTCACCRTGCGCCCT-3' (SEQ ID NO:4) and DUISr3: 5'-AGGCATCCRCCATGCGCCCT-3' (SEQ ID NO:5). Preferably, herein, the BacISf primer is labeled with a fluorescent label. Preferably, herein, the reverse primer is unlabeled.

[0083] In a broad-based community analysis, the intergenic spacer regions of bacteria from the phyla Firmicuta and Actinobacteria can be amplified in a single multiplex reaction with the intergenic spacer regions of bacteria from the phylum Bacteroides. In such a multiplex reaction, both forward primers FirISf and BacISf and all three reverse primers are included in the PCR mixture. Preferably, herein, the FirISf primer is labeled with a first fluorescent label, such as FAM, and the BacISf primer is labeled with a second fluorescent label, such as HEX. Preferably, herein, the reverse primer is not labeled.

[0084] For more extensive or alternative community analysis, the intergenic spacer region of bacteria from the phylum Proteobacteria can be amplified. Suitable primer sets for this purpose include:

[0085] The forward primer was ProtISf: 5'-CCGCCCGTCACACCATGG-3' (SEQ ID NO:6), and the reverse primers were DPISr1: 5'-AATCTCGGTTGATTTCTTTTCCT-3' (SEQ ID NO:7), DPISr2: 5'-AATCTCGGTTGATTTCTTCTCCT-3' (SEQ ID NO:8), DPISr3: 5'-AATCTCTTTTGATTTCTTTTCCTCG-3' (SEQ ID NO:9), DPISr4: 5'-AATCTCATTTGATGTCTTTTCCTCG-3' (SEQ ID NO:10), DPISr5: 5'-AATCTCTTTTGATTTCTTTTCCTTCG-3' (SEQ ID NO:11), and DPISr6: 5'-AATCTCTCTTGATTTCTTTTCCTTCG-3' (SEQ ID NO:12). DPISr7: 5'-AATCTCAATTGATTTCTTTTCCTAAGG-3' (SEQ ID NO:12), and DPISr7: 5'-AATCTCAATTGATTTCTTTTCCTAAGG-3' (SEQ ID NO:13). Preferably, herein, the ProtISf primer is labeled with a fluorescent label. Preferably, herein, the reverse primer is unlabeled.

[0086] Genera within the phylum Bacteroidetes include the genus Bacteroides (organisms abundant in the feces of warm-blooded animals, including humans; e.g., B. acidifaciens, B. distasonis, B. gracilis, B. fragilis, B. oris, B. ovatus, B. proteus, B. purpurea ... The genus Porphyromonas includes B. putredinis, B. pyogenes, B. stercoris, B. suis, B. tectus, B. thetaiotaomicron, and B. vulgatus), and the genus Porphyromonas, a group of organisms present in the human oral cavity.

[0087] Amplification of the Proteobacteria phylum is very beneficially performed by performing multiplex PCR to provide sufficient taxonomic resolution within the Proteobacteria phylum (see WO 2015 / 170979).

[0088] Preferred microorganisms in the context of the present invention are those belonging to the phylum Firmicutes, Fusobacterium, Deferribacteres, Spirochaetes, Cyanobacteria, Acidobacteria, Nitrospina, Nitrospirae, Caldithrix, Haloanaerobiales, Verrucomicrobia, Chlamydiae, Planctomycetes, Gemmimonas, Fibrobacterium, Bacteria, and the like. The bacterium belongs to a bacterial phylum selected from the group consisting of Fibrobacteres, Chlorobi, Bacteroidetes, Proteobacteria, Thermotogae, Coprothermobacter, Synergites, Thermodesulfobacteria, Desulfurobacterium, Aquificae, Deinococcus-Thermus, Chloroflexi, Tenericutes, and Actinobacteria. More preferred phyla targeted in the methods of the present invention include the phyla Bacteroidetes, Firmicutes, Actinobacteria, Proteobacteria, Fusobacteria, and Verrucomicrobia.Highly preferred are the phyla Bacteroidetes, Firmicutes, Actinobacteria and Proteobacteria.

[0089] These phyla are known to the person skilled in the art and are described, inter alia, in Schloss 2004 (Microb. Mol. Biol. Rev. 6 (4): 686-691) or in the Bergey manual of systematics of archaea and bacteria (2015).

[0090] Universal amplification of the internal transcribed spacer (ITS) region of bacterial 16S-23S rRNA of genomic DNA can also be used in the method of the present invention. For such universal amplification of bacterial DNA, a universal bacterial primer set can be used, comprising a combination of a forward primer ITS1FD having the sequence 5'-CGGTGAATACGTTCCCGGIIIIIGTACAC-3' (SEQ ID NO: 14) and a reverse primer ITS2RD having the sequence 5'-CGTCCTTCDTCGVCTBIIIIIGCCARG-3' (SEQ ID NO: 15).

[0091] The samples on which the method of the invention can be carried out can be from any environment containing microorganisms, but are preferably from mammals, such as humans. Suitable sample materials include, for example, samples from humans, plants, animals, water, food (such as dairy products), yeast cultures (for example used in industry) or soil.

[0092] In the IS-pro technique, microbial DNA is preferably and generally isolated by an automated isolation procedure (e.g. EasyMag, Biomerieux, Marcy l'Etoile, France) according to the manufacturer's instructions. In the method of the present invention, microbial DNA is preferably isolated from the sample in the form of genomic DNA. The skilled artisan is well aware of the genomic DNA isolation methods available in the art.

[0093] Amplification of the 16S-23S rRNA ITS region from the isolated microbial DNA with phylum-specific fluorescently labeled PCR primers is then performed using the IS-pro assay (inBiome, Amsterdam, the Netherlands) according to the protocol provided by the manufacturer or by using any of the primer sets described above. IS-pro involves amplifying the 16S-23S rRNA ITS region and subsequently identifying the microbial species as the source of the DNA template from which the amplicon was generated based on the amplicon length. Briefly, appropriately diluted isolated DNA can be amplified in a standardized PCR amplification. One example of standardized PCR amplification involves the use of phylum-specific primer sets to amplify the 16S-23S rRNA ITS region of the combined Firmicutes, Actinobacteria, Fusobacteria, and Verrucomicrobia phyla (FAFV). Another example of standardized PCR amplification involves the use of phylum-specific primer sets to amplify the 16S-23S rRNA ITS region of the Bacteroidetes phylum. Similarly, the 16S-23S rRNA ITS regions of the Firmicutes and Bacteroidetes phyla can be amplified in a single reaction (e.g., using primers of SEQ ID NOs: 1-5 in a single multiplex reaction) from a DNA sample to yield amplicons from any bacterial species from these phyla. Yet another example of standardized PCR amplification includes the use of a phylum-specific primer set to amplify the 16S-23S rRNA ITS region of the Proteobacteria phylum (e.g., using the primers of SEQ ID NOs:6 and 7-13).Yet another example of standardized PCR amplification involves the use of a kingdom-specific primer set to amplify the 16S-23S rRNA ITS region of microorganisms belonging to the kingdom Bacteria (e.g., using the primers of SEQ ID NOs:14 and 15).

[0094] After PCR amplification and generation of the amplicon, a small aliquot of the PCR product can then be separated and analyzed by capillary electrophoresis to determine the 16S-23S rRNA ITS amplicon length, for example, in an ABI Prism 3500 Genetic Fragment Analyzer (Applied Biosystems Carlsbad, California, USA).

[0095] In IS-pro, distinct bacterial species with distinct 16S-23S ITS DNA sequences generate amplicons of different lengths when taxonomically broad or universal amplification primers are used to amplify the 16S-23S rRNA ITS region from a microbial genomic DNA template in a sample, which are easily separated by capillary gel electrophoresis, thereby resulting in a profile of distinct DNA fragments, each of which represents a distinct 16S-23S ITS DNA sequence with a characteristic length (number of nucleotides or base pairs).

[0096] Generally, each fragment is considered to represent a single ITS region from a distinct bacterial species or operational taxonomic unit (OTU). However, it is possible for different bacterial species to produce 16S-23S ITS amplicons of the same length. Furthermore, because a genome may contain multiple rrn operons with different ITS region sequences, not all fragments represent distinct microbial species. Thus, the different peaks (amplicons of distinct lengths) may originate from a single species or distinct species. Finally, the small biological length variation in the ITS sequence may complicate simple species identification based on length measurements alone.

[0097] To improve resolution and species identification, and to better classify and assign amplicons from microbial DNA samples to specific species or strains, the inventors have now found that adding a step of hrMC analysis can result in separate identification of individual species that produce amplicons of the same length (i.e. resulting in a single electrophoretic peak). The hrMC analysis allows for the identification of sequence differences between amplicons of the same length, and therefore allows for the individual identification of the species underlying the same CE peak. Furthermore, by adding the hrMC step, multiple amplicons of different lengths originating from a single species can be unambiguously assigned to the correct species. Also, fragments that may both originate from a single species or from (multiple) distinct species can be classified and assigned as originating from either a specific species or strain, or from multiple species or strains. This can be done in both complex and non-complex microbial populations alike.

[0098] These improvements are of paramount importance for the reliability of the assay for use in clinical practice.

[0099] In addition, it has now been found that certain amplicon peaks in the amplicon length profile, which may now be attributable to human DNA (false positive), cannot be dismissed by the clinician as "non-microbial" based on the amplicon length profile, but rather can actually be dismissed thereafter once hrMC analysis confirms that human DNA is in fact present (false positive identification), thus greatly improving the specificity of the assay in the clinic.

[0100] The specificity of such tests is improved because the hrMC of distinct microbial species are generally easily distinguishable, and individual species that produce identical amplicon lengths in PCR (e.g., C. jeikeium / S. pyogenes) will be unambiguously detected based on differences in the hrMC of their amplicons.

[0101] The methods of the invention include the steps of adding an hrMC dye before, during or after PCR amplification and performing hrMC analysis on the post-PCR mixture, particularly on one or more PCR amplicons.

[0102] Another advantage of using hrMC analysis in diagnostic methods involving amplification reactions using taxonomically broad primers followed by electrophoretic separation and amplicon length analysis is that the hrMC analysis can speed up the procedure. When the hrMC of the reaction products is known, a person skilled in the art can perform electrophoretic separation and amplicon length analysis only for fragments of a predetermined length range based on the hrMC data (e.g., only separate and analyze fragments of 300-500 bp in length). For example, if the hrMC indicates the presence of one of three possible species, one can quickly "zoom in" on the known amplicon lengths for these species to solve the problem.

[0103] The method of the present invention can be used to type, diagnose, investigate, analyze and monitor samples with various microbes, such as those associated with the intestine or gastrointestinal tract, skin, urogenital tract, oral cavity and pulmonary system, as well as to diagnose, monitor or predict diseases. The method can be used for diagnostic purposes, such as diagnosing, monitoring or predicting (including early detection) microbial infections, but also for environmental diagnostics, such as determining the microbial status of a sample source, where the sample source is of environmental, plant, animal or food origin, or a pharmaceutical or chemical product sample intended to be completely free of microbes or microbial DNA, and where a specific profile (or its absence) of the ITS region is indicative of the sterility of the sample, the quality of the environment, the microbial safety of a food, pharmaceutical product, the quality of a chemical product, or the health of a plant or animal.

[0104] An alternative method for determining PCR amplicon length is by a step of DNA sequencing performed on the amplicons in the post-PCR sample. Preferably, the step of DNA sequencing is performed by a next-generation sequencing method capable of determining the sequence length of the amplicons, which have a length of 100 to 1200 bp base pairs, a range that generally resembles the overall variation in length of 16S-23S ITS DNA sequences between different microbial species. Suitable next-generation sequencing methods for determining amplicon length include nanopore-based methods (e.g., devices manufactured by Oxford Nanopore or Pacific-Biosciences) or single molecule real-time sequencing methods (e.g., devices manufactured by Pacific Biosciences).

[0105] When a step of determining PCR amplicon length is performed in the method of the present invention, an amplicon length profile can be generated. Such generated amplicon length profile can be compared to one or more reference or control amplicon length profiles of corresponding 16S-23S rRNA internal transcribed spacer (ITS) regions of known strains or species of microbes. Preferably, said reference amplicon length profiles of corresponding 16S-23S rRNA internal transcribed spacer (ITS) regions of known strains or species of microbes are included in a library or database of reference amplicon length profiles. Such a library or database can be established by (i) generating in vitro PCR amplicons of the target 16S-23S rRNA internal transcribed spacer (ITS) region of genomic DNA of individual known strains and / or species of microbes, (ii) performing a fragment length analysis of said amplicons, and (iii) storing said reference fragment length profiles in a library or database of reference fragment length profiles. Alternatively, a database or library of reference fragment length profiles can be generated by in silico prediction of the fragment length profiles of PCR amplicons of the target 16S-23S rRNA internal transcribed spacer (ITS) region of genomic DNA of individual strains and / or species of microbes.

[0106] It is within the bounds and scope of routine experimentation of one of skill in the art to establish (predetermine) a reference or control amplicon length profile suitable for comparison with the generated amplicon length profile of a test sample. For example, the method of the present invention may include the steps of analyzing the amplification products or amplicons (post-PCR) based on the length differences in the amplification products, thereby resulting in an amplicon length profile of the composition of a population of microorganisms in the microbiome or sample disclosed herein; and comparing the fragment length profile with at least one reference fragment length profile of known micro-organisms.

[0107] For clarity and conciseness of description, features may be described herein as part of the same or separate embodiments, however, it will be understood that the disclosure includes embodiments having all or any combination of the described features.

[0108] The contents of the documents mentioned herein are incorporated by reference.

[0109] Working Example Example 1 Combining melting curve and fragment length data for unambiguous species identification. To detect and identify different micro-organisms, DNA regions that are broadly conserved across taxonomic groups can be used if they have certain characteristics. The important characteristics are the presence of conserved regions in the DNA that can be used to amplify the DNA of many different species using broad-range primers, and the presence of variable regions flanked by conserved regions that can be used to distinguish different species. Different methods exist for analyzing the variable regions, with different suitability based on the specific characteristics of the variable regions. A region of DNA that is present in all life forms and has interspersed conserved and variable regions is ribosomal DNA (rDNA). Within bacterial ribosomal DNA, the 16S-23S interspace (IS) region is of particular interest because it is flanked by highly conserved regions that are present in virtually all bacteria, show great variability in sequence composition and length, and are highly suitable for targeting with broad-range primers.

[0110] Here we demonstrate a highly efficient and accurate method for detecting and identifying bacteria based on a combination of melting curve and fragment length analysis of the 16S-23S IS region.

[0111] Melting curves of 16S-23S interspace (IS) fragments in bacteria can be used to distinguish different bacterial species. In addition, length measurements can also distinguish different bacterial species. Melting curve analysis or fragment analysis alone can be applied to bacterial species identification, but there are many cases where this is not possible. In these cases, the fragment profiles are identical between species or the melting curves do not provide sufficient discrimination between species. When combining fragment length analysis with melting curve analysis and comparing the results to a database, unequivocal species identification can be made. Here we demonstrate that the combination of fragment length analysis and melting curve analysis can unequivocally distinguish bacteria to species level, and that either fragment length analysis alone or melting curve analysis alone cannot.

[0112] Materials and Methods Strain selection and cultivation Clinical isolates of 12 different species were used in this analysis: Bacteroides fragilis, Bacteroides thetaiotaomicron, Enterococcus faecalis, Escherichia coli, Staphylococcus epidermidis, Staphylococcus aureus, Streptococcus cristatus, Streptococcus pyogenes, Corynebacterium jeikeium, and Staphylococcus pneumoniae. The following bacteria were identified: Pseudomonas aeruginosa, Klebsiella pneumoniae, and Enterobacter cloaace. Aerobic bacteria were cultured on sheep blood agar (BioMérieux) at 37°C and incubated aerobically for 2 days, and anaerobic bacteria were cultured on Schaedler agar (Oxoid) and incubated anaerobically for 3 days. Identification of bacterial colonies was performed by MALDI-TOF (VITEK MS syste, Biomérieux).

[0113] DNA isolation DNA was isolated by adding 200 μl of suspended bacteria (0.1 McFarland) to 400 μl AL buffer (Qiagen, Hilden, Germany) and 40 μl proteinase K in an Eppendorf container. The mixture was centrifuged at 9000 g for 10 s, then vortexed and incubated at 56° C. for 1 h with shaking at 1400 rpm. An easyMAG automated DNA isolator (Biomerieux) was used for further DNA extraction.

[0114] The sample mixture (640 μl) was transferred to an 8-well easyMAG vessel provided by the manufacturer and suspended in 2 ml of nucliSens lysis buffer. After 1 h incubation at room temperature, 70 μl of magnetic silica beads provided with the easyMAG machine were added. The mixture was then inserted into the easyMAG machine and the "specific A" protocol was selected, thereby selecting the off-board workflow and eluting the DNA in 110 μl of NucliSens easyMAG extraction buffer 3 provided by the manufacturer (Biomerieux). All DNA was stored at 4°C.

[0115] PCR For the various analyses, three different PCRs were used: two first PCRs were used for phylum-specific amplification of the 16S-23S region and fragment analysis, and a third PCR was used for universal amplification of the 16S-23S region, fragment analysis and melting curve analysis. The first PCR contained two different fluorescently labeled forward primers (SEQ ID NO: 1 and 2) targeting different bacterial groups, and three reverse primers (SEQ ID NO: 3-5) providing universal coverage for these groups. The first forward primer (SEQ ID NO: 1) was specific for the phyla Firmicutes, Actinobacteria, Fusobacteria, and Verrucomicrobia (FAFV), and the second labeled forward primer (SEQ ID NO: 2) was specific for the phylum Bacteroidetes. The second PCR, which used a labeled forward primer (SEQ ID NO:6) in combination with seven reverse primers (SEQ ID NO:7-13), was specific for the phylum Proteobacteria (see Table 1). The third PCR reaction mixture contained a set of primers that recognize a broad collection of bacteria: FOR: 5'CGGTGAATACGTTCCCGGIIIIIGTACAC 3' (SEQ ID NO:14) and REV 5'CGTCCTTCDTCGVCTBIIIIIGCCARG-3' (SEQ ID NO:15). Inosine binds to all four DNA bases, ensuring broad reactivity. The FOR primer contains the ATTO550 fluorescent moiety. The PCR reaction mixture contains EvaGreen, an intercalating dye that fluoresces when bound to double-stranded DNA.

[0116] The two first PCR amplifications were performed using a GeneAmp PCR system 9700 (Applied Biosystems, Foster City, Calif.).

[0117] Melting curve analysis The third PCR included melting curve analysis and was performed in a LightCycler480 (Roche) using the cycle schedule shown in Figure 1 (the cycle schedule was identical for all three PCRs, only the melting curve analysis was added to the lightCycler machine).

[0118] The program ends with a melting curve where all PCR amplicons are slowly heated. The temperature at which the DNA strands separate is sequence and length dependent. The derivative of the fluorescence is then converted to one or more melting temperatures of the PCR products. See FIG. 2 for an example.

[0119] Species identification by melting curve analysis can be performed by comparison to a proprietary melting curve database (inBiome).

[0120] Fragment length analysis After PCR, 5 μl of PCR product was mixed with 20 μl of eMix (inBiome). DNA fragment analysis was performed on an ABI Prism 3500 Genetic Analyzer (Applied Biosystems). Data were analyzed using inBiome's proprietary software suite (inBiome, Amsterdam, the Netherlands) and results were presented as microbial profiles. Automated species calling of fragment length profiles was performed using a proprietary software suite (inBiome), where peaks were linked to a database containing IS profile information of over 500 microbial species.

[0121] Peaks below 128 RFU were considered background noise and discarded from further analysis. The entire procedure, from DNA isolation to data analysis, could be performed within 4 h.

[0122] result To demonstrate the differential potential of the combination of melting curve and fragment length data, a melting curve and fragment analysis of the 16S-23S IS region of eight different species from three different bacterial phyla was performed. The results are shown in Figure 3. Here, it can be clearly seen that the melting curves and fragment length profiles are highly variable between species, and even between different species within the same genus. By comparing these melting curve and fragment length data combinations with a database containing reference profiles of known species (inBiome), an unambiguous identification could be made at the species level for each species in this analysis.

[0123] Example 2. Accurate identification when fragments are of identical length. Situations may arise where either the melting curve or fragment length profile of one species is similar to that of another species. By combining the melting curve data and the fragment length data, a clear identification can still be made. An exemplary practical situation where the fragment length profiles are similar is shown in this example.

[0124] Although the lengths of DNA fragments from the 16S-23S interspace region are highly specific for each individual species, different species may have identical fragment length signatures. In these cases, the fragment lengths may be identical, but the nucleotide sequences within these fragments are necessarily different: modern taxonomy is based mainly on the sequence of ribosomal DNA, and different species always have different nucleotide sequences in this region, especially in the interspace (IS) region, which is the most variable region.

[0125] The melting temperature of a fragment is determined by two factors: the length and the sequence of the fragment. Therefore, if the fragment lengths of two different species are identical, the sequences will always be different and therefore the melting curve signatures must be different. This situation is illustrated in FIG. 4 for two clinically very relevant bacterial species, Streptococcus pyogenes and Corynebacterium jeikeium, for which identification to the species level is crucial for the correct treatment of the patient.

[0126] It has been shown that the two species can be accurately distinguished and identified using the methods of the present invention.

[0127] Example 3. Accurate identification when melting curves are identical. Since the melting curve depends on the length and sequence of the fragment, it is possible that the correlation between length and sequence may result in similar melting curves that are different in length and sequence, nevertheless.

[0128] In addition, similarities in melting curves may be caused by the presence of multiple (PCR) fragments whose individual melting curves are superimposed on each other. This results in complex melting curves that may indicate reduced resolution. This phenomenon may be particularly problematic in closely related species, where each species has multiple rRNA operons and thus 16S-23S IS PCR fragments. In these cases, combining melting curve data with fragment length analysis may nevertheless uniquely identify each bacterial species. This exemplary practical situation is illustrated for the analysis of 16S-23S IS profiles of closely related species in the Enterobacteriaceae family, a bacterial family of cost-challenging in clinical microbiology that includes many human pathogens. In the figure below, melting curves and fragment length analysis of Enterobacter cloacae and Klebsiella pneumoniae are shown. Due to the many similar IS-fragments in these closely related species, the melting curves are difficult to distinguish, but the unique length profiles are still able to distinguish these species (see FIG. 5).

[0129] [Table 1]

Claims

1. 1. A method for identifying microorganisms in a biological sample at the species or strain level by polymerase chain reaction (PCR), comprising: a) providing a biological sample suspected of containing microbes; b) PCR amplifying an internal transcribed spacer (ITS) region of at least one microbial rRNA contained within a nucleic acid sequence contained in the biological sample using a set of broad taxonomic range amplification primers to amplify the at least one microbial rRNA ITS, thereby generating a PCR amplicon, wherein the set of broad taxonomic range amplification primers is a set of universal bacterial amplification primers; c) recording high-resolution melting curves for the PCR amplicons generated in step b) and recording the lengths of the PCR amplicons generated in step b) by capillary electrophoresis or sequencing; d) comparing the high-resolution melting curve recorded in step c) with a database comprising high-resolution melting curves of reference amplicons generated from reference microbial species or strains of known taxonomic identity using the same set of amplification primers, thereby obtaining a first indication of taxonomic identity of the microorganism present in the biological sample; e) comparing each PCR amplicon length, having a distinct length recorded in step c), to a database comprising PCR amplicon lengths of reference amplicons generated from a reference microbial species or strain of known taxonomic identity using the same set of amplification primers, thereby obtaining a second taxonomic identity indication of the microorganisms present in the biological sample; f) identifying the microorganism present in the biological sample to the species or strain level if the first and second taxonomic indexes of identity match. The method comprising the steps of:

2. 2. The method of claim 1, wherein the biological sample suspected of containing microbes is a biological sample suspected of containing archaea, bacteria, viruses, protozoa, or fungi, or a combination thereof.

3. 3. The method of claim 1 or 2, wherein the reference amplicons of the database comprise amplicons generated by in vivo and / or in silico PCR amplification reactions to amplify corresponding rRNA ITS regions of reference microbial species or strains of known taxonomic identity using the same set of broad taxonomic range amplification primers for amplifying the at least one microbial rRNA ITS region.

4. The method according to any one of claims 1 to 3, wherein the databases in steps d) and e) are integrated into a single database.

5. The method of any one of claims 1 to 4, wherein the broad taxonomic range amplification primers are for amplifying microbial rRNA ITS regions of multiple strains or species from a genus, family, order, class, phylum, kingdom and / or domain of microorganisms.

6. 6. The method of any one of claims 1 to 5, wherein the broad taxonomic range amplification primers for amplifying at least one microbial rRNA ITS region comprise forward and reverse primers for amplifying the 16S-23S rRNA ITS region, the 23S-5S rRNA ITS region, the microbial 18S-5.8S rRNA ITS region, or the microbial 5.8S-26S / 28S rRNA ITS region.

7. 7. The method of any one of claims 1 to 6, wherein the biological sample is a subject's body sample selected from bodily fluids or exudates, including but not limited to uterine fluid, whole blood, serum, plasma, lymph, mucus, saliva, sputum, stool / feces, sweat, wound fluid, pus / purulent fluid, gastric contents, ascites deposits / ascites, bile, urine, semen, cerebrospinal fluid / liquid, and breast milk; or a subject's body sample in the form of a swab, biopsy, lavage, or paper point sample, including but not limited to samples from the skin, organs, tissues, oral cavity, urogenital, vaginal tract, gastrointestinal tract, respiratory or pulmonary system, and cardiovascular system.

8. 8. The method of any one of claims 1 to 7, wherein the set of amplification primers of the broad taxonomic range is a set of amplification primers for amplifying at least one rRNA ITS region of bacteria of the phyla Bacteroidetes and / or Firmicutes.

9. 9. The method of any one of claims 1 to 8, wherein the set of amplification primers covering the broad taxonomic region comprises each of the amplification primers in SEQ ID NOs: 1 and 3 to 5, or each of the amplification primers in SEQ ID NOs: 2 to 5, or each of the amplification primers in SEQ ID NOs: 1 to 5.

10. 10. The method of any one of claims 1 to 9, wherein said set of amplification primers covering said broad taxonomic region comprises amplification primers of SEQ ID NOs: 6 and 7 to 13, respectively.

11. 11. The method of any one of claims 1 to 10, wherein said set of amplification primers covering said broad taxonomic region comprises each of the amplification primers of SEQ ID NOs: 14-15.

12. The method of claim 1, wherein the PCR amplification step comprises qPCR.

13. The method according to any one of claims 1 to 12, wherein in step c) the PCR amplicon lengths are recorded by capillary electrophoresis or sequencing.

14. The method according to any one of claims 1 to 13, wherein step c) is carried out in a miniaturized device.

15. 15. The method of any one of claims 1 to 14, wherein the database comprises high-resolution melting curves and PCR amplicon lengths of reference amplicons generated from reference microbial species or strains of known taxonomic identity, and the database further comprises high-resolution melting curves and PCR amplicon lengths of reference amplicons generated from human sequences using the set of amplification primers of the broad taxonomic region as a control for non-specific amplicon generation.

16. The PCR amplification reaction further comprises using a PCR calibrator system, the PCR calibrator system comprising: a set of a plurality of PCR amplification primers, where at least one of the plurality of PCR amplification primers comprises a label; and a set of at least two PCR calibrators, where each PCR calibrator consists of a DNA fragment of a given length flanked by an upstream adaptor DNA sequence and a downstream adaptor DNA sequence, where the adaptor DNA sequence comprises a primer binding site for binding of the PCR amplification primer, where the set of PCR amplification primers binds to all Ps in the set of at least two PCR calibrators.

16. The method of any one of claims 1 to 15, for PCR amplifying DNA sequences of PCR calibrators, wherein spacer region DNA sequences contained in each of said PCR calibrators in said set of at least two PCR calibrators are of different lengths, and each PCR calibrator in said set of at least two PCR calibrators is present in equal or known amounts compared to other PCR calibrators in said set, and wherein said step b) of PCR amplification further comprises PCR amplifying at least two PCR calibrators using PCR amplification primers of a PCR calibrator system.

17. 17. The method of any one of claims 1 to 16, wherein the set of broad taxonomic range amplification primers for amplifying at least one microbial rRNA ITS region comprises a labeled forward primer and / or a labeled reverse primer.

18. The method of any one of claims 1 to 17, wherein a nucleic acid sequence is isolated from the biological sample.

19. The method of claim 4, wherein the database includes rRNA ITS sequences and corresponding taxonomic identity data for bacteria.

20. The method of claim 5, wherein the broad taxonomic range amplification primers are for amplifying the microbial rRNA ITS region of all strains or species from a microbial genus, family, order, class, phylum, kingdom and / or domain.

21. The method of claim 20, wherein the broad taxonomic range amplification primers are for amplifying the microbial rRNA ITS region of all strains or species from a phylum of microorganisms.

22. The method of claim 20, wherein the broad taxonomic range amplification primers are for amplifying the microbial rRNA ITS region of all strains or species from the kingdom of Microorganisms.

23. The method of claim 20, wherein the broad taxonomic range amplification primers are for amplifying the microbial rRNA ITS region of all strains or species from a microbial domain.

24. The method of claim 6, wherein the broad taxonomic range amplification primers for amplifying at least one microbial rRNA ITS region include forward and reverse primers for amplifying the 16S-23S rRNA ITS region.

25. The method of claim 14, further comprising step b) being performed in a miniaturized device.

26. The method of claim 14 or 25, wherein the miniaturized device is a lab-on-chip (LOC) device.

27. The method of claim 17, wherein the set of broad taxonomic range amplification primers for amplifying at least one microbial rRNA ITS region comprises a labeled forward primer.

28. The method of claim 27, wherein the labeled forward primer is a fluorescently labeled forward primer.

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